Download Abstract Book - 27th Annual Meeting of the Society for Virology

27th Annual Meeting of
the Society for Virology
Gesellschaft für Virologie e. V. (GfV) und
DVV
Deutsche Vereinigung zur
Bekämpfung der Viruskrankheiten e. V. (DVV)
22–25 March 2017
Marburg • Germany
Philipps-Universität Marburg
www.virology-meeting.de
Abstract Book
GfV 2017 – Abstracts of keynote speakers
Emerging Viruses ........................................................................................................................ L01 – L03
Structural Virology ....................................................................................................................... L04 – L06
Novel Antiviral Concepts ............................................................................................................. L07 – L09
Innate Immunity .......................................................................................................................... L10 – L12
GfV 2017 – Abstracts of workshops
Virus Receptors and Entry ................................................................................................... OP01 – OP06
Innate Immunity ........................................................................................... OP07 – OP12, OP25 – OP30
Diagnostic Tools................................................................................................................... OP13 – OP18
Viral Replication ........................................................................................... OP19 – OP24, OP31 – OP36
Adaptive Immunity................................................................................................................ OP37 – OP42
Emerging Viruses ................................................................................................................. OP43 – OP48
Trafficking ............................................................................................................................ OP49 – OP54
Virus Vectors and Gene Therapy ......................................................................................... OP55 – OP60
Structure and Assembly ....................................................................................................... OP61 – OP65
Tumor Viruses ...................................................................................................................... OP66 – OP71
Antiviral Therapy and Resistance......................................................................................... OP72 – OP77
Evolution of Viruses – Viruses in Evolution .......................................................................... OP78 – OP83
Vaccines .............................................................................................................................. OP84 – OP89
Viral Pathogenesis and Persistance..................................................................................... OP90 – OP95
Host Cell Factors and Modulation .......................................................... OP96 – OP101, OP114 – OP119
Epidemiology and Public Health....................................................................................... OP102 – OP107
Zoonoses ......................................................................................................................... OP108 – OP113
Clinical Virology................................................................................................................ OP120 – OP125
GfV 2017 – Abstracts of posters
Virus Receptors and Entry ......................................................................................................... P01 – P25
Innate Immunity ......................................................................................................................... P26 – P64
Diagnostic Tools......................................................................................................................... P65 – P79
Viral Replication ...................................................................................................................... P80 – P104
Adaptive Immunity.................................................................................................................. P105 – P133
Emerging Viruses ................................................................................................................... P134 – P165
Trafficking .............................................................................................................................. P166 – P170
Virus Vectors and Gene Therapy ........................................................................................... P171 – P177
Structure and Assembly ......................................................................................................... P178 – P185
Tumor Viruses ........................................................................................................................ P186 – P193
Antiviral Therapy and Resistance .......................................................................................... P195 – P227
Evolution of Viruses – Viruses in Evolution ............................................................................ P228 – P242
Vaccines ................................................................................................................................ P243 – P274
Viral Pathogenesis and Persistence....................................................................................... P275 – P307
Host Cell Factors and Modulation .......................................................................................... P308 – P367
Epidemiology and Public Health............................................................................................. P368 – P380
Zoonoses ............................................................................................................................... P381 – P392
Clinical Virology...................................................................................................................... P393 – P408
Abstracts of keynote speakers
Keynote lectures
L01
Lessons learned from Ebola R&D during a public health emergency
M.-P. Kieny1
1World
Health Organization, Geneva, Switzerland
When the Ebola outbreak in West Africa erupted in the spring of 2014, the global health community was ill prepared to cope.
There were no vaccines, no treatments, few diagnostics, and insufficient medical teams and trained responders.
In spite of this lack of R&D preparedness, the Ebola experience demonstrates that, it is possible to compress R&D timelines
from a decade or longer, to less than a single year. This is mostly to be credited to an unprecedented collaborative effort
building on the availability of a small number of candidate diagnostic tests, drugs and vaccines that could rapidly move into
clinical phase evaluation. A series of international consultations and activities – including the organization of a successful
Ebola vaccine efficacy trial in Guinea - were led by WHO as a contribution to the unprecedented global efforts to control the
Ebola epidemic.
Since September 2015, WHO expert teams, an international scientific advisory board, and partners engaged through global
forums are collaborating to articulate a novel R&D model for emerging pathogens likely to cause severe outbreaks in the
near future, and for which few or no medical countermeasures exist: the WHO R&D Blueprint. Already, several consultations
have been held among national governments and public health agencies, researchers, social scientists and industry. They
have selected priority pathogens on which immediate action is warranted, evaluated several promising platform technologies,
identified major bottlenecks to international collaboration, agreed upon basic data sharing principles, and explored innovative
approaches to conducting clinical trials. New developments on the R&D Blueprint will be presented .
Corresponding author:
Marie-Paule Kieny
[email protected]
Keynote lectures
L02
Vaccines against Emerging Viruses - Insights from Ebola, MERS and beyond
M. Addo
Keynote lectures
L03
Emergence of congenital Zika syndrome in the Americas
S. Weaver1
1University
of Texas Medical Branch, Institute for Human Infections and Immunity and Department of Microbiology & Immunology, Galveston, United States
Zika virus (ZIKV), a mosquito-borne flavivirus, was first isolated from a sentinel rhesus macaque in Uganda in 1947 and soon
thereafter from arboreal Aedes africanus mosquitoes. Although only 14 human infections were documented before 2007,
serosurveys in African and Asia suggested widespread exposure. Following 60 years of obscurity, ZIKV emerged in Yap, a
small Micronesian Island, to cause just over 100 confirmed and thousands suspected cases of febrile illness accompanied by
rash and arthralgia, and epidemiologic studies suggested that up to 73% of the human population was infected. A few years,
larger outbreaks began in French Polynesia and other islands in the South Pacific, with an estimated tens-of-thousands of
infections and an association of some with Guillain–Barré syndrome (GBS), whose incidence increased 20-fold coincident
with the ZIKV outbreak. Then, probably in late 2013, ZIKAV reached Brazil, resulting in 2015 in an explosive outbreak
involving hundreds of thousands of suspected cases, transmitted primarily by the peridomestic mosquito Aedes aegypti and
possibly also the invasive species A. albopictus. Sexual transmission has also been detected in travelers returning to nonendemic regions. In the Americas, Zika virus Infections have again been associated with GBS but also with thousands of
cases of fetal malformations when pregnant women are infected. The virus has spread to the vast majority of countries in
Latin America and the Caribbean, and congenital Zika syndrome has also appeared in many. I will review the current
situation of the epidemic in the Americas, potential explanations for its sudden and unexpected emergence, and
epidemiologic and basic research underway to test these hypotheses to understand these the dramatic spread and predict
future trends. I will also review the prospects for the development better control measures as well as products to protect
against severe outcomes of infection.
Corresponding author:
Scott Weaver
[email protected]
Keynote lectures
L04
New views of virus structure from cryo-EM
J. Briggs1
1MRC
Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom
We are using new cryo-electron microscopy methods to study the structure and assembly of viruses including HIV-1,
influenza A, and Ebola virus. These viruses assemble particles that are heterogeneous in size and shape, making it
challenging to study their structures. Taking advantage of recent developments in electron microscopy, it is now possible to
obtain detailed 3D views of viral components “in situ” within intact virus particles.
I will illustrate how cryo-electron tomography can be used to study irregular virus particles, and the new insights that can be
obtained. I will show our latest data on the maturation process of HIV-1 and the structure of Ebola virus.
Corresponding author:
John Briggs
[email protected]
Keynote lectures
L05
Enveloped virus entry and fusion
G. F. Gao1
1Chinese
Academy of Science, Chinese Center for Disease Control and Prevention, Institute of Microbiology, Beijing, China
Enveloped viruses can cause severe infectious diseases and engender a threat to public health. The process of enveloped
virus infection can be divided into three steps, entry and fusion, replication, assembly and release. Virus entry and fusion is
the first step of viral infection, which is also the target for antiviral intervention. Here we summarized the molecular basis of
entry and fusion for several emerging infectious enveloped viruses, including influenza virus, MERS coronavirus, Ebola virus
and Zika virus. We have showed how the enveloped glycoproteins of these viruses interact with their respective receptors,
providing important information to understand the interspecies transmission and pathogenesis in a decent way. Based on
these information, we have also developed a series of therapeutic antibodies to treat the infectious diseases.
Corresponding author:
George Fu Gao
[email protected]
Keynote lectures
L06
Flavivirus structural heterogeneity – implications for antibody neutralisation and vaccine design
F. Rey1
1Institut
Pasteur, Virology, Paris, France
Zika virus is a member of the flavivirus genus that had not been associated with severe disease in humans until the recent
outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré Syndrome in adults in French
Polynesia. Zika virus is related to dengue virus, and we discovered that a category of antibodies isolated from dengue
patients also potently neutralize Zika virus. We have determined the crystal structures of several of these antibodies in
complex with the envelope protein of Zika virus, which reveal the details of a conserved epitope that is also the site of
interaction with the precursor prM protein during flavivirus maturation. Comparison of the Zika and dengue virus
immunocomplexes (the structures of the latter we had determined previously) provides now a lead for a rational, epitopefocused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect against Zika and
dengue viruses simultaneously.
Corresponding author:
Felix Rey
[email protected]
Keynote lectures
L07
Antiviral therapy of persistent infection using genome editing
J. Hauber
Keynote lectures
L08
Piconnavirus replication – From basic insight to antiviral drug development
F. van Kuppeveld1
1Utrecht
University, Division of Virology, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht, Netherlands
The family of Picornaviruses contains many important pathogens for humans and animals. Well-known human pathogens
are poliovirus, enterovirus-A71, coxsackievirus, enterovirus-D68, and rhinovirus, all of which belong to the Enterovirus genus.
Vaccines are available against poliovirus and -since recently- EV-A71, but development of vaccines against other viruses is
sheer impossible given the large number of virus sero/genotypes (e.g. >100 enteroviruses and >150 rhinoviruses). Hence,
there is a great need for (broad-spectrum) antiviral drugs against enteroviruses. A short overview will be given of the current
status of antiviral drug development against enteroviruses. Then, new insights into the replication strategy and the interaction
of enteroviruses with host factors will be described, which offer novel opportunities to develop “host-targeted” antivirals.
Enteroviruses, like all other (+)RNA viruses, extensively rearrange intracellular membranes to create replication organelles
(ROs), which are generally thought to provide a scaffold for genome replication and lend protection against sensors that can
initiate antiviral host responses. First insights in the 3D structure of the picornavirus replication organelles have been
obtained, but little is known about the identity of the membrane-modifying host cell factors and pathways that are hijacked by
viral proteins to create these unique structures. Our current knowledge on the identity of these factors, which may be targets
for broad-spectrum antiviral therapy, will be summarized, with emphasis on how enteroviruses usurp lipid transfer machinery
(e.g. PI4KB and OSBP) at membrane contact sites to create a lipid environment that drives viral RNA replication.
Furthermore, new data will be presented on the membrane alterations induced by escape mutants that are largely insensitive
to PI4K and OSBP inhibitors, which challenge the dogma that accumulation of cytosolic ROs is indispensable for viral RNA
replication and evasion of innate antiviral host responses.
Corresponding author:
Frank van Kuppeveld
[email protected]
Keynote lectures
L09
HBV-Cure – Fact or fiction?
U. Protzer1
1Technical
University Munich and Helmholtz Centre Munich, Institute of Virology, Munich, Germany
Hepatitis B virus (HBV) establishes a stable nuclear persistence form, the so called cccDNA, in infected hepatocytes.
cccDNA prevents elimination of HBV. Therefore, nucleos(t)ide analogous currently used as antivirals efficiently control HBV
replication but don’t affect its persistence. Thus, hepatitis B requires long-term treatment - unlike HCV infection that can be
cured with directly acting antivirals. And it is under debate whether a complete cure of HBV is possible at all or whether one
should rather aim at a functional cure.
At least a functional cure, however, is desired for the 240-300 million HBV carriers at high risk to develop liver disease and
hepatocellular carcinoma. Taking the HBV life cycle into account, there are only limited options to target nuclear HBV
cccDNA. Capsid assembly modifiers, non-nucleosidic polymerase inhibitors, entry inhibitors and siRNAs are under
development but don’t target cccDNA directly. Talen- or CRIPSR/Cas-based editing of cccDNA are limited by the fact that
they require gene therapy and evoke safety concerns.
Corresponding author:
Ulrike Protzer
[email protected]
Keynote lectures
L10
Enforced viral replication determines outcome of disease
K. S. Lang1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
The innate immune system limits virus replication during systemic infection by producing type I interferons (IFN-I), however
still has to allow viral replication toachieve maximal innate and adaptive immune activation. CD169+ macrophages (antigen
presenting cells) in spleen and lymph node show limited response to IFN-I due to expression of the endogenous interferon
inhibitor Usp18. Therefore virus in these specialized niches replicates despite high levels of IFN-I. This enforced viral
replication leads to an exorbitant propagation of viral antigens and viral RNA. Viral antigen leads to massiveactivation of the
adaptive immune system, whilst viral RNA activates innateimmunity. In contrast to these marginal zone macrophages and
subcapsular sinus macrophages of lymph nodes, liver resident Kupffer cells, take up most of the systemic virus and suppress
it’s replication in response to IFN-I. These mechanisms of enforced viral replication and antiviral immune activation will be
discussed in the lecture
Corresponding author:
Karl Sebastian Lang
[email protected]
Keynote lectures
L11
Induction and suppression of the interferon response by RNA viruses
F. Weber1
1Justus
Liebig University Giessen, Institute of Virology, Giessen, Germany
Release of nucleocapsids into the cytoplasm is one of the first steps of virus infection. At this initial stage, viral copy numbers
are low and the nucleocapsids are especially vulnerable to host cell defenses.
The group of negative-stranded RNA viruses with a segmented genome (s-NSVs) contains several important pathogens like
influenza virus (family Orthomyxoviridae), Rift Valley fever virus (genus phlebovirus, family Bunyaviridae), or Lassa virus
(family Arenviridae). We have previously shown that the “panhandle” promoter of incoming s-NSV nucleocapsids is
recognized by RIG-I, the main cytoplasmic pathogen sensor for RNA viruses. On one hand, this triggers antiviral signalling
and IFN induction as expected. On the other hand, however, the clamping of RIG-I onto the “panhandle” promoter can exert
a direct inhibitory effect on virus propagation. In my presentation, I will sum up these findings, and present viral strategies of
counteraction.
Corresponding author:
Friedemann Weber
[email protected]
Keynote lectures
L12
Innate antiviral defense by PML nuclear bodies
T. Stamminger1
1Friedrich
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
PML nuclear bodies (PML-NBs) are SUMOylation-dependent, highly complex protein assemblies that accumulate in the
interchromosomal territories of the cell nucleus. Research of the last two decades revealed that a considerable number of
viruses have evolved effector proteins that modify PML-NBs. In many cases, this correlates with antagonization of individual
PML-NB components which act as host cell restriction factors. This overview summarizes recent advances concerning the
mechanisms how viral effector proteins modify PML-NBs. Furthermore, the functional consequences of PML-NB modification
will be discussed. In particular, recent studies revealed an emerging role of PML-NBs as coregulatory structures of both type
I and type II interferon responses. This emphasizes that targeting of PML-NBs by viral regulatory proteins has evolved as a
strategy to compromise intrinsic antiviral defense and innate immune responses. Since the interactions of viral effector
proteins like the IE1 protein of human cytomegalovirus with innate host defenses are crucial for the onset of lytic replication,
they may represent promising targets for antiviral strategies.
Corresponding author:
Thomas Stamminger
[email protected]
Abstracts of workshops
Virus Receptors and Entry
OP01
Proteomic analysis of the CD81-receptor complex reveals interactions important for hepatitis C virus and
Plasmodium entry into hepatocytes
J. Brüning1, P. Banse1, S. Kahl1, D. Todt1, F. Vondran2, L. Kaderali3, O. Silvie4, T. Pietschmann1, F. Meissner5, G. Gerold1
1Twincore
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
Medical School, Department of General, Visceral and Transplant Surgery, Hanover, Germany
3Ernst Moritz Arndt University Greifswald, Institute of Bioinformatics, Greifswald, Germany
4Sorbonne Universités, Centre d’Immunologie et des Maladies Infectieuses, Paris, France
5Max Planck Institute of Biochemistry, Martinsried/Munich, Germany
2Hanover
Introduction: Hepatitis C virus (HCV) and Plasmodium spp. are pathogens infecting the liver. Both rely on the tetraspanin
CD81 to enter cells. How CD81 exerts its functions during cell penetration of both pathogens is not completely understood.
However it is known that tetraspanins coordinate protein-protein interactions (PPIs) and thereby modulate signaling. We
hypothesized that PPIs coordinated by CD81 play a critical role in HCV and Plasmodium spp. entry.
Methods: We established co-immunoprecipitation (co-IP) protocols to purify CD81-complexes from primary human
hepatocytes (PHH) and hepatoma cell lines. We analyzed the co-IPs by high-resolution label-free quantitative proteomics
and defined the minimal CD81-interactome as statistically significant hits occurring in all tested sample conditions. Moreover,
we only included proteins which were expressed in PHH. In a functional follow up we evaluated the impact of the CD81interactors on HCV and Plasmodium spp. entry by RNAi. We further characterized the role of two proteins in the HCV life
cycle using CRISPR/Cas9 knock out cell lines.
Results: We identified a total of 44 proteins, including 11 already known CD81-interaction partners and 9 HCV entry factors.
RNAi revealed that knock down of four CD81-interactors significantly reduced HCV entry. Two of these proteins are known
CD81-interactors or HCV co-factors and were excluded from further studies. To further characterize the role of the other two
proteins, both enzymes, in virus infection, we generated CRISPR/Cas9 knock out cell lines. The knock out had no effect on
entry of vesicular stomatitis virus and human coronavirus. However, we observed a clear decrease of infection with all seven
HCV genotypes, suggesting that the two proteins are HCV-specific and pan-genotypic host factors. Furthermore, the knock
down did not affect HCV replication. We did not observe an effect on the entry of lentiviral particles pseudotyped with the
HCV glycoproteins, proposing a role of the proteins in a post-binding or lipoviroparticle-dependent step. Interestingly, RNAi
revealed that one of the two proteins as well as four additional proteins are also critical for entry of P. yoelii into hepatocytes.
Conclusion: We defined the CD81-interactome in human hepatocytes and hepatoma cells and discovered two host cell
proteins as new HCV and five host proteins as Plasmodium entry factors.
Corresponding author:
Janina Brüning
[email protected]
Virus Receptors and Entry
OP02
A novel mechanism of antibody-induced enhancement of flavivirus cell attachment and infection
D. Haslwanter1, D. Blaas2, J. Blazevic1, F. X. Heinz1, K. Stiasny1
1Medical
2Medical
University of Vienna, Department of Virology, Vienna, Austria
University of Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, Vienna, Austria
Introduction: The major surface protein E of flaviviruses can mediate cell attachment as well as membrane fusion after
endocytic uptake. Recent evidence has indicated that the E dimers on the virus surface undergo dynamic movements
leading to the transient exposure of structures that would be cryptic in a rigid viral envelope like the buried fusion peptide
(FP). The FP is known to become exposed and initiate fusion at the low pH of endosomes. Many different molecules have
been described as candidate flavivirus receptors, but their specific roles in virus entry remain obscure in most instances.
Flaviviruses can also infect Fcγ-receptor-positive cells through the internalization of virus-antibody complexes, thereby
bypassing the requirement for a true virus-specific receptor.
Objectives: In this study, we investigated factors that influence E-mediated cell attachment and infectivity of the flaviviruses
tick-borne encephalitis virus (TBEV) as well as dengue virus (DENV). Specifically, we assessed whether structural dynamics
together with antibody-mediated effects can allow the FP to contribute to cell attachment.
Methods: We used TBEV and DENV type 2, TBEV E-specific monoclonal antibodies (mabs) and primary as well as
permanent cells. Virus cell binding was quantified by qPCR and infectivity was measured by focus forming assays. Human
rhinovirus 2 (HRV2), with a known receptor, served as a control.
Results: Binding of TBEV and DENV to permanent (HeLa) and primary skin cells was very low compared to HRV2. DENV
bound efficiently to DC-SIGN bearing cells (in line with published data) whereas no such effect was observed with TBEV.
Pre-incubation of TBEV with one out of several E-specific mabs, however, enhanced binding to cells 5 to 6 fold. Increased
binding also resulted in a 2.5 to 3-fold increase of infectivity. As revealed by biochemical analyses, this mab dissociated the
E dimer and led to the exposure of the FP which we could show to be responsible for enhanced binding and infection.
Conclusion: In this study, we have identified a novel mechanism of antibody-induced enhancement of cell binding and
infection of flaviviruses that is independent of Fcγ receptors but relies on the exposure of the otherwise cryptic FP, allowing
its membrane interaction already at the stage of viral attachment.
Corresponding author:
Denise Haslwanter
[email protected]
Virus Receptors and Entry
OP03
Phosphoproteomic-based kinase profiling of influenza A virus infected cells reveals GRK2 as a crucial host-factor
for viral entry
E. Yangüez1, M. P. Dobay2, S. Stertz1
1University
2Swiss
of Zurich, Zurich, Switzerland
Institute of Bioinformatics, Lausanne, Switzerland
Influenza A viruses (IAVs) harness the cellular endocytic machinery to enter the cell and traffic through the cytoplasm to
reach the replication site in the nucleus. Coordinated early activation of particular signalling pathways has been shown to be
crucial for the entry process and the outcome of viral infection. However, the complexity of these signalling cascades, tightly
connected with each other, requires a broader analysis to identify the main routes, key mediators and direct effectors
involved in the early steps of viral replication. In order to obtain a comprehensive view of the signalling events induced during
IAV entry, we have conducted a proteome-wide SILAC-based quantitative phosphoproteomic screen of A549 cells within
minutes post-infection. We have quantified around 3000 phosphorylation sites from 1300 different proteins and identified
infection-induced changes in the phosphorylation of an important subset. Bioinformatic analysis has revealed that MAPK
signalling is the main pathway activated early during IAV infection and it is accompanied by significant changes in the
phosphorylation of key proteins of the cytoskeleton and the vesicle-mediated transport. Using an algorithm based on the
differential phosphorylation of putative targets, we have identified the G protein-coupled receptor kinase 2 (GRK2) as one of
the key kinases activated during IAV entry. Both silencing or chemical inhibition of GRK2 leads to drastic reduction in viral
replication, which is related with a defect in the virus trafficking through the endosomal pathway. Altogether, our results
indicate that targeting GRK2 kinase activity could be a promising strategy for future antiviral interventions.
Corresponding author:
Emilio Yangüez
yangü[email protected]
Virus Receptors and Entry
OP04
A conserved Eph receptor-binding motif and novel receptor interactions of the gH/gL glycoprotein complexes of
KSHV and RRV – determinants of infectivity and tropism
A. Großkopf1, A. Ensser2, S. Schlagowski1, R. Desrosiers3, A. Hahn1
1German
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
3Miller School of Medicine, University of Miami, Miami, United States
2Friedrich
Kaposi"s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi"s sarcoma and two Bcell proliferative disorders. The rhesus monkey rhadinovirus (RRV) is a closely related γ2-herpesvirus and an animal model
virus for KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complex of
KSHV (Hahn et al, 2012) and RRV (Hahn et al, 2013). Through comparative sequence analysis of the RRV and KSHV gH/gL
complexes and mutational screens we identified conserved residues in the N-terminal domain of gH that are critical for Ephbinding in vitro. Based on these results, we generated RRV and KSHV mutants negative for Eph-binding. Eph-bindingdeficient KSHV and RRV were still found to be infective on many types of cells, exhibiting normal attachment but a defect in
infectivity of at least one log order of magnitude. Eph-binding-negative KSHV and RRV mutants were not inhibited by
blockade of the Eph-interaction, confirming de-targeting from these receptors. While Eph-binding negative RRV mutants
were replication competent on fibroblasts, we found a drastic reduction in the ability of these mutants to infect endothelial
cells: Large subpopulations within endothelial cell cultures remained refractory to infection despite high amounts of input
virus. These results confirm the essential nature of the Eph-interaction for infection of endothelial cells by RRV and establish
the existence of Eph-independent entry mechanisms for RRV and KSHV, suggesting the use of alternative receptors for the
infection of certain cell types. Compatible with the latter hypothesis, we identified the membrane protein Plxdc2 as a
candidate receptor that also binds to the N-terminal region of RRV gH. Currently, we are further investigating the role of
these different interactions in regard to infection and signaling on different cell types. Taken together, our results i) establish
the N-terminal domain of the rhadinoviral gH/gL complex as a conserved receptor-binding domain, ii) identify a critical and
evolutionarily conserved Eph-binding motif present on KSHV and RRV gH/gL, and iii) demonstrate interactions with
additional cellular factors by this very same domain of RRV gH/gL.
Corresponding author:
Anna Großkopf
[email protected]
Virus Receptors and Entry
OP05
A polymorphism within the internal fusion loop of the Ebola virus glycoprotein modulates host cell entry efficiency
M. Hoffmann1, L. Crone1, E. Dietzel2, J. Paijo3, M. González Hernández1, U. Kalinke3, S. Becker2, S. Pöhlmann1
1German
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
University Marburg, Institute of Virology, Marburg, Germany
3Twincore GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany, a joint venture between the Helmholtz Centre for Infection
Research and the Hanover Medical School, Institute of Experimental Infection Research, Hanover, Germany
2Philipps
Introduction: Ebola virus (EBOV) infection can cause a life-threatening disease, Ebola virus disease (EVD), with casefatality rates of up to 90 %. The viral glycoprotein (GP) facilitates the first step in EBOV infection, entry into target cells. The
large scale of the EVD outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the
responsible EBOV strain differed from those of other ebolaviruses. We had previously reported that the GP of the virus
circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two non-human primate (NHP)derived cell lines relative to the GP of a virus detected during the outbreak in Zaire in 1976 (EBOV1976) (Hofmann-Winkler et
al., 2015; Hoffmann et al., 2016).
Objectives: The goal of our study was to identify the molecular determinants underlying the differential entry efficiencies of
EBOV1976 and EBOV2014.
Material & Methods: We compared replication kinetics of authentic EBOV1976 and EBOV2014 and investigated the
capacity of wildtype and mutant GPs to mediate entry into a variety of human, NHP and fruit bat cell lines, employing
rhabdoviral pseudotypes.
Results: EBOV2014-GP-driven entry into diverse NHP-derived cell lines as well as human macrophages and dendritic cells
was reduced as compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was
comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976. Mutagenic
analysis revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell
line-independent manner but did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast,
polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry
phenotype.
Conclusion: Our results show that position 544 is an important determinant of EBOV infectivity for NHP and certain human
target cell lines.
Corresponding author:
Markus Hoffmann
[email protected]
Virus Receptors and Entry
OP06
ErbB2 as part of the secondary and entry receptor complex
S. Mikulicic1, K. D. Scheffer1, F. Boukhallouk1, S. Tenzer2, K. Reiß3, L. Florin1
1University
Medical Center Mainz, Institute of Microbiology and Hygiene, Mainz, Germany
Medical Center Mainz, Department of Immunology, Mainz, Germany
3University Hospital Schleswig-Holstein Campus, Department of Dermatology and Allergology, Kiel, Germany
2University
Introduction: Human Papillomaviruses (HPV) infect skin and mucosa and induce warts and cancers. Infection with high-risk
type HPV16 may cause carcinomas of the cervix and other anogenital sites. HPV16 enters their host cell by a novel clathrinindependent pathway via tetraspanin-enriched microdomains.
Objectives: HPV16 attaches to primary receptors on the cell surface from which it is transferred to the entry receptor
complex in tetraspanin-assemblies. The composition of the entry receptor complex is still a matter of debate.
Material & Methods: We performed proteome analysis by quantitative mass spectrometry to identify components of the
HPV16 entry receptor complex and therefore potentially enriched in endosomes after virus endocytosis. We found that the
growth factor receptor ErbB2 was enriched in endosomes after infection. In order to characterize the function of ErbB2 in
HPV16 infection, immunofluorescence, loss-of-function studies, phosphorylation assays and pseudovirus (PsV) infection
assays were performed.
Results: Mass spectrometry showed strong enrichment of growth factor receptor ErbB2 in endosomes after infection. This
result implicates co-internalization of ErbB2 and the virus particles thereby suggesting ErbB2 as part of the entry receptor
complex. Immunofluorescence analyses confirmed close association of ErbB2 and HPV16 on the cell surface. Subsequently,
knock-down of ErbB2 caused strong inhibition of HPV16 pseudovirus infection and confirmed functional requirement of the
growth factor receptor. In addition, we observed phosphorylation of ErbB2 after PsV cell binding what brings us to the
conclusion that binding of HPV16 to the cell surface triggers ErbB2-mediated signalling events.
Conclusion: Our results showed that ErbB2 plays a role in viral entry and triggers yet unknown signalling events. Defining
the role of ErbB2 in HPV16 infection could advance our understanding of a novel endocytic pathway that is used by HPV16
during infection.
Corresponding author:
Snjezana Mikulicic
[email protected]
Innate Immunity
OP07
Nuclear RIG-I as an influenza A virus restriction factor
M. Gerlach1,2,3, W. L. Yao2,3, J. M. Cabrejas 2,3, A. Makino4, K. Tomonaga4,5,6,7, H. Kato2,3, F. Weber1, T. Fujita2,3
1Justus
Liebig University Giessen, Institute of Virology, Giessen, Germany
University, Laboratory of Molecular Genetics, Institute of Virus Research, Kyoto, Japan
3Kyoto University, Laboratory of Molecular Cell Biology, Graduate School of Biostudies, Kyoto, Japan
4Kyoto University, Department of Viral Oncology, Institute of Virus Research, Kyoto, Japan
5Kyoto University, Center for Emerging Virus Research, Institute of Virus Research, Kyoto, Japan
6Kyoto University, Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto, Japan
7Kyoto University, Department of Tumor Viruses, Graduate School of Medicine, Kyoto, Japan
2Kyoto
RIG-I is a well-described cytoplasmic sensor of viral RNA molecules. RIG-I interaction with its cognate RNA agonist
stimulates downstream signaling via the adapter molecule MAVS, culminating in activation of the antiviral type I interferon
(IFN-alpha/beta) response. Besides this established function of RIG-I in transmitting cytoplasmic signaling, less is known
whether RIG-I exhibits antiviral activity in the nucleus.
Here, we report the presence of nuclear RIG-I in various immortalized and primary human cell lines. A bipartite nuclear
localization signal (NLS) within the RIG-I helicase domain thereby promotes importin-alpha binding and nuclear import.
Mutation of RIG-I-NLS accordingly retains RIG-I in the cytoplasm. During viral infection, nuclear RIG-I accumulation was
triggered by the presence of 5`triphosphorylated (5`ppp) influenza A virus (FLUAV) and Thogoto virus (THOV) nucleocapsids
in the nucleus. However, RIG-I remained in the cytoplasm during infection with viruses replicating in the cytoplasm like La
Crosse virus (LACV), Rift valley fever virus or New Castle Disease virus (NDV). Despite nuclear replication,
5`monophosphorylated (5`p) nucleocapsids of Borna disease virus (BDV) did not trigger nuclear RIG-I translocation.
Immunoprecipitation of nuclear fractions confirms an interaction of RIG-I with 5`ppp-FLUAV nucleocapsids in the nucleus.
Furthermore, a RIG-I mutant excluded from the nucleus results in a reduced antiviral effect against FLUAV whereas enforced
nuclear RIG-I localization by an artificial NLS limits viral replication. The nuclear antiviral effect was also observed in the
background of the RIG-I-K270A mutant that binds RNA but deficient in downstream signaling function, and in cells lacking
the signaling adapter MAVS. This indicates an antiviral RIG-I activity in the nucleus independent of IFN response initiation.
In summary, in addition to the well-studied activities of cytoplasmic RIG-I, we identified RIG-I as a nuclear factor that directly
interferes with FLUAV at the site of virus replication.
Corresponding author:
Michaela Gerlach
[email protected]
Innate Immunity
OP08
The evolutionary stand-off between STAT2-mediated antiviral activity and virus-encoded STAT2 antagonism
determines host as well as virus survival
V. T. K. Le-Trilling1, K. Wohlgemuth1, M. Rückborn1, A. Jagnjic1, L. Timmer1, B. Katschinski1, M. Trilling1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Interferons (IFN) are pleiotropic cytokines which are induced upon pathogen encounter and signal via janus kinases and
STAT transcription factors to establish an antiviral state. However, host and pathogens are situated in an ongoing arms race
in which the selective pressure elicited by pathogens continuously drives the evolution towards enhanced host immune
responses while the antiviral activity of the immune system shapes viral evolution towards more effective antagonists.
Human and mouse cytomegalovirus (HCMV and MCMV) both counteract IFN signalling by reducing the amounts of STAT2.
The MCMV-encoded protein pM27 induces (poly-) ubiquitination and subsequent proteasomal degradation of STAT2.
Using MCMV mutants lacking M27 and mice lacking STAT2 as well as their respective wildtype counterparts, we studied the
opposing relation between antiviral activities and viral antagonistic countermeasures. ΔM27-MCMV exhibits extreme
susceptibility towards IFNs in vitro. This effect was fully reverted in the absence of STAT2 or pharmacologic inhibition of
janus kinase activity, indicating that IFN antagonism is the relevant function of pM27. In vivo, ΔM27-MCMV replication was
efficiently cleared from all organs after 3 days in BALB/c, C57BL/6 and 129 mice, highlighting a mouse strain-independent
importance of STAT2 antagonism for MCMV replication.
Despite this clear impact of viral STAT2 antagonism, STAT2-deficient mice are highly susceptible to MCMV replication. The
viral replication was increased in all organs and was even disseminated to the brain, and within one week STAT2-deficient
mice became moribund. Taken together, our study reveals the central role of STAT2 for virus control as well as the
importance of cytomegaloviral IFN antagonism in vitro and in vivo. This mutual influence resulted in an evolutionary stand-off
situation with severe effects when the balance is disturbed.
Corresponding author:
Vu Thuy Khanh Le-Trilling
[email protected]
Innate Immunity
OP09
Identification of potent restriction factors of HCV infection and their contribution to viral species-tropism
R. Brown1, C. Ginkel1, M. Engelmann1, A. Kusuma1, J. Sheldon1, T. Khera1, K. Welsch1, R. Weller1, D. Todt1, P. Perin1, K.
Hüging1, G. Vieyres1, G. Gerold1, D. Bankwitz 1, E. Steinmann1, Q. Yuan1, M. Ott1, E. Michailidis2, V. L. Dao Thi2, M. Saeed2,
Q. Ding3, B. Winer3, C. Rice2, A. Ploss3, T. Pietschmann1
1Twincore
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
University, Laboratory of Virology and Infectious Disease, New York, United States
3Princeton University, Department of Molecular Biology, Princeton, United States
2Rockefeller
Introduction: Hepatitis C virus (HCV) has a narrow host range and mice are refractory to infection. Humanized mice with
blunted innate immunity support only low level viral replication.
Objectives: This study aimed to identify restriction factors which suppress HCV replication in mice.
Material & Methods: A liver cDNA library from an IFN-treated mouse was transduced it into human hepatoma cells which
were modified to undergo apoptosis upon HCV replication. Iterative rounds of selection with HCV resulted in cell population
that was 200-fold less permissive for HCV infection. RNAseq revealed this phenotype resulted from enrichment of a subset
of delivered murine liver genes.
Results: Lentiviral over-expression of individual mouse genes identified two potent restriction factors of HCV genotype 1-7
infection. Combined over-expression of both factors resulted in a 2.5 log drop in the ability to infect permissive cells and RNA
silencing of over-expressed factors rescued infectivity. These factors were also able to restrict infection of permissive cells
with primary HCV isolates when combined with over-expression of human SEC14L2. RNAseq revealed transcripts of both
factors were highly abundant in murine livers from different laboratory mouse strains, and immunofluorescence microscopy
and FACS indicate this correlated with high levels of protein expression. Both factors were non-interferon inducible in primary
mouse hepatocytes. The human homologs showed differential levels of restriction: one homolog was also a potent HCV
restriction factor whereas the second had no effect. Levels of human homolog expression in human hepatocytes also
differed from the pattern observed in murine livers.
Conclusion: Mechanistic investigation of how these factors mediate their effect and their liver-specific ablation in mice using
CRISPR/Cas9 technology is currently underway. In summary, newly identified murine restriction factors contribute to the
human: mouse species barrier in HCV infection, and represent targets for gene knock-out to facilitate the generation of a fully
permissive immunocompetent small animal model for HCV.
Corresponding author:
Richard Brown
[email protected]
Innate Immunity
OP10
Secretion of Hepatitis C virus replication intermediates in extracellular vesicles reduces TLR3 response in
hepatocytes
O. Grünvogel1, V. Klöss2, A. Reustle1, K. Esser-Nobis1, I. K. Stöck1, J. Y. Lee1, R. Bartenschlager1, A. Dalpke2, V. Lohmann1
1University
2University
of Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
of Heidelberg, Department of Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg, Germany
Introduction: Replication of positive strand RNA viruses results in a constant production of viral replication intermediates, in
particular double-stranded RNA (dsRNA), representing potent inducers of innate immune responses sensed by pattern
recognition receptors (PRRs) like TLR3. TLR3 is activated by hepatitis C virus (HCV) within endosomes of infected
hepatocytes and induces strong antiviral and inflammatory responses.
Objectives: We aimed to characterize the fate of HCV-derived replication intermediates in HCV-positive cells, and to
investigate the activation of PRRs as well as viral avoidance mechanisms in the context of persistent viral replication.
Material & Methods: HCV positive and negative strand RNA was quantified by strand-specific RT-qPCR.
Immunofluorescence and correlative light electron microscopy was used to analyze the localization of dsRNA and HCV
replication complexes within cells. PRRs were ectopically expressed in hepatoma cells and activation by HCV was monitored
by RT-qPCR.
Results: dsRNA and entire HCV replication complexes were detected in the late endosomal and lysosomal compartment,
also co-localizing with exosome markers. We therefore hypothesized that dsRNA can be secreted in extracellular vesicles
(EVs) to avoid intracellular detection by PRRs. Indeed, HCV-positive EVs contained large amounts of HCV positive and
negative strand RNA, with ratios shifted strongly towards dsRNA when compared to intracellular HCV RNA. A significant
portion of negative strand HCV RNA was secreted into the apical compartment of polarized cells, arguing that part of the
secreted dsRNA can avoid basolateral secretion and exposure to immune cells in the liver sinusoids. Block of exosome
release in HCV-positive cells increased intracellular dsRNA levels and led to an increased activation of TLR3, strongly
inhibiting HCV replication. In contrast, hepatitis A virus replication intermediates do not activate or reach TLR3, indicating that
a balanced TLR3 induction might help to establish persistence.
Conclusion: Our results indicate that part of the HCV dsRNA intermediates are released from infected cells in extracellular
vesicles to attenuate activation of TLR3 in HCV-infected cells. This mechanism might represent a strategy to balance the
innate immune response to maintain chronic infection.
Corresponding author:
Oliver Grünvogel
[email protected]
Innate Immunity
OP11
Polarized innate immune response against enteric viruses reveals novel mechanisms of immune tolerance in the
human gut
M. Stanifer1, D. Albrecht2, K. Pervolakari2, S. Bartfeld3, S. Boulant1,2
1University
of Heidelberg, Infectious Disease: Virology, Heidelberg, Germany
Heidelberg, Germany
3University of Würzburg, Würzburg, Germany
2DKFZ,
Question: Polarized intestinal epithelial cells (IECs) lining the surface of our gastrointestinal tract have their apical sides in
constant contact with the lumenal commensal flora. As such, IECs are faced with a major challenge as they must tolerate the
presence of the microbiota while maintaining full responsiveness against enteric pathogens. How IECs achieve such tailored
immune response remains unclear but it is known that an inappropriate response against the microbiota participates in
inflammatory bowel diseases. Here, we are identifying and characterizing the IECs-specific molecular mechanisms that
govern this unique immune response and ultimately lead to gut immune homeostasis.
Methods: The antiviral innate immune response developed by the gut epithelia was investigated in human primary nontransformed IECs by using mini-gut organoids. To address whether the polarized nature of the intestinal epithelia participates
in immune homeostasis, IECs were infected with enteric viruses, in a polarized manner using transwell inserts and sidespecific microinjection of organoids. How hIECs sense and respond to viral infection was addressed by confocal microscopy,
q-PCR, Western blot, and genetic and pharmacological ablation of innate immune functions.
Results: We found that IECs mount a distinct immune response as a function of infection side. Viral infection of IECs from
the basolateral side triggers a stronger innate immune response characterized by prolonged production of type III interferon
compared to infection emanating from the apical side. We identified that the asymmetric immune response is mediated by
the RIG-I like receptors (RLRs). Importantly, we demonstrated that prolonged production of type III IFN during basolateral
infection is fully dependent on the polarized nature of IECs which specifically regulate TLR3 signaling.
Conclusion: We have identified novel mechanisms developed by hIECs to regulate their immune response as a function of
infection side. This polarized response would represent a strategy to maintain gut immune homeostasis by avoiding
excessive response against microbes incl. viruses located in the lumenal side while maintaining full responsiveness against
invasive pathogens that have passed the epithelium barrier.
Corresponding author:
Megan Stanifer
[email protected]
Innate Immunity
OP12
LGP2 plays an essential role in HCV infection-induced interferon responses
L. Hei1, J. Zhong1
1Chinese
Academy of Sciences, Institut Pasteur of Shanghai, Shanghai, China
Introduction: RIG-I-like receptors (RLRs) are cytosolic pattern recognition receptors (PRR) that detect non-self RNA and
activate downstream interferon (IFN) signaling. One of the RLRs, LGP2 was originally thought to be a negative feedback
regulator in RIG-I signaling pathway, but growing evidence indicates LGP2 is one co-factor of MDA5 in MDA5-mediated IFN
signaling activation. The role of LGP2 in HCV infection-induced IFN signaling has not been elucidated.
Objectives: The aim of this work is to study the role of LGP2 in HCV infection-induced IFN signaling.
Material & methods: The previously established Huh7-MAVSR cell, in which the NS3/4A cleavage site in MAVS (amino acid
residue position 508) was mutated from cysteine to arginine, was used to study the IFN signaling during HCV infection. HCV
infection induced robust IFN response in Huh7-MAVSR cells, allowing for investigation of IFN signaling pathway in the
context of HCV infection. The CRISPR/Cas9 technology was employed to generate LGP2 knockout Huh7-MAVSR cells. The
LGP2 knockout cells were tested for the activation of IFN signaling in response to HCV infection, the transfection of HCV
3UTR RNA or double-stranded RNA mimics Poly(I:C). Biochemical assays were performed to probe the LGP2/MDA5
interaction and MDA5 binding to viral RNA during HCV infection. The role of LGP2s ATPase activity in its function in HCVinduced IFN signaling activation was determined.
Results: Knockout of LGP2 in hepatocytes significantly diminished the IFN production in response to HCV infection but not
to HCV 3UTR RNA transfection. Mechanistic studies showed that LGP2 exerted its function at a step upstream of MDA5 in
the IFN signaling. HCV infection promoted the molecular interaction between LGP2 and MDA5, which in turn enhanced
MDA5-HCV RNA association. Finally we showed that the ATPase activity of LGP2 was critical for assisting MDA5-HCV RNA
interaction and activating IFN signaling during HCV infection.
Conclusion: In summary, our work demonstrated that LGP2 plays an essential role in activating IFN signaling against HCV
infection by promoting MDA5 recognition of HCV RNA.
Corresponding author:
Jin Zhong
[email protected]
Diagnostic Tools
OP13
Effective detection of porcine cytomegalovirus (PCMV) in piglets using samples collected by non-invasive methods
V. Morozov1, G. Heinrichs2, J. Denner1
1Robert
Koch Institute, Berlin, Germany
Minipigs, Aachen, Germany
2Aachen
Background: The shortage of human organs forced the development of xenotransplantation using cells, tissues and organs
from pigs. Xenotransplantation may be associated with the transmission of porcine zoonotic microorganisms, among them
the PCMV. PCMV belongs to the Herpesviridae family, subfamily Betaherpesvinae, genus Roseolavirus. In adult animals
PCMV is latent and the virus titer in body fluids and organs is very low or even undetectable. The low virus titer is a principal
obstacle in PCMV diagnostic by PCR. PCMV significantly reduces the survival time of porcine transplants in non-human
primates. To perform regular follow-ups, breeding programs and reduce stress in animals, samples should be collected by
non-invasive methods. Here, we compared PCMV load in biological samples that were collected from piglets by low-invasive
and non-invasive means.
Material & Methods: Blood, ear biopsies and non-invasively taken samples, such as oral and anal swabs, were collected
from ten 10 days-old minipigs from Aachen. Extracted DNA specimens were investigated using sensitive nested PCR,
uniplex real-time PCR [Morozov et. al., Arch.Virol., 2016] and duplex PCR, which targeted simultaneously the DNA
polymerase gene of PCMV and the porcine cyclophilin A. The sensitivity of detection methods was 5 (nested PCR) and 1-5
(real-time PCR) genome equivalents (g.e.)/reaction.
Results: PCMV was detected in 10/10 oral swabs, 7/10 anal swabs and 6/10 sera. The highest virus load was detected in
oral swabs. Contribution of milk to virus dissemination is possible. A duplex real-time PCR demonstrated reduced sensitivity
compared with the uniplex PCR when amount of targets were below 200 g.e./reaction. If there were less than 15
g.e./reaction of PCMV were present in samples the virus was undetectable by duplex real-time PCR.
Conclusion: All ten 10 days-old Aachen minipigs were exposed to PCMV. Testing of oral and anal swabs by sensitive
uniplex real-time PCR is recommended for virus early diagnostic. Testing of animal samples collected by non-invasive
means right before the xenotransplantation should be mandatory.
Acknowledgments: This study was supported in part by the German Research Foundation (Deutsche
Forschungsgemeinschaft), TRR 127.
Corresponding author:
Vladimir Morozov
[email protected]
Diagnostic Tools
OP14
Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes
M. Hassemer1, M. Finkernagel1, K. H. Peiffer2, D. Glebe3, S. Akhras1, A. Reutter4, H. Scheiblauer5, L. Sommer2, M. Chudy1,
C. M. Nuebling1, E. Hildt1,6
1Paul
Ehrlich Institute, Virology, Langen, Germany
Wolfgang Goethe University Frankfurt, Frankfurt a.M., Germany
3Justus Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
4Paul Ehrlich Institute, Allergology, Langen, Germany
5Paul Ehrlich Institute, IVD, Langen, Germany
6German Centre for Infection Research (DZIF), Braunschweig, Germany
2Johann
For the human hepatitis B virus (HBV) eight distinct and two candidate genotypes are currently described. These genotypes
differ with respect to their geographic distribution, molecular virology and virus-associated pathogenesis. HBV surface
antigen (HBsAg) represents the principal marker for the diagnosis of HBV infections. Mandatory HBsAg screening of blood
donors has led to a drastic reduction of transfusion-transmitted HBV infections. Highly sensitive HBsAg assays are crucial for
optimal detection of HBV-positive blood donors and for ensuring blood safety. With respect to the different HBV genotypes it
has to be ensured that the analytical sensitivity established for different assays does not depend on HBV genotypes.
Based on sera obtained from infected patients, the International Consortium for Blood Safety (ICBS) established HBsAg
master panels, which include the major HBV genotypes A-F and HBsAg subtypes adw2-4, ayw1-4 and adr. The availability of
these materials is limited and cannot be reproduced exactly. In light of this it was investigated whether recombinant HBsAg
can serve as standard material.
Comparative analysis of HBsAg derived from the different HBV genotypes revealed, with exception of HBV/G that shows
impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular
distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of
L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different
posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, the detectability of
purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated,
showing similar sensitivities for the genotypes included in this analysis. Moreover these data demonstrate that recombinant
HBsAg reproducibly purified following a defined protocol can be used as an alternative to the reference materials currently
established.
Corresponding author:
Eberhard Hildt
[email protected]
Diagnostic Tools
OP15
Differentiation of flavivirus infections using a multi antigen ELISA based on recombinant envelope proteins with
mutations in the conserved fusion loop domain
A. Rockstroh1, B. Moges1, F. von Daak1, L. Barzon2, M. Niedrig3, S. Ulbert1
1Fraunhofer
IZI, Immunology, Leipzig, Germany
of Padova, Molecular Medicine, Padova, Italy
3Robert Koch Institute, Berlin, Germany
2University
Introduction and Objective: The high cross-reactivity of antibodies derived from flavivirus infections is a challenging fact in
serodiagnosis of important human pathogens such as Dengue, Zika, Yellow fever, Japanese encephalitis, and Tick-borne
encephalitis viruses (DENV, ZIKV, YFV, WNV, JEV, and TBEV, respectively). Moreover, areas of co-circulating flaviviruses
are increasing and clinical symptoms are similar in many cases which raises the need of a reliable serological differentiation
of these infections. The envelope (E) protein is a major target of the humoral immune response and its highly conserved
fusion loop (FL) domain binds the majority of cross-reacting antibodies. Therefore, we inserted mutations in the FL domains
of DENV, ZIKV, WNV and TBEV E proteins.
Methods: Recombinant quadruple E protein mutants (Equad) of DENV (Serotype 1-4), ZIKV, WNV and TBEV were stably
expressed in Drosophila S2 cells and purified from culture supernatants with affinity and size exclusion chromatography.
Antibody responses were measured in IgM- and IgG- based ELISAs with DENV, ZIKV, WNV, TBEV infected, YFV
vaccinated and flavivirus negative human serum samples.
Results: All serum samples from infected individuals were detected as positive for at least one flavivirus; hence sensitivity
and specificity are 100 %. Many DENV-positive sera displayed residual cross-reactivity, mostly to ZIKV Equad. Sera from
DENV infected patients bound strongly the DENV antigen and less to the others, including ZIKV, whereas ZIKV positive
serum samples behaved the opposite way. By simultaneous measurement of the same serum sample with antigens from
several viruses a specific differentiation of all tested flaviviruses could be achieved.
Conclusion: Here, we present a multi antigen based ELISA system for the simultaneous testing of human serum samples
for DENV, ZIKV, WNV and TBEV IgM- and IgG antibodies which allows their discrimination with high specificity and
sensitivity.
Corresponding author:
Alexandra Rockstroh
[email protected]
Diagnostic Tools
OP16
Development of a novel highly specific ZIKV immune complex binding IgG ELISA
A. Rackow1, C. Ehmen1, A. M. Mallmann1, J. Sievertsen1, H. Schmitz2, J. Schmidt-Chanasit2, C. Deschermeier1, A. Mika1
1Bernhard
2Bernhard
Nocht Institute of Tropical Medicine, Diagnostics Development, Hamburg, Germany
Nocht Institute of Tropical Medicine, Virology, Hamburg, Germany
Recently we developed a novel immune complex binding (ICB) ELISA to detect Zika virus specific human IgG antibodies.
The test principle is based on the binding of immune complexes formed between IgG antibodies from Zika patients and
recombinant antigens by CD32, an Fcɣ receptor. Recombinant antigens were cloned using Zika virus RNA. Polyhistidine
fusion proteins were expressed in E. coli, purified by affinity chromatography and size exclusion and labeled with horseradish
peroxidase. In addition, purified recombinant homologous proteins from other flaviviruses were added to the assay as
competitors in order to suppress interference by other flaviviral antibodies.
Pre-validation with more than 200 human sera from a range of healthy blood donors with diverse flaviviral vaccinations and
flaviviral infections (ZIKV, DENV, TBEV, JEV, WNV, YFV) collected in Europe but also in South American and Asian Dengue
endemic regions showed a very high specificity of the test system. In addition, a comparison with a commercial ZIKV IgG
ELISA was performed.
Corresponding author:
Anne Rackow
[email protected]
Diagnostic Tools
OP17
How to manage the change of measurement method for viral load in an ISO 15189 accredited laboratory using the
Panther platform?
H. Le Guillou-Guillemette1, A. Pivert1, C. T. Tran1, A. Ducancelle1, F. Lunel-Fabiani1
1University
Hospital of Angers, Laboratory of Virology, Angers, France
Question: The objective was to verify in a short time the routinely performance of the Panther platform (Hologic) for the HIV,
HCV ad HBV viral load in an ISO 15189 accredited laboratory of molecular virology. Previous accreditation was obtained with
the M2000 platform (RealTime m2000, Abbott Molecular Diagnostics). Time for evaluation before production was limited to 2
weeks as the switch of method occurred in a process of bidding for public procurement contract.
Methods: Each following criteria was evaluated for the 3 viruses. Repeatability was evaluated with a panel of certification.
Reproducibility was determined using the 2 positive internal quality controls from the manufacturer. Pre-production data were
collected to obtain minimal evaluation (N= 12 to 17). Results obtained thereafter in production period were added to improve
the pertinence of the evaluation (N=36 to 72). Our results were compared to our previous results obtained in the routinely
use with the Abbott platform. A minimal comparison of method with our previous automated platform was performed with 10,
24 and 41 samples for HIV, HCV and HBV respectively representing the genetic diversity. Contamination was also
evaluated. The exactitude has been determined a posteriori with the next external quality control run.
Results: All the coefficients of variation (CV) and measurement uncertainty (MU) were correct and similar to our previous
data (CV<10%, MU<0,2 log copies or UI/ml for low level and under the 0.5 log copies or UI/ml corresponding to the clinical
significance level). Any contamination was observed. Correlations were excellent: r=0,98/0,96/0,96 for HIV, HCV and HBV
respectively
Conclusion: Despite a reduced time, the evaluation of the Panther platform performances can be performed as required to
maintain our ISO 15189 accreditation for viral load. Results were reliable, reproducible and similar to the previous results
achieved on the Abbott platform. The workflow of the lab is greatly improved. Now the random access loading coupled with
the simultaneous assays of the three viral allows an a priori viral qualification of each donor of organs. Measure of the HIV
viral load in the cerebrospinal fluid and HCV RNA stability before centrifugation (limited to 6H in the notice) are under
evaluation.
Corresponding author:
Hélène Le Guillou-Guillemette
[email protected]
Figure 1
Diagnostic Tools
OP18
Deciphering the viral origin of acute febrile illnesses in Uganda using a metagenomic next-generation sequencing
approach
H. Jerome1, T. Byaruhanga2, B. Kigozi2, H. Bukenya2, J. Salazar-Gonzalez3, M. Salazar3, A. Nankya2, G. Kharod4, S.
Shadomy4, D. Blaney4, W. Bower4, J. Bwogi2, R. Downing2, J. Kayiwa2, J. Lutwama2, P. Kaleebu2,3, E. Thomson1
1University
of Glasgow, MRC - Centre for Virus Research, Glasgow, United Kingdom
Virus Research Institute, Entebbe, Uganda
3MRC / Uganda Virus Research Institute , Uganda Research Unit, Entebbe, Uganda
4Centers for Disease Control and Prevention, Atlanta, United States
2Uganda
Background: A high percentage of infectious diseases in East Africa remain undiagnosed (33-64%; Crump et al. 2013) and
viral pathogens in particular are suspected to circulate in the human population beneath the radar of local health care
systems. We investigated 255 serum samples from Ugandan patients with fever of unknown cause. The majority of samples
were obtained from the Acute Febrile Illness Study (n=218); originating from patients in three distinct regions of Uganda who
remained undiagnosed after extensive serological tests targeting common causes of acute febrile illness including malaria,
chikungunya virus, rickettsioses (typhus and spotted fever), dengue virus and West Nile virus.
Methods: We analysed plasma samples using an unbiased metagenomic RNA next-generation sequencing approach on the
Illumina MiSeq platform, with the aim of identifying nucleotide sequences from undiagnosed viral pathogens. We used
mapping and de novo assembly software (Tanoti and DipSPADES) to identify viral pathogens.
Results: We found evidence for viral pathogens in 40 of 255 samples. These included hepatitis A virus (HAV) (n=16) and
hepatitis E virus (HEV) (n=8), measles virus (MeV) (n=5) and human immunodeficiency virus 1 (HIV-1) (n=5). In addition we
identified the following pathogenic viruses in one sample respectively: Le Dantec virus (LEDV) which was last described
clinically in 1977, Saffold virus (SAFV) which is a cardiovirus recently discovered in stool and respiratory samples of feverish
children, Crimean Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Dengue virus 3 (DENV3) and
Yellow fever virus (YFV) which was an Ugandan strain and unrelated to the current outbreak in Angola and DRC. We did not
find evidence for the circulation of Zika virus in Uganda.
Conclusion: Knowledge about pathogens circulating in humans In East Africa is essential for health care authorities to
prevent future infectious disease outbreaks. Unbiased metagenomic next-generation sequencing on sentinel patients reveals
low-level transmission of pathogens of epidemic potential. Further studies using PCR-based tests are necessary in order to
validate our findings, estimate the prevalence of these viruses in the Ugandan population and to be prepared for infectious
disease outbreaks caused by them.
Corresponding author:
Hanna Jerome
[email protected]
Viral Replication
OP19
The 5'-terminal stem-loop 2 is a structurally and functionally conserved cis-acting RNA element in coronavirus
genomes
R. Madhugiri1, N. Karl1, D. Petersen1, M. Fricke2, M. Marz2, J. Ziebuhr1
1Justus
Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
Schiller University Jena, Faculty of Mathematics and Computer Science, Jena, Germany
2Friedrich
Introduction: Coronaviruses (CoV) are enveloped plus-strand RNA viruses that have very large genomes and employ
complex genome expression and replication strategies. Previous studies have identified essential replicative proteins and, in
a few cases, cis-acting RNA elements involved in coronavirus RNA synthesis. Most of these studies focused on
representative Beta-CoVs while there is limited information on (i) cis-acting RNA elements in Alpha-CoVs and (ii) the
possible conservation of these structural elements among members of the Coronavirinae.
Objectives: To identify and characterize cis-acting RNA elements that are conserved across different coronavirus genera.
Material & Methods: A combination of computational, biochemical and reverse-genetic analyses was used to characterize
RNA structures in the human coronavirus 229E (HCoV-229E) 5'-terminal genome regions.
Results: RNA structures that we previously predicted to be conserved among Alpha-CoVs and Beta-CoVs were further
characterized by RNA structure probing analyses including the 600-nt 5'-terminal genome regions of HCoV-229E and HCoVNL63. Based on these data, HCoV-229E mutants carrying replacements of specific RNA structural elements with their
presumed counterparts from Beta-CoVs were designed and generated by reverse genetics. Analysis of these mutants
provided strong evidence that the 5'-terminal cis-acting RNA element SL2 is functionally conserved among alpha- and betaCoVs. Serial passaging of selected HCoV-229E mutants in cell culture allowed us to isolate stable virus clones that
replicated with near-wildtype growth kinetics. Specific nucleotide substitutions (pseudoreversions) identified by sequence
analysis in some of the mutants provided interesting insight into (presumably) relevant local and long-range RNA interactions
in coronavirus genomes.
Conclusion: Our data show that RNA structures in the 5'-terminal region are largely (but not completely) conserved across
different coronavirus genera, supporting their functional significance in RNA synthesis. More specifically, our reverse
genetics data provide strong evidence to suggest that the structure of the SL2 present in the 5'-UTRs of alpha- and betaCoVs is conserved and plays an essential role in viral replication.
Corresponding author:
Ramakanth Madhugiri
[email protected]
Viral Replication
OP20
Role of RNA secondary structures within the transcription start sites of the Ebola virus (EBOV) genome
R. K. Hartmann1, S. Bach1, H. Huber1, A. Grünweller1, S. Becker2, N. Biedenkopf2
1Philipps
2Philipps
University Marburg, Pharmaceutical Chemistry, Marburg, Germany
University Marburg, Institute of Virology, Marburg, Germany
Ebola virus (EBOV) genome replication is mediated by a protein complex comprising the viral RNA polymerase L, the
nucleoprotein (NP) and viral protein 35 (VP35). To accomplish transcription of viral mRNAs, the replication complex needs to
be supplemented with viral protein 30 (VP30), an EBOV-specific transcription factor.
Previous findings [1, 2] have given rise to a working model according to which VP30, via interaction with RNA and VP35,
increases the rate of productive transcription initiation at sites prone to termination due to RNA secondary structure
formation. The transcriptional start regions of all EBOV genes are embedded in 5'-UTR secondary structures of varying
stability on the genomic (-) RNA as well as (+) mRNA level. In this study, we used replication-proficient and -deficient EBOV
minigenomes (mono- and bicistronic) to investigate the role of these secondary structures in viral transcription and its
dependency on VP30. Analyzing these structures in the context of the first NP gene is complicated by interdigitation of the
transcriptional start region and the replication promoter. Testing the same structural elements in the context of the first versus
the second gene aims at disentangling effects on transcriptional initiation in the regulatory 3'-leader region and transcriptional
reinitiation at internal termination sites. First results indicate that the spacer between the two replication promoter elements
PE1 and 2, which includes the transcription start of the first NP gene, has to be a multiple of 6 nt not only for efficient
replication [3] but also efficient transcription initiation 56 nt downstream of the genome 3'-end. This spacer length "rule of six"
suggests that the replication promoter directs genome replication and mRNA transcription. This was inferred from the
observation that the 5'-terminal hairpin structure of the NP 5'-UTR could be replaced with corresponding structural elements
derived from internal genes (VP35, VP40 or GP) as long as the replacement followed the "rule of six". This principle was
attenuated when the VP24 5'-element was substituted for the NP element. As the VP24 element represented the longest
insertion variant tested so far, we are currently addressing if transcriptional efficiency decreases upon exceeding a certain
length limit of the spacer separating PE1 and PE2.
References:
[1] Schlereth J et al. (2016) RNA Biol.13:783-98
[2] Biedenkopf N et al. (2016) J Virol 90:7481-96
[3] Weik M et al. (2005) J Virol 79: 10660-71.
Corresponding author:
Roland K. Hartmann
[email protected]
Viral Replication
OP21
Hepatitis E virus replication and interferon response in human placental-derived cells
L. Knegendorf1, S. A. Drave1, Y. Debing2, M. Engelmann1, D. Todt1, J. Neyts2, P. Behrendt1,3, E. Steinmann1
1Twincore
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany, a joint venture between the Helmholtz Centre for Infection
Research and the Hanover Medical School, Institute of Experimental Infection Research, Hanover, Germany
2Rega Institute of Medical Research, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium
3Hanover Medical School, Department of Gastroenterology, Hepatology und Endocrinology, Hanover, Germany
The Hepatitis E virus (HEV) is one major cause of viral hepatitis and shows high epidemic potential in developing countries.
Notably, HEV infection has a high incidence among pregnant women and might take a serious course. High maternal
mortality rates of about 25 % in association with fulminant hepatic failure have been observed in HEV-infected pregnant
women. However, the underlying pathogenic mechanisms remain largely unexplained and the cases have only been
reported for infections with genotype (gt) 1 HEV, while gt 3 infections remain often subclinical without severe impairment. In
this study, subgenomic replicons (SGR) for both genotypes and a full-length HEV clone for gt 3 were used to investigate,
whether HEV is capable of performing the whole viral life cycle in the human placental-derived cell line Jeg3. Viral replication
was compared to the human hepatoma cell line HepG2. Jeg3 cells supported HEV RNA replication of both SGRs and the
full-length gt 3 clone and showed high replication capacity. We could confirm the functioning of virus assembly and release
by the detection of intra- and extracellular viral capsid through an HEV antigen ELISA and of viral entry by infection with cell
culture-acquired genotype 3 virions. Furthermore, we could show that both genotypes in placental cells have a similar
sensitivity to Ribavirin compared to HEV in hepatoma cells. In contrast, we experienced HEV gt 1 SGR to respond
significantly lower to the antiviral activity of Interferon (IFN)-alpha-a1 in Jeg3 cells compared to HEV gt 3 SGR. However, this
effect cannot be seen in HepG2 cells. Simultaneous determination of interferon-stimulated gene expression levels
demonstrated an efficient downregulation by both HEV genotypes, although the gt 1 SGR replicates less efficient in tissue
culture in comparison to the gt 3 SGR. In conclusion, we found that HEV is capable of performing the full viral life cycle in a
placental cell line in vitro and identified a potential pathogenic mechanism in host interferon response contributing to severe
outcomes of HEV infection during pregnancy. Further studies should address the identification of genotype-dependent
differences and a follow up on the host interferon response in HEV infection. Jeg3 cells could serve as an in vitro model for
future research in this field.
Corresponding author:
Leonard Knegendorf
[email protected]
Viral Replication
OP22
Mutational analysis suggests that coordinated acetylation of the influenza A virus nucleoprotein is required for
efficient budding of viral particles and polymerase activity
S. Giese1, K. Ciminski1, H. Bolte1,2,3, E. Moreira1,2,3, S. Lakdawala4, Q. David1, L. Kolesnikova5, V. Götz1, Y. Zhao6, E. Chin6,
K. Xu7, M. Schwemmle1
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
Ludwigs University, Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany
3Albert Ludwigs University, Faculty of Biology, University of Freiburg, Freiburg, Germany
4Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219, Pittsburgh, United
States
5Philipps University of Marburg, Institute of Virology, Marburg, Germany
6Chinese Academy of Sciences, Institute of Health Sciences, Shanghai Institutes for Biological SciencesShanghai, China
7Chinese Academy of Sciences, Institut Pasteur of Shanghai, Shanghai, China
2Albert
Influenza A viruses (IAV) harbour a segmented RNA genome (vRNAs) in negative polarity. Each vRNA segment is organized
into an antiparallel double helix, which is encapsidated by multiple copies of the viral nucleoprotein (NP) and terminally
bound by the viral polymerase. The architecture of these viral ribonucleoprotein (vRNP) complexes is similar to cellular
nucleosomes. In addition, NP, like histones, are known to be post-translationally modified, however, whereas histone
acetylation is known to have a regulatory function, it had remained elusive whether NP acetylation occurs in infected cells
and if it is required to regulate the function of NP. Here, we identified surface-exposed NP acetylation sites (K229 and K273)
of different IAV, including A/SC35M, which are highly conserved in all IAV strains. Unlike SC35M wild-type, viral growth of
both mutant viruses encoding either NP-K229R or NP-K273R (mimicking non-acetylated lysine residues) was severely
attenuated across different cell types. This attenuation was neither the result of decreased polymerase activity, altered
protein expression or disordered vRNP co-migration in the cytoplasm but rather inefficient budding likely due to the inability
to incorporate the bundle of eight segments. Intriguingly, a similar attenuated phenotype was observed with the mutant virus
SC35M-NP-K229Q mimicking constant acetylation at this site. However, mimicking NP hyper-acetylation by introducing both
K229Q and K273Q substitutions in NP severely diminished the viral polymerase activity, whereas mimicking NP hypoacetylation at these sites (K229R and K273R) had only minor effects. Based on these results we speculate that NP
acetylation must be tightly controlled to allow efficient viral budding as well as to sustain viral polymerase activity.
Corresponding author:
Sebastian Giese
[email protected]
Viral Replication
OP23
Mechanistic studies of NS2-3 independent virion morphogenesis in pestiviruses
F. Schlotthauer1, D. Dubrau1, N. Tautz1
1University
of Luebeck, Institute of Virology and Cell Biology, Lübeck, Germany
Members of Flaviviridae use non-structural (NS) proteins not only for RNA replication but also for virus assembly.
Pestiviruses, like bovine viral diarrhea virus (BVDV), are dependent on uncleaved NS2-3/4A for virion morphogenesis while
free NS3 is a prerequisite for viral RNA replication. In contrast, the related Hepatitis C virus (HCV) does not rely on
uncleaved NS2-3/4A for virion formation but uses NS3/4A for both processes. BVDV could be adapted to NS2-3 independent
virion morphogenesis with two amino acid exchanges (NS2/E440V, NS3/V132A). One hypothesis is that differences in
NS3/4A complexes dictate if NS3/4A can function either in replication or virus assembly. We could recently demonstrate that
such alternative conformations of BVDV NS3/4A are used to switch between RNA replication and virion morphogenesis.
Structure-function analysis showed that alternative interactions at the NS3/4A interface involving NS3/V132A are the
molecular basis for this regulation.
To characterize these alternative interactions at the NS3/4A interface which promote NS2-3 independent virion
morphogenesis in more detail, we analyzed which minimal NS4A features are required for this regulation. In a transcomplementation assay using different E2-4A derivatives we could show that a C-terminal NS4A deletion comprising the kink
region and the acidic domain still allows for virion assembly in this context. Furthermore, in the absence of the C-terminal
part of NS4A one of the critical mutations for NS2-3 independent virion morphogenesis (NS3/V132A) is not required for
successful trans-complementation. These interesting observations indicate that a general feature supporting virus assembly
is an accessible NS3 surface around V132. However, the presence of adaptive mutation NS2/E440V was still critical. To
better understand the role of NS2/E440V we analyzed protein-protein interactions in more detail. Interestingly, NS2/E440V
promoted the interaction between NS2 and NS3 only when the NS3 surface was accessible. These results implicate the
different architecture of NS3/4A-containing complexes supporting either RNA replication or virion morphogenesis and help to
understand differences between replicase- and packaging-competent protein complexes on the molecular level.
Corresponding author:
Norbert Tautz
[email protected]
Viral Replication
OP24
Membrane alterations induced by nonstructural proteins of human Norovirus
S. Doerflinger1,2, M. Cortese1, I. Romero Brey1, Z. Menne1, T. Tubiana3, R. Bartenschlager1, S. Bressanelli3, G. S.
Hansman3,2, V. Lohmann1
1University
of Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
Heidelberg, Germany
3Institut de Biologie Intégrative de la Cellule, GIF-SUR-YVETTE, France
2DKFZ,
Introduction: Positive strand RNA viruses induce membrane alterations harboring the viral replication complexes. In case of
human Noroviruses (HuNoV), these are induced by the ORF1 polyprotein, which is post-translationally processed into the
functional NS proteins 1-2, 3, 4, 5, 6 and 7. The replication sites of murine Norovirus (MNV) have been shown to consist of
accumulations of vesicles. Nothing is known so far about membrane alterations induced by HuNoV belonging to genogroup
II.4 (GII.4), due to the lack of efficient cell culture models, or about the contribution of individual NS proteins to their
biogenesis.
Objectives: Ultrastructural analysis of membrane alterations induced by ORF1 proteins of HuNoV and of the NS proteins
involved in their generation, compared to those found in MNV infected cells.
Methods: ORF1 polyprotein consensus sequences of medically relevant GII.4 strains and individual NS proteins fused to
GFP were expressed using a T7 RNA based expression model. Subcellular localization was analyzed by
immunofluorescence and eGFP autofluorescence. Ultrastructural changes induced by ORF1 or NS protein expression were
studied by electron microscopy and by correlative light and electron microscopy (CLEM).
Results: Expression of ORF1 proteins of three HuNoV GII.4 strains induced vesicular membrane alterations similar to those
found in MNV infected cells, but being more complex than previously described. In addition to single membrane vesicles we
identified double- and multi-membrane vesicles (DMVs and MMVs), similar to picornaviruses. NS1-2 induced tubular
protrusions of the smooth ER, distinct from MNV. NS3 was found mainly in proximity to lipid droplets. Sole expression of NS4
generated several distinct membrane structures, including SMVs and DMVs as well as crystalline ER structures similar to
those reported for Hepatitis A virus 2C. We furthermore generated models of the putative membrane association of human
NS1-2, NS3 and NS4 and experimentally verified the proposed domain organization of NS1-2.
Conclusion: Our work provides first insights into the morphology of HuNoV induced replication complexes, consiting of
SMVs, DMVs and MNVs and identifies NS4 as the most important determinant in their generation. We furthermore provide
experimental evidence for functions of the enigmatic protein NS1-2.
Corresponding author:
Sylvie Doerflinger
[email protected]
Innate Immunity
OP25
SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, is required for the antiviral activity of
MxA
D. Dornfeld1, A. Dudek2,3,1, T. Vausselin4, S. Günther1, J. Hultquist5, A. Garcia-Sastre4,6,7, M. Shaw4, M. Schwemmle1
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
Ludwigs University, Faculty of Biology, Freiburg, Germany
3 Albert Ludwigs University, Spemann Graduate School of Biology and Medicine, Freiburg, Germany
4Icahn School of Medicine at Mount Sinai, Department of Microbiology, New York, United States
5University of California, San Francisco, Department of Cellular and Molecular Pharmacology, San Francisco, United States
6Icahn School of Medicine at Mount Sinai, Global Health and Emerging Pathogens Institute, New York, United States
7Icahn School of Medicine at Mount Sinai, Department of Medicine, Division of Infectious Diseases, New York, United States
2Albert
The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting a broad antiviral activity
against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype. The mechanism
of how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this
activity.
To identify such MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549)
constitutively expressing MxA using an H5N1 reporter virus. This approach identified SMARCA2, the ATPase subunit of the
BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. We provide
evidence that SMARCA2 is essential for type I IFN-mediated signaling and the establishment of an antiviral state.
Interestingly, SMARCA2 activity is not required for induction of MxA, suggesting that the effect of SMARCA2 on MxA activity
is indirect. Furthermore, SMARCA2 was still required for the antiviral activity of MxA after inhibition of Janus kinase 1 and 2
signaling, suggesting that the MxA co-factor is an interferon induced gene (ISG) activated by virus infection.
Together, these findings suggest that the SMARCA2-containing BAF complex regulates the expression of ISGs required for
the antiviral activity of MxA.
Corresponding author:
Dominik Dornfeld
[email protected]
Innate Immunity
OP26
The interferon-γ-inducible protein 16 (IFI16) restricts HIV replication by inhibiting LTR promoter-driven viral gene
expression
D. Hotter1, K. L. Jønsson2, D. Krnavek1, K. Regensburger1, M. Bosso1, D. Sauter1, F. Kirchhoff1
1Ulm
University Medical Center, Institute of Molecular Virology, Ulm, Germany
University, Department of Biomedicine, Aarhus, Denmark
2Aarhus
Introduction: The human interferon γ-inducible protein 16 (IFI16) acts as sensor of exogenous viral DNAs and also directly
restricts replication of herpes simplex virus 1 as well as cytomegalo- and papillomaviruses by binding and/or modifying the
viral promoter regions (Johnson et al., PLoS Pathog. 2014; Gariano et al., PLoS Pathog. 2012; Lo Cigno et al., J Virol. 2015).
Notably, IFI16 also senses human immunodeficiency virus (HIV) DNA species (Jakobsen et al., 2014) and reduced infectious
HIV-1 yield in a screen for novel antiviral factors (McLaren et al., Retrovirol., 2015).
Objective: To elucidate the mechanisms underlying IFI16-mediated restriction of HIV-1.
Material & Methods: The effect of IFI16 on infectious HIV-1 yield was examined in transfected HEK293T cells as well as
infected THP1 cells and primary target cells of HIV-1 using both overexpression and siRNA knockdown conditions.
Luciferase assays, FACS, ELISA and Western blot analyses were applied to define which steps of the HIV replication cycle
are affected by IFI16. Specific mutant and chimeric forms of IFI16 were used to determine the motifs required for its antiretroviral activity.
Results: Experiments in transfected HEK293T cells revealed that IFI16 inhibits HIV infectious virus production in a dosedependent manner, independently of its sensing activity and the activation of the STING-IRF3/7 pathway. In agreement with
this, virus production increased upon knockout of IFI16 in THP1 cells and siRNA-mediated depletion in macrophages.
Notably, the antiviral activity of IFI16 was conserved among different mammalian species and not limited to HIV-1, as related
simian immunodeficiency viruses (SIV) were also restricted. Decreased infectious virus production was associated with
reduced expression of viral structural proteins and impaired activity of the viral LTR promoter. Mapping experiments showed
that the N-terminal PYRIN domain of IFI16 is sufficient to restrict HIV. Interestingly, the HIN domains are crucial for the
sensing activity of IFI16 but dispensable for suppression of LTR-driven viral gene expression.
Conclusion: Our data demonstrate that IFI16 directly restricts infectious HIV and SIV production by targeting the viral LTR
promoter, and highlight the importance of this multifunctional protein in antiviral immunity.
Corresponding author:
Dominik Hotter
[email protected]
Innate Immunity
OP27
pH-optimum of hemagglutinin-mediated membrane fusion determines influenza virus sensitivity to interferoninduced antiviral state
T. Gerlach1, L. Hensen1, T. Matrosovich1, J. Wolf1, M. Winkler2, C. Peteranderl3, H. D. Klenk1, F. Weber4, S. Herold3, S.
Pöhlmann2, M. Matrosovich1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
3Universities Giessen & Marburg Lung Center, Department of Internal Medicine II, Giessen, Germany
4Justus Liebig University Giessen, Institute of Virology, Giessen, Germany
2German
Introduction: Replication and pathogenicity of influenza viruses (IAVs) critically depend on their ability to tolerate the type I
interferon (IFN) antiviral response. However, the roles of different viral proteins in IFN evasion are not fully defined.
Objectives: In this study, we wanted to test whether properties of the viral hemagglutinin (HA) can affect the sensitivity of
IAVs to the IFN-induced antiviral state.
Material and Methods: We studied restriction of infection in IFN-β-treated human epithelial cells and in MDCK cells stably
expressing IFN-inducible antiviral proteins IFITM2 or IFITM3 using a panel of recombinant 2:6 reassortant IAVs with HAs and
NAs of representative avian, human and zoonotic viruses and 6 other gene segments from PR8.
Results: Analyses of infection inhibition in IFN-b-treated human epithelial cells showed that viruses having a higher pH
optimum of HA-mediated membrane fusion (such as human-infecting H5N1 and H7N9 viruses) are less susceptible to the
IFN-induced antiviral state than their counterparts with HAs from duck and human viruses which fuse at a lower pH. The
effects of HA fusion activity on viral sensitivity to IFN were substantiated further by experiments with single point HA fusion
mutants of A/Hong Kong/1/1968-PR8 (H3N2). A higher pH optimum of fusion correlated with a lower virus restriction in
MDCK-IFITM cells expressing IFITM2 or IFITM3 proteins, which are known to interfere with viral endosomal fusion. Our
findings and their biological relevance were supported by similar observations obtained using i) primary human type II
alveolar epithelial cells and differentiated cultures of human airway epithelial cells, ii) non-recombinant zoonotic and
pandemic IAVs and iii) preparations of IFN-α and IFN-λ1.
Conclusion: Our results for the first time demonstrate that sensitivity of IAVs to IFN-induced state is modulated by
membrane fusion activity of viral HA, and that this effect depends, at least in part, on interplay between the HA and IFITM2
and IFITM3 proteins. Our data imply that high pH optimum of fusion typical for zoonotic IAVs of gallinaceous poultry, such as
H5N1 and H7N9, may contribute to their enhanced virulence in humans.
Corresponding author:
Thomas Gerlach
[email protected]
Innate Immunity
OP28
Effect of allelic variations in the human MxA protein on its antiviral activity
L. Graf1,2,3, F. Sendker1,2, A. Dick4,5, E. Barth6, M. Marz6, O. Daumke4,5, G. Kochs1,2,3
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
Ludwigs University, Faculty of Medicine, Freiburg, Germany
3Albert Ludwigs University, Spemann Graduate School of Biology and Medicine, Freiburg, Germany
4Max Delbrück Centre for Molecular Medicine, Berlin, Germany
5Free University Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany
6Friedrich Schiller University Jena, Faculty of Mathematics and Computer Science, Jena, Germany
2Albert
Question: Most people infected by influenza A virus suffer from a mild to moderate respiratory illness. Only a minority of
patients show a severe or fatal progression of the disease. A key influenza virus restriction factor in humans is the myxovirus
resistance protein A (MxA). The interferon-induced GTPase has an N-terminal GTPase domain (G domain) and a C-terminal
stalk responsible for oligomerization and viral target recognition. GTPase activity as well as oligomerization are necessary for
the antiviral function of MxA.
In order to gain further insight into the mechanisms underlying inter-individual variability in influenza susceptibility, we
investigated the influence of naturally occurring allelic variations in human MxA on its antiviral function
Methods: Online genome and exome databases were used to search for allelic variations in the MX1 gene, encoding the
MxA protein, of non-infected individuals. Selected variants were characterized with regards to antiviral activity as well as
GTPase activity and oligomerization.
Results: Two coding variations in the G domain caused a complete loss of antiviral and GTPase activity. They disrupt the
interface responsible for G domain dimerization which is necessary for GTP hydrolysis. Allelic variations in the stalk were
neutral or abolished MxA antiviral function except for one variant in an oligomerization interface that actually showed
increased antiviral activity. Remarkably, two other stalk variants even altered the antiviral specificity of MxA. Most variations
causing the loss of antiviral activity occurred heterozygously. Surprisingly, some of these antivirally inactive variants had no
negative effect on the activity of co-expressed wt MxA indicating that heterozygous carriers are still protected.
Conclusion: Our data demonstrate that naturally occurring variations in the human MX1 gene influence MxA function and
contribute to a better understanding of the antiviral mechanism of MxA. Future analysis of MxA variations will reveal whether
these are enriched in patients suffering from severe influenza.
Corresponding author:
Laura Graf
[email protected]
Innate Immunity
OP29
Exploitation of the immune-regulatory function of ADAR1 by measles virus
C. Pfaller1, R. Donohue1,2, R. Cattaneo1,2
1Mayo
2Mayo
Clinic, Molecular Medicine, Rochester, United States
Graduate School, Virology and Gene Therapy Program, Rochester, United States
Introduction: The adenosine deaminase acting on RNA 1 (ADAR1) catalyzes C6-deamination of adenosine in doublestranded RNA substrates to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. Two
ADAR1 isoforms exist: constitutively expressed ADAR1p110, and interferon-inducible ADAR1p150. Lack of ADAR1 catalytic
activity is associated with autoimmune disorders such as the Aicardi-Goutières syndrome. Remarkably, despite being
interferon-inducible, ADAR1 enhances the replication efficiency of several viruses, including the negative-strand RNA virus
measles (MV).
Objectives: Here we seek to understand the mechanisms by which MV exploits ADAR1 as a proviral factor.
Material & Methods: We characterized ADAR1 editing of MV ribonucleocapsids (RNPs). We operated with a vaccineequivalent (MVvac2) and a mutant virus (MVvac2-CKO) that generates high levels of immune-stimulatory defective interfering
RNAs (DI RNAs; Pfaller et al., 2015, J. Virol. 89. 7735-7747). We characterized viral replication on ADAR1-expressing,
p150KO or complete ADAR1KO cells and performed next generation sequencing of purified viral RNPs and whole cellular
transcriptomes.
Results: We detected in both viruses clusters of A-to-G transitions characteristic of those favored by ADAR1. These clusters
particularly accumulated near both genome termini of MVvac2-CKO, suggesting that DI RNAs were the primary source of
ADAR1-edited reads. Titers of both MVvac2 and MVvac2-CKO were reduced up to 1000-fold in p150KO and ADAR1KO cells
compared to parental cells. No significant differences were observed between p150 KO and ADAR1KO cells. Remarkably,
p150KO and parental cells produced similar quantities of MV RNPs.
Conclusion: The proviral activity of ADAR1 is mostly conferred by ADAR1p150 in case of MV infection. We propose two
mechanisms: (1) Modification of immunostimulatory DI RNA reduces innate immune responses to MV infection. (2) Since
viruses produced similar amounts of RNPs in parental and p150KO cells, but viral titers were strongly reduced only in p150KO
cells, ADAR1p150 must favor a late step of the viral life cycle. To investigate the underlying mechanism, we are currently
identifying cellular genes differentially expressed in knock-out cell lines that may influence viral replication.
Corresponding author:
Christian Pfaller
[email protected]
Innate Immunity
OP30
The role of TRIM proteins in virus-induced autophagy
K. M. Sparrer1, S. Gableske1, F. Full1, G. J. Baumgart1, J. Kato2, G. Pacheco-Rodriguez2, C. Liang3, O. Pornillos4, J. Moss2,
M. Vaughan2, M. U. Gack1
1University
of Chicago, Department of Microbiology, Chicago, United States
Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, Bethesda, United States
3University of Southern California, Department of Molecular Microbiology and Immunology, Los Angeles, United States
4University of Virginia, Department of Molecular Physiology and Biological Physics, Charlottesville, United States
2NIH,
Recent studies demonstrated that members of the TRIM protein family are critical modulators of both cytokine- and
autophagy-mediated antiviral defenses. While the molecular mechanisms by which TRIM proteins regulate antiviral interferon
responses have been well characterized, significantly less is known about the role of TRIM proteins in autophagy in response
to specific viral pathogens. Using both cDNA and RNAi screens, we systematically assessed the role of TRIM proteins in
autophagy induction in response to RNA virus and DNA virus infections. From these screens TRIM23 emerged as a major
hit. Depletion of endogenous TRIM23 reduced viral-induced autophagy to near-basal levels, comparably to the knockdown of
the key autophagic proteins ATG5, ATG7 or ATG12, demonstrating that TRIM23 is essential for autophagy activation in
response to viral infection. Mutational analysis showed that the autophagy-promoting activity of TRIM23 is dependent on
both its N-terminal RING E3 ubiquitin ligase and C-terminal ADP-ribosylation factor (ARF) GTPase activity. Mechanistically,
unconventional non-degradative auto-ubiquitination of the ARF domain is essential for the GTP hydrolysis activity of TRIM23,
its recruitment to autophagosomes, and subsequent TBK1-mediated antiviral autophagy. Our study thus provides novel
mechanistic insight into the activation of TRIM proteins during virus-induced autophagy and identifies TRIM23 as a core
component of the selective autophagy machinery.
Corresponding author:
Konstantin MJ Sparrer
[email protected]
Viral Replication
OP31
Herpes Simplex Virus 1 induces antisense transcription from the host cell genome
E. Wyler1, J. Menegatti2, V. Franke1, C. Kocks1, A. Rutkowski3, L. Baer2, L. Münster1, A. Boltengagen1, K. Theil1, T. Hennig3,
A. Akalin1, N. Rajewsky1, L. Dölken3, M. Landthaler1, F. Grässer2
1Max
Delbrück Center for Molecular Medicine, Berlin, Germany
University, Institute of Virology, Homburg, Germany
3Julius Maximilians University Würzburg, Institute of Virology and Immunbiology, Würzburg, Germany
2Saarland
Question: Herpes simplex virus 1 (HSV-1) is an important human pathogen. An emerging feature of how HSV-1 modulates
host cell biology is transcription outside of or antisense to annotated genes, possibly yielding stable and functional long noncoding RNAs (lncRNAs). Our study addresses the question to what extent HSV-1 infection induces expression of noncanonical transcripts, and how they may be regulated and function.
Methods: We performed high-throughput RNA sequencing on primary human fibroblasts at different timepoints after lytic
infection with HSV-1. Using a novel computational approach, we defined antisense transcripts in our and published data
base (Rutkowski et al. Nat Commun. 2015 May 20;6:7126). Predicted transcripts were validated (qPCR,
Nanostring/nCounter) and localized by single molecule RNA-FISH. Functional studies were performed using CRISPR/Cas9mediated promoter knockout, pulse RNA labeling and reporter assays. Factors activating antisense transcription are
investigated using knockout viruses and overexpression of candidate proteins.
Results: Antisense transcripts were defined using a novel algorithm. More than thousand antisense transcripts were found to
be upregulated upon infection, of which most are not found in existing genome annotations. Validation of twelve of these
predicted transcripts show an induction very early in infection. In one case, we observed localization in strong nuclear foci,
indicating a potential cis-regulatory function. The putative promoter region of this antisense transcript was furthermore shown
to be transcriptionally activated upon HSV-1 infection in a reporter assay. Knockout of the promoter of this antisense
transcript suggests transcriptional silencing of the corresponding pro-apoptotic host gene. Infection with knockout viruses
and overexpression of viral proteins indicates that viral factors can induce the antisense transcripts.
Conclusion: During lytic infection, HSV-1 shuts down and reprograms host cell gene expression for virion production. Our
results reveal surprising effects also at the transcriptional level: HSV-1 infection induces hundreds of completely novel
antisense transcripts. Our results on one hand indicate that some of them may be functional lncRNAs, adding another aspect
to our knowledge of virus infections. On the other hand, they show the value of viruses to study transcriptional and posttranscriptional regulation in human cells.
Corresponding author:
Friedrich Grässer
[email protected]
Viral Replication
OP32
Role of virus replication in Marek's disease virus integration
R. Lopes Previdelli1, L. D. Bertzbach1, T. Faflíková1, B. B. Kaufer1
1Free
University Berlin, Department of Veterinary Medicine, Berlin, Germany
Introduction: Marek"s disease virus (MDV) is a highly oncogenic alphaherpesvirus that integrates its genetic material into
the telomeres of tumor and latently infected cells. Telomeric repeats present at the ends of the MDV genome facilitated
integration. However, it remains unknown which viral or cellular proteins are needed and if viral replication is required for this
process.
Objectives: The objective of this study is to determine if MDV replication is required for genome integration in latently
infected cells using a recombinant virus with an inducible viral DNA polymerase (UL30).
Material & Methods: To facilitate an inducible expression of the UL30 polymerase, we fused the dihydrofolate reductase
destabilization domain (ddDHFR) to the C-terminus of UL30 in the MDV genome (pUL30-DHFR), resulting in the degradation
of the viral polymerase and abrogation of viral replication. Upon addition of the stabilizing ligand trimethoprim (TMP), pUL30
expression could be efficiently restored. To determine the role of MDV replication in the integration process, we infected
CU91 T-cells with pUL30-DHFR in the presence and absence of TMP. Fluorescent in-situ hybridization (FISH) technique and
qPCR will allow us to determine if the MDV integration frequency is affected in the absence of MDV replication.
Results: pUL30-DHFR replicated in a TMP concentration-dependent manner. Replication was comparable to wild type virus
(RB-1B) in the in the presence of 10 mM TMP, while removal of TMP from the medium completely abrogated MDV
replication. CU91 cells were infected with pUL30-DHFR and the integration frequency is currently under investigation by
FISH and qPCR.
Conclusion: Our study provides the first evidence that the ddDHFR system can be used to modulate the expression of
herpesvirus genes. This system will allow us to determine if MDV replication is required for virus genome integration into host
telomeres.
Corresponding author:
Renato Lopes Previdelli
[email protected]
Viral Replication
OP33
Three-dimensional ultrastructural analysis reveals rate-determining steps of HCMV egress
C. Villinger1,2, G. Neusser3, P. Walther2, T. Mertens1, J. von Einem1
1Ulm
University Medical Center, Institute of Virology, Ulm, Germany
University, Central Facility for Electron Microscopy, Ulm, Germany
3Ulm University, Institute of Analytical and Bioanalytical Chemistry, Ulm, Germany
2Ulm
Electron microscopy (EM) of high-pressure frozen and freeze-substituted samples permits unambiguous identification and
detailed analysis of various morphogenesis stages during human cytomegalovirus (HCMV) virion maturation. Although EM
allows investigation of fixed samples only, it can provide some insight into HCMV egress dynamics by applying quantitative
analysis, since EM captures slow and/or frequent events with a higher chance than quick and/or rare events. Additionally,
application of three-dimensional (3D) EM techniques enables quantifications with higher accuracy.
The aim of this study was to quantify HCMV morphogenesis of entire cells by applying two different 3D EM techniques focused ion beam-scanning electron microscopy (FIB-SEM) tomography and serial sectioning transmission electron
microscopy (TEM) - and reassessment of two-dimensional micrographs from over 40 TEM experiments. We evaluated the
number of capsids (I) within the nucleoplasm, (II) budding (primary envelopment) at the inner nuclear membrane into the
perinuclear space (PNS), (III) within the PNS, (IV) leaving the PNS by de-envelopment at the outer nuclear membrane and
(V) at the viral assembly complex (vAC), which is the site of secondary envelopment. Capsids at the vAC were also
quantified regarding their stage of secondary envelopment (free, budding and enveloped). These results were then
incorporated into a snapshot model together with dynamic data from life cell imaging studies.
Analysis of our data identified nuclear egress and release of virions at the plasma membrane as bottlenecks for the
production and release of infectious virions. We also noticed much higher numbers of cytoplasmic capsids that were either in
the process of secondary envelopment or already enveloped at the vAC compared to numbers of nuclear capsids in the
process of primary envelopment and numbers of primary enveloped capsids in the PNS, although both envelopment
processes might occur with similar velocities. This underlines the importance of the vAC to provide an optimized environment
for secondary envelopment by concentrating host cell membranes at a distinct site in the cytoplasm.
Altogether, this knowledge could be useful to identify new targets for future development of antiviral drugs.
Corresponding author:
Clarissa Villinger
[email protected]
Viral Replication
OP34
Non-plaque-forming virions of Modified Vaccinia virus Ankara express viral genes
A. T. Lülf1, A. Freudenstein1, L. Marr1, G. Sutter1, A. Volz1
1Ludwig
Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
In cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of
plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia virus Ankara
produce only about 20-30% plaque-forming virions in fully permissive cell cultures.
To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing
fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and
automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral
gene expression.
Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected
activation of late viral transcription in 10-20% of single infected cells.
Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus
vaccines.
Corresponding author:
Anna-Theresa Lülf
[email protected]
Viral Replication
OP35
Covalent SUMO attachment to HAdV E2A/DBP by host-cell cascades is beneficial for productive virus replication
M. Terzic1, J. Berscheminski2, R. T. Hay3, T. Dobner2, S. Schreiner1
1Technical
University Munich and Helmholtz Centre Munich, Institute of Virology, Munich, Germany
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
3University of Dundee, Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, Dundee, United Kingdom
2Heinrich
Human Adenoviruses (HAdV) are non-enveloped viruses containing a linear, double-stranded DNA genome surrounded by
an icosahedral capsid with type-specific antigens. HAdV represent the causative agent for infections, associated with clinical
symptoms such as gastroenteritis, keratoconjunvtivitis, pneumonia, hepatitis and encephalitis. These viruses are often nonpathogenic for healthy individuals with protective immunity. However, in the absence of protective immunity, HAdV infections
can become lethal due to uncontrolled immunopathology and processes, which are still far from being understood in detail.
To ensure proper replication, DNA viruses express early viral genes to degrade or displace key regulators of cellular antiviral
measures and immunopathology. In contrast, host-cells repress incoming viral genomes through a network of transcriptional
repressors and activators that normally control cellular homeostasis. The nuclear domains thought to be responsible for
repressing viral DNA genomes are PML-NBs, interferon inducible, dot-like nuclear structures representing the hotspots of
host SUMO posttranslational modification. In HAdV infected cells such SUMO factories are found in close proximity to newly
established replication centers indicated by the viral marker protein E2A. This early HAdV DNA-binding protein is essential
for efficient viral replication, regulation of viral gene expression, mRNA stability and virion assembly. Here, we show that E2A
represents a novel target of the host SUMOylation machinery, orchestrating essential E2A functions. Mimicking removal of
SUMOylated E2A from the infected cell by generation of HAdV variants with mutations in the SUMO conjugation motif, we
found that SUMO is covalently attached to the viral determinant, repressing viral replication properties. Our data clearly
illustrate the influence of E2A SUMOylation on PML-NBs and proper establishment of HAdV replication centers. Taken
together, abrogation of E2A SUMO modification by novel inhibitors or compounds might represent a future step in virus
intervention strategies.
Corresponding author:
Miona Terzic
[email protected]
Viral Replication
OP36
Refined characterization of Cowpox virus replication and virus-host interaction using human skin equivalents as a
3D infection model
M. Neumann1, R. Koban1, F. Groeber2, H. Walles3,2, H. Ellerbrok1
1Robert
Koch Institute, Centre for Biological Threats and Special Pathogens (ZBS) 1 - Highly Pathogenic Viruses, Berlin, Germany
Institute of Interfacial Engineering and Biotechnology (IGB), Translational Center Würzburg, Würzburg, Germany
3Julius Maximilians University Würzburg, Department for Tissue Engineering and Regenerative Medicine, Würzburg, Germany
2Fraunhofer
Zoonotic Cowpox viruses (CPXV) transmitted from infected animals to humans through minor skin lesions result in localized
infections in immune-competent individuals. 3D organoid models may facilitate the examination of viral replication,
pathogenicity and virus-cell interactions in a more physiological manner, thus bridging the gap between 2D cell culture and
animal experiments. For this purpose a 3D infection model of CPXV in human skin equivalents was established. The effect of
CPXV infection on primary human skin cells in a 3D setup was further characterized via RNA-Seq-based transcriptome
analysis of infected cells and compared to classical 2D cultivation.
Differentiated skin equivalents were generated from primary keratinocytes and fibroblasts. Infection was performed with a
fluorescent CPXV strain puncturing the outer layer. Viral replication was monitored via qPCR and immunohistochemistry
(IHC). RNA-Seq analysis of infected keratinocytes either from the epidermal part of the equivalent or from 2D cultures was
performed on the Illumina HiSeq1500 platform in biological triplicates. Downstream gene specific analysis was carried out
with PartekFlow.
CPXV caused permissive infections in human skin equivalents. Increasing amounts of viral nucleic acids and viral particles
were detected over time. Cell-to-cell spread of infection was slower compared to 2D culture systems. The sequencing runs of
selected 2D and 3D samples generated high-quality reads, representing around 15.000 different human and all 229
annotated viral genes. For skin samples 4% of genes were significantly regulated in infected samples compared to mockinfected controls. Comparison of different time points and cultivation setups reveals cell- or model-specific regulated genes.
Changes in expression were confirmed via qPCR analysis, IHC and Western Blotting for selected genes involved e.g. in
microtubule formation.
In conclusion infection experiments in cells exploiting the particular characteristics of a 3D skin equivalent combined with
RNA-Seq transcriptome analysis have provided new insights in virus spread and regulation of cellular genes by CPXV. With
this approach candidate genes involved in virus replication and pathogenesis were identified and are further analysed as
potential targets for antiviral therapies.
Corresponding author:
Markus Neumann
[email protected]
Adaptive Immunity
OP37
Investigating into the "antibody bipolar bridging hypothesis" by the HCMV-encoded Fc-gamma receptors gp34 and
gp68
P. Kolb1, D. Gütle1, H. Hengel1
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
Human cytomegalovirus (HCMV) is known for multiple highly sophisticated immune evasion strategies, including the evasion
from antibody mediated control by counteracting host Fc-gamma receptor (FcγR) activation. To this end, HCMV encodes a
set of IgG-Fc binding glycoproteins expressed as plasma membrane-resident receptors (viral Fc-gamma receptors, vFcγRs)
which include the vFcγRs gp34 and gp68 (Atalay et al., 2002). While their most obvious molecular mechanism of inhibition
would be competitive IgG-Fc binding of gp34 and/or gp68 according to the common "antibody bipolar bridging (ABB)
hypothesis", experimental evidence for this mode of action is still incomplete. Importantly, the ABB hypothesis includes the
implicit presupposition that due to the presence of vFcγRs the host FcγRs are prevented from recognizing the Fc domain of
IgG. While the formation of ternary ABB complexes consisting of antigen, IgG and the vFcγRs gp34 or gp68 could be
demonstrated (Corrales-Aguilar et al., 2014), the consequences for the interaction of host FcγRs with IgG of ABB complexes
on the cell surface have not been experimentally approached.
To address this important issue we compared IgG-Fc binding of host FcγRs in the presence or absence of gp34 or gp68.
Specifically, we developed a cell based test system using CD20 as a model antigen and anti-CD20 Rituximab to allow ABB
complex formation upon transient expression of gp34 or gp68. Binding of soluble host FcγR CD16 to these ABB-complexes
was readily detected by FACS and found unimpaired in the presence of gp34 but strongly reduced by gp68. This observation
was made with variants of gp34 and gp68 that were optimized for enhanced and equalized membrane-residency by
replacement of the respective transmembrane domains and cytosolic tails with appropriate sequences from human CD4.
Conversely, when monitoring surface antigen removal over time using wildtype molecules, gp34 induced significantly faster
internalization of CD20-Rituximab complexes than gp68. Overall, our data suggest distinct modes of action for both vFcγRs
and a division of labor between gp34 and gp68. Altogether, the data support a novel concept of non-redundant but
cooperative immune evasion mechanisms from IgG-Fc mediated antibody effector responses.
Corresponding author:
Philipp Kolb
[email protected]
Adaptive Immunity
OP38
Fas Ligand-mediated cytotoxicity of CD4+ T cells control chronic Friend Retrovirus Infection
A. Malyshkina1, U. Dittmer1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
CD4+ helper T cells and cytotoxic CD8+ T cells are key players for adaptive immune responses against acute infections with
retroviruses. The depletion of CD4+ T cells during acute Friend Virus (FV) infection mainly results in reduced antibody
responses and in earlier decline of the CD8+ T cell responses. Thus, the most important function of CD4+ T cells during an
acute retrovirus infection seems to be their helper function for other immune cells.
Whereas there was no direct anti-viral activity of CD4+ T cells during acute FV infection, they were absolutely required for
the control of chronic virus and for preventing onset of leukemia. We therefore investigated, if FV-specific CD4+ T cells have
cytotoxic potential against FV-infected cells. During chronic FV infection a population of virus-specific CD4+ T cells was still
detectable. Interestingly, these cells did not express cytotoxic molecules (e.g. granzymes), but they expressed Fas Ligand
that can induce Fas-induced apoptosis in target cells.
Using an MHC II-restricted in vivo CTL assay we demonstrated that these FV-specific CD4+ T cells indeed mediated
cytotoxic effects on FV epitope peptide loaded targets. Since chronic FV mainly hides in MHC class II expressing B cells and
monocytes these cells can be targets for killing by CD4+ T cells. CD4+ CTL killing was also detected in granzyme B
knockout mice confirming that the exocytosis pathway was not involved. However, killing could be blocked by antibodies
against FasL, which identified the Fas-FasL pathway as critical cytotoxic mechanism during chronic FV infection.
The data indicate that cytotoxicity of CD4+ T cells is a critical factor for the immune control of chronic retroviral infection and
that this cytotoxicity can be augmented by immunotherapy to achieve superior suppression of chronic virus.
Corresponding author:
Anna Malyshkina
[email protected]
Adaptive Immunity
OP39
NKG2Cpos NK cells associated with CMV infection inhibit expansion of activated virus-specific CD8 T cells
C. Thöns1, E. Bäcker1, A. Zimmermann1, M. Uhrberg2, P. Lang3, J. Timm1
1Heinrich
Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
Heine University Dusseldorf, Department of Transplantation Diagnostics and Cell Therapeutics, Dusseldorf, Germany
3Heinrich Heine University Dusseldorf, Department of Molecular Medicine II, Dusseldorf, Germany
2Heinrich
Background & Aims: Infection with the human Cytomegalovirus (CMV) imprints both the T cell and the NK cell
compartment often resulting in expansion of CMV-specific CD8+ T cells and NK cells expressing NKG2C, an activating NK
cell receptor binding to HLA-E. In the elderly, infection with CMV has been associated with impaired immunity. Here, we
analyzed the impact of CMV-associated changes in the NK cell compartment on antiviral CD8 T cells.
Results: To analyze the impact of NK cell activation on virus-specific T cells, PBMCs of CMV seronegative and seropositive
donors were cultivated in the absence or presence of irradiated NK cell target cells (K562). Virus-specific CD8 T cells
targeting HLA-A*02-restricted epitopes of CMV or influenza A virus (IAV) were identified with dextramers. In CMV
seropositive donors, antigen-specific expansion of CMV- or IAV-specific memory CD8 T cells was inhibited in the presence of
K562 cells. In contrast, expansion of IAV-specific CD8 T cells was not inhibited in CMV seronegative donors. As previously
described, CMV was associated with high levels of the activating receptor NKG2C on NK cells. We therefore hypothesized
that expression of the NKG2C-ligand HLA-E on CD8 T cells may play a role in negative regulation. Indeed, expression of
HLA-E increased with CD8 T cell differentiation to effector memory cells and was upregulated upon unspecific T cell
activation and upon stimulation of CMV-specific or IAV-specific CD8 T cells with the cognate antigen in vitro. Importantly, this
upregulation of HLA-E on activated virus-specific CD8 T cells was decreased in the presence of K562 cells in CMV
seropositive donors but not in CMV seronegative donors, suggesting that negative regulation of CD8 T cells by activated NK
cells is promoted in CMV infected donors. To address if cytolytic effector functions of NKG2C+ NK cells against HLA-Ehigh
cells play a role in negative regulation of CD8 T cells, an HLA-Ehigh K562 target cell line was used. Upon activation with
HLA-Ehigh target cells expression of the degranulation marker CD107a was significantly higher on NK cells from CMV
seropositive donors compared to CMV seronegative donors and was associated with high NKG2C levels.
Conclusion: The CMV-associated expansion of NKG2Cpos NK cells promotes negative regulation of virus-specific CD8 T
cells presumably via the interaction between NKG2C and HLA-E on activated CD8 T cells. This enhanced negative
regulatory effect on T cells may contribute to the immune senescent state observed in elderly CMV seropositive individuals.
Corresponding author:
Christine Thöns
[email protected]
Adaptive Immunity
OP40
Down regulation of Matrix protein 2 levels is a novel immune escape mechanism of influenza A virus
S. Xavier1, S. Van den Hoecke1, B. Vrancken1, L. Deng1, E. Job1, K. Roose1, B. Schepens1, P. Lemey1
1Ghent
University and VIB, Ghent, Belgium
The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. We
investigated the potential evasion strategies of influenza A viruses under in vivo M2e-based immune selection pressure
imposed by passively transferred M2e-specific IgG antibodies. A/Puerto Rico/8/34 virus infected SCID mice were chronically
treated with anti-M2e mouse IgG monoclonal antibodies that differ in epitope and/or antibody isotype. Treatment of
challenged SCID mice with these monoclonal antibodies significantly prolonged survival compared to control IgG treatment.
Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and could even clear the challenge virus in the
SCID mice. Whole virus genome next-generation sequence analysis of the virus population that persisted in anti-M2e
monoclonal antibody treated mice revealed that viruses emerged with a proline to histidine or leucine mutation at position 10
and/or an isoleucine to threonine mutation at position 11 in M2. These mutations abolished recognition by some of the
monoclonal antibodies occurred at diverse frequencies, either alone or combined in the viral population. Polyclonal anti-M2e
serum, induced by vaccination with an M2e-virus-like particle vaccine, still recognized these M2e variants. Remarkably,
viruses with a wild type M2 sequence but with non-synonymous mutations in the polymerases and/or the hemagglutinin were
frequently isolated in anti-M2e IgG treated mice. Some of these mutations were associated with a delay in M2 expression
relative to other structural proteins. We conclude that immune protection by M2e-specific mAbs selects for viruses with
limited variation in M2e, which can still be recognized by polyclonal anti-M2e serum. Our results also suggest that influenza A
viruses can undergo an alternative escape route from M2e-specific mAbs by acquiring mutations elsewhere in their genome
that results in delayed M2 expression.
Corresponding author:
Saelens Xavier
[email protected]
Adaptive Immunity
OP41
Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus
Q. Wang*1,2, H. Yang*1,3, X. Liu*4, L. Dai*5, T. Ma1,3, J. Qi6, G. Wong2,6,7, R. Peng6, S. Liu6,8, J. Li1, S. Li6, J. Song6, J. Liu9, J.
He10, H. Yuan4, Y. Xiong4, Y. Liao11, J. Li11, J. Yang11, Z. Tong1,2,6, B. D. Griffin12,13, Y. Bi2,6,7, M. Liang14, X. Xu15, C. Qin16, G.
Cheng9, X. Zhang17,18, P. Wang19, X. Qiu12,13, G. Kobinger12, Y. Shi2,6, J. Yan1,2,3,6,20, G. F. Gao2,5,6,7,8,14,21,22
1Chinese
Academy of Sciences, CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Beijing, China
Third People's Hospital, Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen, China
3Anhui University, School of Life Sciences, Hefei, China
4Jiangxi Province Center for Disease Control and Prevention, Nanchang, China
5Chinese Academy of Sciences, Beijing Institutes of Life Science, Beijing, China
6Chinese Academy of Sciences, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Beijing, China
7Chinese Academy of Sciences, CAS Center for Influenza Research and Early-warning (CASCIRE), Beijing, China
8University of Science and Technology of China, School of Life Sciences, Hefei, China
9Tsinghua University, Tsinghua-Peking Center for Life Sciences, School of Medicine, Beijing, China
10Chinese Academy of Sciences, Shanghai Institute of Applied Physics, Shanghai, China
11Ganzhou Prefecture Center for Disease Control and Prevention, Ganzhou, China
12Public Health Agency of Canada, Special Pathogens Program, National Microbiology Laboratory, Winnipeg, Canada
13University of Manitoba, Department of Medical Microbiology, Winnipeg, Canada
14Chinese Center for Disease Control and Prevention (China CDC), National Institute of Viral Disease Control and Prevention, Beijing, China
15University of Oxford, MRC Human Immunology Unit, Oxford, United Kingdom
16Chinese Academy of Medical Sciences, Key Laboratory of Human Diseases Comparative Medicine, Ministry of Health, Institute of Medical Laboratory
Animal Science, Beijing, China
17Chinese Academy of Sciences, National Laboratory of Biomacromolecules, Institute of Biophysics, Beijing, China
18Chinese Academy of Sciences, Center for Biological Imaging CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Beijing, China
19University of Leeds, Faculty of Biological Science, Leeds, United Kingdom
20Chinese Academy of Sciences, College of Life Sciences, Beijing, China
21Zhejiang University, Collaborative Innovation Center for diagnosis and treatment of infectious diseases, Hangzhou, China
22Chinese Academy of Sciences, Savaid Medical School, Beijing, China
2Shenzhen
*These authors contributed equally to this work.
The 2015-2016 outbreak of Zika virus (ZIKV) disease has affected many countries and is a major public health concern.
ZIKV is associated with fetal microcephaly and neurological complications, and medical countermeasures are needed to treat
and prevent ZIKV infection. Here, we report the isolation of 13 specific human monoclonal antibodies from a single imported
patient infected with ZIKV in China. Two of the isolated antibodies (Z23 and Z3L1) demonstrated potent ZIKV-specific
neutralization in vitro without binding or neutralizing activity against dengue virus (DENV) 1-4, the closest relative to ZIKV,
and provided complete post-exposure protection to mice in vivo. Structural studies revealed that Z23 and Z3L1 bound to
tertiary epitopes covering envelope protein domains I, II and/or III, indicating potential targets for ZIKV-specific therapy. Our
results suggest the potential of antibody-based therapeutics and provide a structure-based rationale for the design of future
ZIKV-specific vaccines.
Corresponding author:
Qihui Wang*
[email protected]
Adaptive Immunity
OP42
Antigen-dependent competition shapes the local repertoire of tissue-resident memory CD8+ T cells
A. Muschaweckh1,2, V. Bucholz3, A. Fellenzer4, C. Hessel4, P. A. König2, S. Tao5, R. Tao5, M. Heikenwälder2, D. Busch3, T.
Korn1, W. Kastenmüller6, G. Gasteiger4, I. Drexler5
1Technical
University Munich and Helmholtz Centre Munich, Institute of Virology, Munich, Germany
University Munich, Institute of Virology, Munich, Germany
3Technical University Munich, Institute of Medical Microbiology, Immunology and Hygiene, Munich, Germany
4University Medical Center Mainz, Institute of Medical Microbiology and Hygiene, Mainz, Germany
5Heinrich Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
6University of Bonn Medical Center, Institute of Virology, Bonn, Germany
2Technical
Tissue-resident memory CD8+ T cells (TRM) constitute a major component of the immune-surveillance system in
nonlymphoid organs. Local, noncognate factors are both necessary and sufficient to support the programming of TRM cell
fate in tissue-infiltrating T cells. Recent evidence suggests that TCR signals received in infected nonlymphoid tissues
additionally contribute to TRM cell formation. Here, we asked how antigen-dependent pathways influence the generation of
skin-resident memory T cells that arise from a polyclonal repertoire of cells induced by infection with an antigenically complex
virus and recombinant vaccine vector. We found that CD8+ T cells of different specificities underwent antigen-dependent
competition in the infected tissue, which shaped the composition of the local pool of TRM cells. This local cross-competition
was active for T cells recognizing antigens that are coexpressed by infected cells. In contrast, TRM cell development
remained largely undisturbed by the presence of potential competitors when antigens expressed in the same tissue were
segregated through infection with antigenically distinct viral quasispecies. Functionally, local cross-competition might serve
as a gatekeeping mechanism to regulate access to the resident memory niche and to fine-tune the local repertoire of antiviral
TRM cells.
Corresponding author:
Georg Gasteiger
[email protected]
Emerging Viruses
OP43
Uukuniemi virus as a tick-borne virus model
M. Mazelier1, B. Brügger2, L. Bell-Sakyi3, P. Y. Lozach1
1University
Hospital Heidelberg, Infectious Diseases, Heidelberg, Germany
of Heidelberg, Biochemistry Center (BZH), Heidelberg, Germany
3The Pirbright Institute, The Tick Cell Biobank, Pirbright, United Kingdom
2University
Novel tick-borne pathogenic phleboviruses in the Bunyaviridae family have lately emerged through distinct continents, all
closely related to Uukuniemi virus (UUKV). To recapitulate tick-mammal switch in vitro, we established a reverse genetics
system to rescue UUKV (rUUKV) from plasmid DNAs. IRE/CTVM19 cell line, made from tick Ixodes ricinus, and IDE8 cell
line, made from the tick Ixodes scapularis, are sensitive to rUUKV and enabled the production of the virus after several
weeks, indicating that infection is persistent. Although UUKV was originally found in Ixodes ricinus ticks, our results suggest
that the virus might use other hard ticks for transmission. Our recent results show that UUKV derived from tick vector cells
presents original structural and glycosylation characteristics. Moreover, the amount of structural protein per infectious particle
units appear to be lower in rUUKV particles produced from tick cells than in those derived from mammalian cells, suggesting
that arthropod vector-derived viruses are more infectious. When rUUKV produced in tick or mammalian cells is analyzed by
mass spectrometry for the lipid composition, our preliminary results suggest that tick cell-derived rUUKV has a high amount
of cholesterol, which would also confer a different feature to the virus. Electron microscopy of rUUKV seem to show smaller
particles for the tick cell-derived viruses although this has to be confirm by tomography. Together, our results indicate that
tick cell-derived viruses have a unique fingerprint that make them more infectious. This study also highlights the importance
to work with viruses originating from arthropod vector cells in investigation into the cell biology of arbovirus transmission and
entry.
Corresponding author:
Magalie Mazelier
[email protected]
Emerging Viruses
OP44
Novel reassorted highly pathogenic H5N8 influenza A viruses cause massive outbreaks in wild birds and poultry in
Germany, 2016/2017
A. Pohlmann1, E. Starick1, T. C. Harder1, C. Grund1, D. Höper1, A. Globig2, C. Staubach2, K. Dietze2, G. Strebelow1, R. G.
Ulrich3, J. Schinköthe3, J. P. Teifke3, F. Conraths2, T. C. Mettenleiter4, M. Beer1
1Friedrich
Loeffler Institute, Institute of Diagnostic Virology, Greifswald - Isle of Riems, Germany
Loeffler Institute, Institute of Epidemiology, Greifswald - Isle of Riems, Germany
3Friedrich Loeffler Institute, Department of Experimental Animal Facilities and Biorisk Management, Greifswald - Isle of Riems, Germany
4Friedrich Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
2Friedrich
On November 7th 2016 large numbers of tufted ducks were found dead at Lake Plön in Northern Germany, and at Lake
Constance in Southern Germany. The vast majority of these cases were tested positive for a novel type of HPAIV H5N8
different from the virus that caused the 2014/2015 outbreaks. The epidemic continued and also spread towards the centre of
Germany.
Here, we report about the epidemiology of the outbreak, genetic analysis, the in vitro and in vivo characterization of this
novel HPAIV.
As of January 2017, over 550 infected wild birds and over 30 outbreaks in kept birds were diagnosed. In most cases, birds
were found dead or exhibited severe clinical signs with a sudden increase of mortality. Genetic virus characterization
identified as closest relatives H5N8 strains, which were first detected in wild birds at the Russian-Mongolian border in late
May 2016. They belong to group B (Gochang) H5N8 clade 2.3.4.4 first detected in Asia and are clearly distinct from group A
(Donglim) viruses that caused the outbreaks in 2014/2015. The novel German 2016 viruses further evolved and now harbor
two reassorted segments (PA, NP).
Pathological changes in deceased H5N8-infected birds resembled findings in dead tufted ducks and were dominated by
severe lesions like multifocal necrosis in liver, brain and pancreas. The presence of influenza virus antigen was proven by
immunohistology. High pathogenicity for gallinaceous poultry was confirmed with an intravenous pathogenicity index of 2.93.
In addition, experimental infection of domestic ducks revealed a high virulence, which is in line with the mortality in wild duck
species, but in clear contrast to the German H5N8 strains of 2014/2015.
In conclusion, a novel reassortant H5N8 virus is responsible for the recent massive HPAIV epizootic in Germany. The
observed differences in pathogenicity for waterfowl in comparison to the 2014/2015 H5N8 viruses correlate with a new
genome composition. It can be hypothesized that these new strains are more efficiently shed and transmitted to other wild
and domestic birds. This parallels the large number of cases in wild birds and poultry. Nevertheless, at present, there is no
indication for natural infection of mammals or humans by these novel strains.
Corresponding author:
Martin Beer
[email protected]
Emerging Viruses
OP45
Zika virus NS2B-NS3 protease – Structures, cleavage specificity, and inhibitors
R. Hilgenfeld1, L. Zhang1, Y. Gül1, M. Onwuatuegwo1, W. Rut 2, M. Drag2, J. Lei1
1University
2Wroclaw
of Lübeck, Institute of Biochemistry, Lübeck, Germany
University of Technology, Department of Bioorganic Chemistry, Wroclaw, Poland
The viral NS2B-NS3 protease is an established target for the design of antiviral agents against flaviruses. We have
determined the crystal structure of the ZIKV protease in complex with a potent (Ki = 42 ± 5 nM) boronic-acid inhibitor. The P2
4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the unique Asp83 of the NS2B polypeptide chain,
providing a possible explanation for the unusally low Km and high catalytic activity of the ZIKV protease towards standard
substrates. In the crystal, we observe an unusual dimer with two-fold non-crystallographic symmetry between two ZIKV
NS2B-NS3:inhibitor complexes, which may serve as a model for aggregates formed at high local concentrations of protease
at the endoplasmatic reticulum membrane, the site of polyprotein processing and replication.
The crystal structure is now being used to design inhibitors with improved specificity and membrane permeability. In addition,
we have characterized all NS2B-NS3 protease cleavage sites in the viral polyprotein and derived the substrate specifity from
these and other data.
References:
[1] Lei, J., Hansen, G., Nitsche, C., Klein, C.D., Zhang, L. & Hilgenfeld, R.: Crystal structure of Zika virus NS2B-NS3 protease
in complex with a boronate inhibitor. Science 353, 503-505 (2016).
[2] Nitsche, C., Zhang, L., Weigel, L.F., Schilz, J., Graf, D., Bartenschlager, R., Hilgenfeld, R. & Klein, C.D.: Peptide-boronic
acid inhibitors of flaviviral proteases: Medicinal chemistry and structural biology. J. Med. Chem., in press (E-published: Dec
14, 2016). (DOI: 10.1021/acs.jmedchem.6b01021)
[3] Rut, W., Zhang, L., Kasperkiewicz, P., Poreba, M., Hilgenfeld, R. & Drąg, M.: Extended substrate specificity and first
potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease. Antiviral Res., in press (Epublished: Dec 26, 2016). (DOI: 10.1016/j.antiviral.2016.12.018)
Corresponding author:
Rolf Hilgenfeld
[email protected]
Emerging Viruses
OP46
Epidemic strains of Zika virus infect cytotrophoblasts and Hofbauer cells modeled in explants of chorionic villi from
early-gestation human placentas
L. Pereira1, T. Tabata1, M. Petitt1, H. Puerta-Gardo2, D. Michlmayr2, E. Harris2
1U
California San Francisco, Cell & Tissue Biology, San Francisco, CA, United States
of California Berkeley, Division of Infectious Disease and Vaccinology, Berkeley, United States
2University
Zika virus (ZIKV), a member of the Flavivirus family is responsible for the recent pandemic in the Americas. Women infected
during pregnancy have prolonged viremia and an increased risk of transmitting virus to the fetus, which can lead to
devastating birth defects. Intrauterine growth restriction, a placental defect, is often present in congenital ZIKV infection. How
virus disseminates to the placenta and undermines development is unknown. We recently reported that ZIKV infects primary
cells isolated from the human placenta and fetal membranes from mid- and late-gestation and explants of chorionic villi from
first trimester, ZIKV E and NS3 proteins were expressed and infectious progeny produced suggesting different routes of
transmission. Here, we modeled ZIKV infection in explants of human chorionic villi in the early stages of development,
comparing isolates from the Nicaraguan epidemic (Nica-16) with the African prototype (MR766) in the tissue environment.
Our results are as follows. (i) Consistent patterns of infection were observed in foci of cytotrophoblasts (CTBs) proximal to
the villus core in columns of proliferating cells and expression of Ki67 was reduced in cells infected with both strains. (ii)
CTBs in Nica-16 infected chorionic villi, migrated from distal cell columns and invaded the extracellular matrix; but migration
was limited in CTBs infected with MR766 and many cells appeared apoptotic. (iii) Infected Hofbauer cells in the villus core
were located near cell columns and downregulated the macrophage markers CD163 and LYVE-1. In contrast, floating villi
lacking cell columns seldom contained infected Hofbauer cells except near focal villus sprouts. (iv) Cytokines secreted into
the conditioned medium by control chorionic villi reduced the titers of infectious progeny 5- to 20-fold, or more, and
suppressed NS3 expression. In summary, we identified a transient stage in early placental development vulnerable to ZIKV
infection suggesting transmission could occur in the intervillous blood space, reduce CTB proliferation and Hofbauer cell
functions, and that host factors could limit the spread of ZIKV and bolster protection of the placenta. Funding: This work was
supported by grants from the National Institutes of Health, Institute of Allergy and Infectious Diseases, R01AI046657 (LP),
P01AI106695 (EH) and R01124493 (EH).
Corresponding author:
Lenore Pereira
[email protected]
Emerging Viruses
OP47
Structural biology of Zika virus – drug targets from NS1 and NS5
Y. Shi1, 2, G. F. Gao1,2
1Chinese
2Chinese
Academy of Sciences, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Beijing, China
Academy of Sciences, Beijing, China
Introduction: The association of Zika virus (ZIKV) infections with microcephaly and neurological diseases has highlighted an
emerging public health concern. The ZIKV nonstructural protein 1 (NS1) and nonstructural protein 5 (NS5) function in viral
replication, pathogenesis, and immune evasion, and are the promising targets for antiviral intervention.
Objectives: To solve the crystal structures of ZIKV NS1 and NS5, and provide structural information for drug design.
Material & Methods: We solved the crystal structures of NS1 and NS5 proteins from the ZIKV involved in the 2015 outbreak
in Brazil by X-ray diffraction method.
Results: ZIKV NS1 and NS5 proteins have structural similarities to those found in closely related viruses such as the dengue
and West Nile viruses. But NS1 has some key differences from these related proteins - most notably, a different pattern of
electric-charge distribution in a region that interacts with host cells. In addition, a long-intertwined loop is observed in the
wing domain of ZIKV NS1, and forms a hydrophobic “spike”, which can contribute to cellular membrane association. The
spike region is indispensable for virus infection and can be new target for drug design. Moreover, the tertiary complex
structure of ZIKV methyltransferase (MTase) with S-adenosyl-L-methionine (SAM) and RNA analogue (m7GpppA) reveals
both conserved and specific features on the substrate-binding site and cap-binding site for antiviral inhibitor design. ZIKV
NS5 RNA-dependent RNA polymerase (RdRp) structure reveals a tighter and more closed conformation compared to other
flaviviruses. ZIKV NS5 RdRp possesses conserved drug binding sites including the RNA template entry tunnel and N-pocket,
implicating current available drugs targeting Dengue virus RdRp should be tested for anti-ZIKV activity.
Conclusion: We elucidated a differential electrostatic potential in the host-interaction interface, and found an extended
membrane association interface contributed by the hydrophobic “spike” of a long-intertwined loop, providing important
information for ZIKV pathogenesis. We have identified the conserved features of ZIKV NS5 MTase and RdRp structures that
could lead to development of current antiviral inhibitors used against flaviviruses, including dengue virus and West Nile virus,
to treat ZIKV infection. These results should inform and accelerate the structure-based design of antiviral compounds against
ZIKV.
Corresponding author:
Yi Shi
[email protected]
Emerging Viruses
OP48
Several novel orthobunyaviruses detected in mosquitoes from Central-American rainforests
M. Marklewitz1,2, K. Hermanns1, J. Schmid2,3, G. Eibner2,3, F. Zirkel1,4, J. R. Loaiza2,5, P. Trippner1, S. Sommer3, C.
Drosten1,4, S. Junglen1,4
1University
of Bonn Medical Centre, Institute of Virology, Bonn, Germany
Tropical Research Institute (STRI), Panama City, Panama
3University of Ulm, Institute of Evolutionary Ecology and Conservation Genomics, Ulm, Germany
4German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, Bonn, Germany
5Instituto de Investigaciones Científicas y Servicios de Alta Tecnología, Centro de Biodiversidad y Descubrimiento de Drogas, Panama City, Panama
2Smithsonian
Introduction: The genus Orthobunyavirus (family Bunyaviridae) comprises more than 170 arthropod-transmitted viruses that
can cause severe diseases in humans and animals, like La Crosse virus and Schmallenberg virus. Little is known about the
diversity of orthobunyaviruses existing in sylvatic amplification cycles.
Objectives: Here we tested mosquitoes from Central-American tropical forests for infections with orthobunyaviruses.
Material & methods: Mosquitoes (n=13.978) were collected in the Panama Canal zone in 2013-14 and tested for
orthobunyaviruses by generic RT-PCR. Insect and vertebrate cells were used for virus isolation and growth kinetics. Full
genome sequencing was performed by NGS and RACE-PCR.
Results: Five diverse and previously unknown orthobunyaviruses and a novel strain of Gamboa virus (GAMV) were detected
by RT-PCR. The new viruses showed identities of 54-79% in the palm motif of the RNA-dependent RNA polymerase (RdRp)
to known orthobunyaviruses suggesting the identification of five new species. One of the viruses was isolated from Culex
Melanoconion sp. in C6/36 and VeroE6/7 cells and named Gigante virus (GIGV). The GIGV RdRp, nucleocapsid (N), and
glycoprotein precursor (GPC) proteins showed maximal identities of 70%, 57% and 26%, respectively, to Brazoran virus
(BRZV). Distinct to orthobunyaviruses but similar to BRZV the ORF of the putative non-structural protein NSs is preceding
the N ORF. Phylogenetic analyses suggest that GIGV may belong to a new serogroup. Human-, bird- and rodent cells were
susceptible to GIGV infection. Virus replication peaked in rodent cells suggesting that rodents may play a role in the
amplification cycle. Serum samples from rodents collected along with the mosquitoes are currently tested for infections with
GIGV.
Conclusion: Our data show that sylvatic mosquitoes are infected with a high genetic diversity of previously unknown
orthobunyaviruses including viruses with atypical genome organisations.
Corresponding author:
Marco Marklewitz
[email protected]
Trafficking
OP49
Identification of syntaxin 4 as an essential factor for the hepatitis C virus life cycle
H. Ren1, F. Elgner1, K. Himmelsbach1, S. Akhras1, B. Jiang1, R. Medvedev1, D. Ploen1, E. Hildt 1,2
1Paul
Ehrlich Institute, Virology, Langen, Germany
Centre for Infection Research (DZIF), Braunschweig, Germany
2German
Introduction: The release of hepatitis C virus (HCV) from the infected cell is not fully understood. Although it is well
established that the release of HCV involves structures of the secretory pathway, there is evidence that at least a fraction of
HCV can be released by multivesicular bodies (MVBs) in exosomes. Syntaxin 4 belongs to the t-SNARE protein family and
mediates vesicles fusion.
Objectives: Our aim is to characterize the release pathway of HCV and to study the relevance of syntaxin 4 for the release
of HCV.
Material & Methods: HCV J6-replicating Huh7.5 cells were used to study the release of HCV, while HCV subgenomic
replicon Huh7 cells (Huh9-13) were used to study the impact on HCV replication in the absence of virion formation. Cells
transfected with syntaxin 4 expression construct or syntaxin 4-specific siRNA were analyzed by qPCR, Western blot,
confocal immunofluorescence microscopy, and TCID50.
Results: HCV-replicating cells possess an increased amount of syntaxin 4 protein, although the amount of syntaxin 4specific transcripts is decreased in HCV-replicating Huh7.5 cells and in HCV-infected primary human hepatocytes. In HCVreplicating cells, a significant longer half-life of syntaxin 4 was found that overcompensates the decreased expression and
leads to the elevated level of syntaxin 4. Overexpression of syntaxin 4 reduces the intracellular amount of infectious viral
particles by facilitating viral release, while silencing of syntaxin 4 expression using specific siRNAs increases the intracellular
amount of viral particles by inhibiting HCV particles release. This indicates that HCV uses a SNARE-dependent pathway for
viral release. Confocal immunofluorescence microscopy revealed a colocalization of syntaxin 4 with LDs, exosomes and
MVBs which are essential structures for viral morphogenesis and release.
Conclusion: Our data reveal that the t-SNARE syntaxin 4 facilitates the release of HCV from MVBs by the SNAREdependent pathway, which suggests that syntaxin 4 acts as a novel essential cellular factor for HCV release.
Corresponding author:
Huimei Ren
[email protected]
Trafficking
OP50
Cytomegalovirus pUL50, the multi-interacting determinant of the core nuclear egress complex (NEC), is
phosphorylated by viral and cellular protein kinases
E. Sonntag1, J. Milbradt1, M. Marschall1
1Friedrich
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
Introduction: Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear
egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membraneanchored pUL50 and its nuclear cofactor pUL53, tightly associated through heterodimeric corecruitment to the nuclear
envelope. For multimeric extension, cellular proteins such as p32/gC1qR, emerin, protein kinase C (PKC) and cyclindependent kinase 1 (CDK1), are additionally recruited by pUL50. As a functionally important aspect, an indirect recruitment
of the viral protein kinase pUL97 leads to site-specific lamin phosphorylation and nuclear lamina disassembly. Additionally,
phosphorylation of core NEC proteins is considered as an essential step in NEC formation and capsid egress.
Objective: The aim of the current study is to analyze the pUL50 phosphorylation by viral and cellular protein kinases.
Results: Multiple phosphorylated pUL50 varieties were detected by Western Blot analyses in combination with Phos-tag, as
resulting from pUL97 and/or cellular kinases. In order to clarify the impact of cellular protein kinases, we defined the PKCα
interaction domain within pUL50 (residues 100-280). By means of an in vitro kinase assay (IVKA), we confirmed earlier
evidence that pUL50 is a PKCα substrate. To narrow down the phosphorylation site, additional IVKAs are presently
performed. Moreover, our group previously demonstrated that core NEC formation is sensitive to CDK inhibitors in HCMVinfected cells. Seeking for further NEC-associated protein kinases, CoIP analysis revealed an interaction of pUL50 with
CDK1. This kinase possesses lamin-phosphorylating activity during mitosis, but its role in pUL50 or other NEC protein
phosphorylation is yet unknown. This question is currently under investigation, hence data on pUL50 modification will be
presented. In line with pUL50, we also provided evidence that pUL53 can be phosphorylated by cellular kinases.
Phosphatase treatment of pUL53 revealed a loss of possibly phosphorylation-derived modifications of pUL53.
Conclusion: Summarized, our findings provide first evidence that the HCMV egress protein pUL50 is modified by cellular
protein kinases, hence functional significance needs to be determined in the ongoing project.
Corresponding author:
Eric Sonntag
[email protected]
Trafficking
OP51
A tyrosine-based trafficking motif of the tegument protein pUL71 is crucial for human cytomegalovirus virion
envelopment
A. Dietz1, C. Villinger2,3, S. Becker2, L. Wettstein2, M. Frick1, J. von Einem2
1University
of Ulm, Institute of Gerneral Physiology, Ulm, Germany
University Medical Center, Institute of Virology, Ulm, Germany
3University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany
2Ulm
The final step for generation of infectious human cytomegalovirus (HCMV) particles requires the functional interaction of viral
proteins and their placement at the site of secondary envelopment, the Golgi-derived viral assembly complex (vAC). The
HCMV tegument protein pUL71 is localized at the vAC during infection and known to be required for efficient secondary
envelopment. Analysis of various C-terminally truncated pUL71 fused to EGFP identified amino acids 23-34 to harbor an
important determinant for its Golgi localization. Sequence analysis as well as mutational verification revealed the presence of
an N-terminal tyrosine-based trafficking motif (YxxΦ) in pUL71. Due to its function in trafficking and the reported role of
pUL71 in HCMV secondary envelopment, we hypothesized a requirement of the YxxΦ-motif for generation of infectious
particles. This was addressed by generation of mutant viruses. Mutation of both the entire motif and the tyrosine residue
resulted in a plasma membrane and cytoplasmic localization of pUL71 with very little signal at the vAC in mutant virus
infections, whereas in wild-type virus infected cells, pUL71 strongly localized at the vAC. Both mutant viruses exhibited an
impaired growth, which is characterized by a decreased focal growth and reduced virus yield in the supernatants.
Ultrastructurally, mutant virus infected cells showed a severe defect in secondary envelopment as well as increased numbers
of capsids at the vAC. Additionally, clusters of capsid accumulations surrounding the vAC were observed similarly to the
ultrastructural phenotype of a UL71-deficient mutant. Therefore, our results indicate that the YxxΦ-motif determines the
intracellular localization of pUL71 both in transfection and infection and is required for the function of pUL71 during late
stages of secondary envelopment. A requirement of the YxxΦ-motif for targeting pUL71 to the Golgi was further
demonstrated when clathrin-mediated endocytosis was inhibited by co-expression of AP180-C and treatment with Methylbeta-cyclodextrine. Altogether, these data show the presence of a functional N-terminal endocytosis motif that determines
the intracellular localization of pUL71 during infection and thus its function during HCMV secondary envelopment.
Corresponding author:
Andrea Dietz
[email protected]
Trafficking
OP52
The importins of nuclear targeting of herpes simplex virus in neurons
K. Döhner1, A. Buch1, S. Hügel2, F. Rother3, R. Lebbink4, E. Wiertz4, E. Hartmann2, M. Bader3, B. Sodeik5
1Hanover
Medical School, Institute of Virology, Hanover, Germany
of Lübeck, Department of Biology, Lübeck, Germany
3Max Delbrück Center of Molecular Medicine, Berlin, Germany
4University Medical Center Utrecht, Department of Medical Microbiology, Utrecht, Netherlands
5Hanover Medical School, Institute of Virology, Hanover, Germany
2University
Introduction: Incoming capsids of alphaherpesviruses utilize dynein-mediated transport from the periphery to the cell center
and dock via importin (imp) β at the nuclear pores to inject their genome into the nucleoplasm for transcription and
replication. Similarly, neuronal signaling complexes exploit dynein-mediated axonal transport to the soma, and this transport
process depends on imp α and imp β, suggesting that importins might act as adaptors to recruit dynein.
Objectives: We want to determine, whether herpes simplex virus (HSV1) requires imp α isoforms of the α-S family (α5 & α7)
for retrograde axonal transport and nuclear capsid targeting in neurons.
Material & Methods: Viral gene expression and the subcellular localization of incoming viral particles were analyzed by
confocal laser fluorescence microscopy in neurons of wt, imp-α5-/- or imp-α7-/- mice or after depleting imp-α7 with shRNAs.
Retrograde axonal transport was examined by live cell imaging in compartmentalized chambers which allow for selective
inoculation of axons.
Results: In primary neurons from dorsal root ganglia of adult mice, incoming axonal HSV1 particles colocalized with dynein,
its cofactor dynactin and imp β. After simultaneous infection of somata and axons, nuclear capsid targeting and viral gene
expression were severely impaired after depolymerizing microtubules or interfering with dynein function. However, nuclear
capsid targeting and viral gene expression were just moderately decreased in neurons derived from imp-α5-/- or imp-α7-/knockout mice or after knockdown of imp-α7. In contrast, after selective infection of axons, retrograde axonal transport was
severely impaired in neurons simultaneously depleted for imp-α5 and imp-α7.
Conclusion: In neurons, efficient retrograde axonal transport of HSV1 depends on the presence of at least one imp α-S
family member, suggesting that import factors might cooperate with microtubule motors during axonal transport of HSV1.
Corresponding author:
Katinka Döhner
[email protected]
Trafficking
OP53
A novel live-cell imaging system reveals essential viral factors for the transport of Ebola virus nucleocapsids
Y. Takamatsu1, S. Becker1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
The filoviruses Marburg (MARV) and Ebola (EBOV) cause severe hemorrhagic fever with high case-fatality rates in humans
and nonhuman primates. No approved specific therapy is available and therefore further understanding of the filovirus life
cycle is essential for the development of novel therapeutic options.
EBOV particles contain the non-segmented negative-sense RNA genome and seven viral proteins: NP, VP35, VP40, GP
(glycoprotein), VP30, VP24, and the RNA-dependent RNA polymerase (L). Necessary for EBOV transcription and replication
are the nucleocapsid proteins NP, VP30, VP35, and L. VP24 is an additional factor required for nucleocapsid (NC) assembly.
A layer of the matrix protein VP40 connects the NC with the viral membrane in which GP is inserted. It is known that NP,
VP24 and VP35 are the main structural components of the filoviral nucleocapsids. Recent live-cell imaging studies of MARVand EBOV-infected cells revealed a long-distance actin-dependent transport of NCs from viral inclusions to the plasma
membrane. In order to identify the essential viral proteins for the intracellular NC transport, we have constructed a set of
fluorescently tagged viral proteins (e.g., VP30-GFP, VP35-GFP, VP24-TagRFP) and developed novel systems to visualize
transport of NC-like particles in the background of filovirus-specific virus-like particle systems. We detected that transport of
NC-like particles was dependent on the polymerization of actin and proceeded with similar speed as NC in EBOV- or MARVinfected cells. Moreover, using this system, we identified the viral factors essential for the transport of NC-like particles. The
newly developed non-infectious live-cell imaging system will further contribute to our understanding of molecular interactions
between NCs and cellular proteins, and the development of anti-viral drugs.
Corresponding author:
Yuki Takamatsu
[email protected]
Trafficking
OP54
The CD63-syntenin-1 complex controls post-endocytic trafficking of oncogenic human papillomaviruses
L. Fast1, L. Gräßel1, K. D. Scheffer1, F. Boukhallouk1, G. A. Spoden1, S. Tenzer2, K. Boller3, R. Bago4, S. Rajesh4, M.
Overduin5, F. Berditchevski4, L. Florin1
1University
Medical Center Mainz, Institute of Microbiology and Hygiene, Mainz, Germany
Medical Center Mainz, Department of Immunology, Mainz, Germany
3Paul Ehrlich Institute, Virology, Langen, Germany
4The University of Birmingham, School of Cancer Sciences and Department of Pathology, Birmingham, United Kingdom
5University of Alberta, Department of Biochemistry, Faculty of Medicine and Dentistry, Edmonton, Canada
2University
Introduction: Human papillomaviruses (HPV) are small DNA viruses that infect the basal cells of skin and mucosa. While
low risk HPV types cause benign warts, high risk HPV types are etiologic agents of skin, oropharynx and anogenital cancers.
Objectives: HPV entry involves a novel endocytic pathway that is clathrin, caveolin and dynamin independent. Although
recent studies have shown that HPVs enter host cells by associating with tetraspanin proteins, post-endocytic trafficking
required for virus capsid disassembly remains unclear.
Methods: The role of tetraspanin CD63 during HPV infection was established by siRNA knockdown and re-expression
experiments. To functionally analyse the role of CD63 and its interaction partner syntenin-1 during post endocytic HPV
trafficking, immunofluorescence and electron microscopy followed by endosomal preparations and co-immunoprecipitation
were performed. Interaction of CD63, syntenin-1 and ALIX as a prerequisite for post-endocytic HPV trafficking was analysed
by mutant experiments.
Results: This study demonstrates that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63,
syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive
endosomes recruiting CD63-interacting protein syntenin-1. Mutant analyses show that CD63 and syntenin-1 form a complex
that controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types
16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or
syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and
CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly.
Conclusion: Our results identify the CD63-syntenin-1 complex as a key regulatory component in post-endocytic HPV
trafficking.
Corresponding author:
Laura Fast
[email protected]
Virus Vectors and Gene Therapy
OP55
Construction of large capacity gene transfer vectors based on murine cytomegalovirus
A. Riedl1, D. Bojková1, P. König1, P. Zimmermann1, S. Naumann1, Z. Ruzsics1
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
We investigated the basic vectorological characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using
BAC technology, we constructed a replication competent recombinant MCMV vector lacking 24% of the wildtype genome
and carry combined deletions of the five gene blocks m01-m017, m106-m109, m129-m141, m144-m158, and m159-m170
encompassing 55 kbp in size. While deletion mutants, lacking 24-18% of wildtype genome were attenuated in fibroblasts,
smaller deletions (up to 16%) grown like wildtype virus. By means of a new methodology we inserted two independent large
genomic DNA segments (33 and 42 kbp in size) as stuffer along with reporter genes into an m01-m017, m106-m109, m144m158, and m159-m170 deficient vector (Q4). Surprisingly, the insertion of both foreign DNAs rescued the growth phenotype
of Q4 and the large inserts were stably maintained during serial passages in vitro. Using reporter gene expressing
recombinant MCMVs, we could successfully transduced, beside of mouse cell lines, non-rodent mammalian cells with
human, monkey, bovine, bat origin, and even non-mammalian cell lines derived from chicken. We also tested toxicity of the
MCMV mediated gene transfer and investigated the abortive replication cycle of the MCMV-vectors in human cells upon
transduction.
Corresponding author:
Zsolt Ruzsics
[email protected]
Virus Vectors and Gene Therapy
OP56
Oncolytic viruses retargeted to the tumor stem cell marker CD133
D. Kleinlützum1,2, A. G. Muik1, J. D. S. Hanauer1, S. K. Kays1, C. Ayala-Breton3, K. W. Peng3, C. J. Buchholz1,2
1Paul
Ehrlich Institute, Molecular Biotechnology and Gene Therapy, Langen, Germany
Cancer Research Center (DKFZ), Heidelberg, Germany
3Mayo Clinic, Molecular Medicine, Rochester, United States
2German
Introduction: Therapy resistance and tumor recurrence are often linked to small refractory and highly tumorigenic
subpopulations of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1).
We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to
specifically eliminate CD133+ tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV
hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody
fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133+ tumors than the unmodified
MV-NSe despite being highly selective for its target cells.
Objectives: To further enhance the antitumoral activity of CD133-targeted MV we here pursued arming technologies,
receptor extension and chimeras between MV-CD133 and vesicular stomatitis virus (VSV).
Material & Methods: Specificity and efficacy were analyzed by cell culture techniques using conventional cell viability
assays and flow cytometry. Preclinical feasibility was assessed in a subcutaneous (s.c.) hepatocellular carcinoma (HCC)
xenograft model and an orthotopic glioma model. Viral distribution and spreading were quantified by immunofluorescence
staining of cryo slices.
Results: The data show that all oncolytic viruses (OVs) selectively infected and lysed their target cells. In terms of
antitumoral activity, MV-CD133-CD46, which can use both CD46 and CD133 as entry receptor, and VSV-CD133 performed
best in vitro and in vivo. Further, while immunostaining revealed replicating virus in all tumors of all treatment groups, the
numbers and sizes of infectious centers in VSV-CD133 treated mice were up to 2-log steps higher than within MV-treated
tumors. Unexpectedly, however, VSV-CD133 caused neurotoxicity in the orthotopic glioma model. Use of CD133 as receptor
could be excluded as being causative.
Conclusion: Our data reveal new concepts and approaches towards enhancing the oncolytic activity of tumor-targeted OVs
but also raise awareness about careful toxicity testing of novel OV types.
Corresponding author:
Dina Kleinlützum
[email protected]
Virus Vectors and Gene Therapy
OP57
Measles virus-derived oncolytic tumor vaccines inhibit metastasis and are effective during therapeutic treatment
B. Bodmer1, S. Hutzler1, S. Erbar2, C. Nürnberger1, J. Schnotz1, R. Jabulowsky2, U. Sahin2, M. Muehlebach1
1Paul
Ehrlich Institute, Veterinary Medicine, Langen, Germany
RNA Pharmaceuticals GmbH, Mainz, Germany
2BioNTech
Question: Immunotherapeutic strategies for cancer treatment aim to break tolerance of the patients´ immune system to
induce specific responses against mostly endogenous tumor-associated antigens. Vaccine strain-derived measles virus (MV)
is under clinical development as an oncolytic virus directly killing tumor cells, but can also be used as a vaccine platform. MV
is considerably immunogenic, since both damage- and pathogen-associated molecular patterns are released during MV
replication. To take advantage of these properties, we constructed oncolytic MVs that additionally present a selected tumorassociated antigen in a highly immunogenic format to induce specific anti-tumoral immune responses, which should enhance
therapeutic efficacy by an immunotherapeutic mode of action.
Methods: Moraten vaccine strain-derived recombinant MVvac2 were generated that present full length muGNTP01, a recently
described tumor-associated antigen (TAA), in infected cells or on virus-like particles. After determining stable antigenexpression in vitro, immunogenicity of the oncolytic vaccines was tested in MV-susceptible IFNAR-/--CD46Ge mice. Efficacy
and synergy of induced anti-tumoral immune responses were then tested in a prophylactic model for lung metastasis or in a
therapeutic model employing subcutaneous tumors, both models utilizing syngenic B16 melanoma cells that were stably
transduced to express muGNTP01 and the MV receptor CD46. Tumor burden, survival, infiltration of leucocytes into the
tumor tissue, and induction of GNTP01-specific immunity in the therapeutic setting were monitored.
Results: Both MV-derived vaccines as well as controls revealed normal virus growths, were genetically stable over 10
passages, and presented the antigen in cells or on particles. Significant tumor antigen-specific humoral or cellular immune
responses were induced in mice, which inhibited metastasis formation and synergistically enhanced the therapeutic effect of
oncolysis. Prolonged survival correlated with enhanced leucocyte infiltration into tumor tissue of the oncolytic vaccine-treated
mice, while TAA-specific T cells became detectable after therapeutic treatment.
Conclusion: Oncolytic MVs presenting TAAs induce effective immune anti-tumoral immune responses that synergize with
oncolysis.
Corresponding author:
Michael Muehlebach
[email protected]
Virus Vectors and Gene Therapy
OP58
Foamy virus particles – Adaptable nucleic acid delivery vehicles to provide tools for genetic engineering
F. Lindel1, N. Weidner1, C. Dodt1, F. Bergemann1, M. Hamann1, D. Lindemann1
1Technical
University of Dresden, Institute of Virology, Dresden, Germany
Foamy viruses (FVs), like other retroviruses, can be used to efficiently transfer nucleic acids (DNA and RNA) into a broad
range of mammalian cells. Since technologies for specific engineering and modification of genomic DNA rapidly advanced in
recent years we developed a FV vector system for transduction and transient expression of CRISPR/Cas9 components
(Cas9 and sgRNA) to specifically inactivate genes in mammal target tissues. Our approach makes use of a FV capability we
recently described; the efficient Gag-dependent encapsidation of non-viral RNAs into viral particles that enables a transient
genetic modification of target tissues in vitro and in vivo (Hamann et al., Molecular Therapy 2014). By a single, simultaneous
co-transduction of Cas9 mRNA and a non-integrating GFP-specific sgRNA DNA expression cassette (integrase-deficient FV
particles) into human bone osteosarcoma indicator cells (U2OS-GFP), harboring a stably integrated single copy of GFP, we
achieved an efficient gene inactivation (>95%). The promising results encouraged us to further advance our system for
homology directed repair (HDR)-mediated gene editing. Therefore we provided an additional DNA repair template in particles
transferring the sgRNA coding sequence. We were able to demonstrate HDR events in U2OS-GFP cells by a flow cytometric
assay, detecting a change of green to red fluorescence as a consequence of Cas9 induced disruption of the GFP ORF and
subsequent HDR-mediated integration of a red fluorescent protein (RFP) ORF provided by the viral-delivered repair matrix.
Only recently, we have also successfully applied our new FV gene editing system in an endogenous protein tagging
approach. Endogenous human polo-like kinase 1 protein was labeled with GFP to study its interaction with PFV Gag under
physiological conditions by fluorescent imaging approaches. Our results show that the new FV vector system can be easily
adopted for efficient and transient delivery of genetic engineering tools into various target tissues.
Corresponding author:
Fabian Lindel
[email protected]
Virus Vectors and Gene Therapy
OP59
CD30-targeted oncolytic viruses as novel therapeutic approach against Hodgkin Lymphoma
J. D. S. Hanauer1, B. Rengstl2, T. Friedel1, I. Schneider1, S. Newrzela2, C. J. Buchholz1, A. G. Muik1
1Paul
Ehrlich Institute, Molecular Biotechnology and Gene Therapy, Langen, Germany
Wolfgang Goethe University Frankfurt, Institute of Pathology, Frankfurt a.M., Germany
2Johann
Introduction: Hodgkin lymphoma (HL) is a hematopoietic malignancy with a characteristic cellular composition. The tumor
mass is made up of infiltrated lymphocytes and other cells of hematologic origin but only very few neoplastic cells (<2%) that
are identified by the diagnostic marker CD30. While most patients can be cured by standard therapy, approximately 20%
relapse, suffer from progressive disease and develop secondary cancers. Here, we suggest a novel therapeutic concept
relying on oncolytic viruses that selectively destroy the CD30-positive HL tumor cells.
Objectives: To selectively destroy CD30-positive HL tumor cells and to assess the performance of CD30-targeted measles
virus (MV-CD30) and vesicular stomatitis virus (VSV-CD30) for this task.
Material & Methods: The coding sequence of a recently described CD30-specific scFv was displayed on the MV
hemagglutinin. The H gene in the MV genome was substituted with this construct and MV-CD30 was rescued. For VSVCD30 the VSV glycoprotein G reading frame was replaced by those of MV-F and H-CD30. Specificity and efficacy of the
viruses were analyzed by cell viability assays and flow cytometry. Preclinical feasibility was assessed in a subcutaneous
(s.c.) HL xenograft model. Viral replication was analyzed by an overlay of explanted tumor tissue onto target cells.
Results: MV-CD30 and VSV-CD30 were found to be highly selective for CD30-positive cells as demonstrated by infection of
co-cultures of target and non-target cells as well as through blocking infection by soluble CD30. Notably, VSV-CD30
replicated significantly faster to much higher titers than MV-CD30 and resulted in a more rapid and efficient killing of
cultivated HL cells. Data from mouse tumor models show that intratumorally as well as systemically injected VSV-CD30
replicated in s.c. HL xenografts and significantly slowed down tumor growth resulting in a substantially prolonged survival of
tumor-bearing mice.
Conclusion: Taken together, the data support further preclinical testing of VSV-CD30 as novel therapeutic agent for the
treatment of HL.
Corresponding author:
Julia D. S. Hanauer
[email protected]
Virus Vectors and Gene Therapy
OP60
Efficient delivery of a multiplex CRISPR/Cas9 machinery for the correction of mutations underlying Duchenne
Muscular Dystrophy (DMD) using one single high capacity adenoviral vector
E. Ehrke-Schulz1, M. Gebbing1, R. A. Kley2, A. Ehrhardt1
1Witten/Herdecke
2Ruhr
University, Institute of Virology and Microbiology, Witten, Germany
University Bochum, Department of Neurology, Neuromuscular Centre Ruhrgebiet, University Hospital Bergmannsheil, Bochum, Germany
Introduction: Recent advances in the field of designer nuclease directed genome editing hold great promise to correct
underlying mutations leading to Duchenne Muscular Dystrophy (DMD). The CRISPR/Cas9 system provides an easy way to
design and assemble RNA guided nucleases offering the potential to develop personalized treatments to correct different
mutations leading to this fatal disease. Recent studies showed efficient genome editing in a myoblast cell line derived from
DMD patients and mdx mice. Nevertheless viral delivery of all required CRISPR/Cas9 components including Cas9 and one
or multiple guide RNA (gRNA) expression units has not been fully exploited. Gene deleted high-capacity adenoviral vectors
(HCAdVs) offer the packaging capacity to deliver the complete CRISPR/Cas9 machinery including several gRNA expression
units using one single viral vector.
Objectives: We aimed at delivering a multiplex CRISPR/Cas9 machinery for the correction of mutations underlying DMD
using one single HCAdV.
Material & Methods: By using a new toolbox facilitating customization, cloning and production of CRISPR-HCAdVs, we
assembled a HCAdV genome containing a spCas9 gene including two guide RNA (gRNA) expression units specific for
intronic sequences flanking the mutated exon 51 of the DMD gene. CRISPR-HCAdV was amplified in medium scale using a
shortened protocol.
Results: We successfully assembled the DMD specific CRISPR-HCAdV genome and produced the vector to high titers.
Infection of primary human myoblasts with DMD specific CRISPR-HCAdV resulted in strong locus specific deletion efficiency
for DMD exon 51 as shown with locus specific PCR and sequencing. Therefore this strategy could be exploited to develop a
novel treatment option for DMD patients.
Conclusion: Our CRISPR-HCAdV platform enables cloning and production of gene deleted adenoviral vectors for the
delivery of a DMD specific CRISPR/Cas9 system providing a valuable tool for viral delivery of customized CRISPR/Cas9 for
the correction of the manifold mutations causing DMD. Guide RNAs with other specificities can be included in the allowing
personalized molecular design of the vector. We expect that this may pave the way towards broader applications of the
CRISPR technology for DMD treatment including preclinical and eventually clinical studies.
Corresponding author:
Eric Ehrke-Schulz
[email protected]
Structure and Assembly
OP61
Labeling of the HIV-1 envelope protein by genetic code expansion and click chemistryfor live-cell imaging and
super-resolution nanoscopy
V. Sakin1, J. Hanne1,2, M. Anders-Össwein1, V. Laketa1,3, I. Nikić4,5, H. G. Kräusslich1,3, E. A. Lemke4, B. Müller1
1University
Hospital of Heidelberg, Department of Infectious Diseases, Virology, Heidelberg, Germany
Optical Nanoscopy Division, Heidelberg, Germany
3German Centre for Infection Research (DZIF), Heidelberg, Germany
4European Molecular Biology Laboratory, Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, Heidelberg, Germany
5University of Tübingen, Werner Reichard Centre for Integrative Neuroscience, Tübingen, Germany
2DKFZ,
The envelope glycoproteins (Env) of human immunodeficiency virus (HIV-1) mediate cell entry through fusion of the viral
envelope with a target cell membrane. Env incorporation into budding virions is known to be mainly mediated by the matrix
domain of the Gag polyprotein. Although the molecular details of this process are still controversial, the advent of superresolution fluorescence microscopy (SRFM) techniques opened novel possibilities to study the nanoscale organisation of Env
and Gag both on the surface of virus expressing cell as well as on the viral envelope. Previous live-cell and super-resolution
microscopy studies showed that intramembrane mobility and clustering of Env trimers at the viral budding site are essential
for its function. However, these studies were limited by lack of a functional fluorescent Env derivative, requiring antibody
labeling for detection. Introduction of bio-orthogonal amino acids by genetic code expansion, combined with click-chemistry,
offers novel possibilities for site-specific, minimally invasive labeling. Using this approach, we established efficient
incorporation of non-canonical amino acids within HIV-1 Env in mammalian cells. The engineered protein retained plasma
membrane localization, glycosylation, virion incorporation and fusogenic activity and could be rapidly and specifically labeled
with synthetic dyes. This strategy allowed us to revisit Env dynamics and nanoscale distribution at the plasma membrane
close to its native state, applying fluorescence recovery after photobleaching and STED nanoscopy, respectively.
Corresponding author:
Volkan Sakin
[email protected]
Structure and Assembly
OP62
Structural insights into arenavirus cap-snatching machinery
M. Rosenthal1, N. Gogrefe1, J. Reguera2, D. Vogel1, B. Rauschenberger1, S. Cusack3, S. Günther1, S. Reindl1
1Bernhard
Nocht Institute of Tropical Medicine, Virology, Hamburg, Germany
et Fonction des Macromolécules Biologiques, Marseille, France
3European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France
2Architecture
Arenaviruses can cause hemorrhagic fevers and are thus a human thread. For transcription of viral mRNAs the virus
depends on a mechanism called cap-snatching, where viral proteins cleave off 5'-cap structures of host mRNAs and use
those to prime viral mRNA synthesis. This mechanism is essential and virus specific and therefore an attractive drug target.
Available data is mainly on the related influenza virus, where cap-snatching is mediated by the viral polymerase and takes
place in the nucleus of the host cell. For arenaviruses and closely related bunyaviruses this mechanism is thought to be
carried out by the viral >200 kDa L protein in the cytoplasm of the host cell. The essential components for the cap-snatching
mechanism are a cap-binding site to "snatch" the cap and an endonuclease to cleave it off. So far only the endonuclease has
been identified in the very N-terminal region of arena- and bunyavirus L proteins and was structurally and functionally
characterized. The location of the cap-binding site, however, is not known.
We present novel crystal structures of two domains of an arenavirus L protein: the endonuclease positioned at the very Nterminus as well as a C-terminal fragment. We provide the first structural evidence for a potential cap-binding site in the
arenavirus L protein. Our structures open the floor for discussions about the cap-snatching mechanism of arena- and
bunyaviruses in relation to influenza virus.
Corresponding author:
Maria Rosenthal
[email protected]
Structure and Assembly
OP63
RNA structure in Influenza A virus ribonucleoproteins
D. Ferhadian1, V. Vivet-Boudou1, G. Bec1, P. Wolff1, R. Marquet1
1IBMC
/ CNRS, UPR9002, Strasbourg, France
The Influenza A virus (IAV) genome consist of eight (-) sense viral RNA (vRNA) segments of different length, each of them
coding for at least one essential viral protein. A heterotrimeric polymerase complex is bound at the extremities of each vRNA,
while internal parts of the vRNAs are associated with multiple copies of the viral nucleoprotein (NP) in order to form viral
ribonucleoproteins (vRNP). NP forms a double helix on which vRNA binds. However, while the structure of the NP scaffold
has been partially elucidated by cryo-EM, the structure of the vRNA structure within vRNPs remains elusive.
In an attempt to address this question, we performed chemical probing of in vitro transcribed vRNAs 6, 7, and 8 of the
A/Puerto Rico/8/34 (PR8) strain using SHAPE (selective 2"-hydroxyl acylation analyzed by primer extension). While these
vRNAs appear highly structured, most helices are fairly short and they are devoid of very long and stable double helices:
depending on the vRNA, 70 to 85 % of helices are ≤6 base-pairs. We next compared the SHAPE reactivity of "naked"
vRNAs 7 and 8 with those of vRNA/NP complexes formed in vitro. To this aim, we overexpressed His-tagged PR8 NP in E.
coli and purify it to homogeneity by two HPLC steps (Ni-NTA and heparin columns). Finally, we also analyzed the vRNA
structure after incubation with NP and removal of the protein. Altogether these experiments should allow us to address the
following questions: 1) does NP recognize specific sequence or/and structural motifs? 2) to what extent does NP destabilize
the vRNA secondary structure? 3) does NP permanently modify the vRNA structure? (i.e. does NP possess an RNA
chaperone activity). We observed 12 and 26 protected sites in single stranded regions of vRNAs 7 and 8, respectively (for
947 and 817 analyzed nucleotides, respectively). These sites tend to be pyrimidine-rich and less than half of them are
located in apical loops. Interestingly, regions with increased SHAPE reactivity were systematically observed in the vicinity of
the protected regions, strongly suggesting that NP destabilizes the helices adjacent to its binding sites. Another possibility is
that NP not only binds to single-stranded region but also to helices, in an asymmetric manner.
Corresponding author:
Damien Ferhadian
[email protected]
Structure and Assembly
OP64
Crystal structure of the HCMV inner tegument protein pUL77
P. Naniima1,2, E. Borst1, B. Sodeik1,2, M. Messerle1,2, T. F. Schulz1,2, T. Krey1,2
1Hanover
2German
Medical School, Institute of Virology, Hanover, Germany
Center for Infection Research (DZIF), Hanover, Germany
Human herpesviruses give rise to severe diseases, especially in children, immunocompromised and elderly individuals.
Unsatisfactory current treatment options together with few safe available vaccines result in an unmet demand for novel
antiviral drugs. One way to develop small molecule inhibitors are structure-based approaches, such as molecular docking or
in silico screening.
α- and γ-Herpesvirus capsids are stabilized during virus maturation by an essential protein complex at the capsid vertices
known as the capsid vertex-specific component (CVSC; pUL25 and pUL17 in herpes simplex virus 1 (HSV-1) and their
homologs ORF19 and ORF32 in Kaposi's sarcoma-associated herpesvirus (KSHV)). Interestingly, recent electron
microscopy studies suggested that β-herpesviral capsids (e.g., human cytomegalovirus (HCMV)) are stabilized by the viral
protein pp150 via an interaction with the smallest capsid protein (SCP).
We expressed and biochemically characterized a soluble N-terminally truncated fragment of pUL77 and ORF19, the pUL25
homologs of HCMV and KSHV, respectively, revealing monodisperse monomeric proteins that gave rise to diffraction quality
crystals of both proteins. We determined the crystal structure of pUL77 at 1.9Å resolution and are currently in the process of
structure determination for ORF19. A comparison of our pUL77 structure with the reported structure of pUL25 revealed a
striking structural conservation despite low sequence similarity (~25% amino acid identity). Our results suggest strong
evolutionary constraints on the overall fold of this herpesviral tegument protein and highlight structurally conserved (and thus
likely functionally important) regions. In addition, the obtained crystal structure together with recently reported cryo-EM
reconstructions of herpesviral capsids will allow the identification of potential protein-protein interfaces that could serve as
targets for novel anti-herpesvirus drugs.
Corresponding author:
Thomas Krey
[email protected]
Structure and Assembly
OP65
HCMV nuclear capsid motility is neither directed nor dependent on nuclear F-actin
E. M. Borst1, M. Holthaus2, T. Günther3, R. Reimer4, C. Schneider4, M. Messerle1, A. Grundhoff3, K. Grünewald4, J. B. Bosse2
1Hanover
Medical School, Institute of Virology, Hanover, Germany
Pette Institute, Structural Biology of Viruses - Quantitative Virology, Hamburg, Germany
3Heinrich Pette Institute, Leibniz Institute of Experimental Virology
4Heinrich Pette Institute, Structural Biology of Viruses, Hamburg, Germany
2Heinrich
Cellular life is based on the steady state of a myriad of interconnected, dynamic interactions and transport processes,
allowing the cell to respond rapidly to cues. Herpesviruses hijack this web of dynamic interactions to fulfill their life cycles. A
large body of work clearly shows that long-range transport of cytosolic herpesvirus particles is mediated through microtubulebased, active transport. However, herpesvirus capsids assemble in the host nucleus and must reach the nuclear envelope
for nuclear egress. Early work had suggested that nuclear HSV-1 capsids use F-actin mediated, active transport to bridge the
nucleoplasm.
In discordance with this model, we found that neither HSV-1, PRV, MCMV nor MHV-68 induce detectable, nuclear F-actin.
We also analyzed the mode of nuclear capsid motion by single particle tracking for HSV-1 and PRV and found that nuclear
remodeling allowed capsids to bridge the nucleoplasm by diffusion. Recently, another report (Wilkie et al. mBio 2016)
suggested that HCMV induces nuclear F-actin in HFF cells expressing LifeAct-EGFP-NLS and that these filaments are
involved in transport of capsids to the nuclear periphery for nuclear egress.
Using a combination of imaging approaches, we show here that HCMV capsids do not use nuclear actin filaments for
intranuclear transport. First, a novel, capsid-tagged HCMV mutant allowed us to perform single particle tracking with high
temporal resolution. Our analysis of hundreds of tracks confirms that also HCMV remodels the nuclear chromatin structure to
facilitate capsid diffusion and does not use directed intranuclear transport. Second, correlation of nuclear filaments with
capsid tracks at superresolution precision shows no evidence for directed transport along these filaments. This evidence is
further supported by ultrastructural data using serial block face scanning EM allowing us to quantify the effects of nuclear Factin-depolymerizing drugs on the capsid distribution in whole cells.
Corresponding author:
Jens Bernhard Bosse
[email protected]
Tumor Viruses
OP66
Induction of dormancy in hypoxic human papillomavirus (HPV)-positive cancer cells
K. Hoppe-Seyler1, F. Bossler1, C. Lohrey1, J. Bulkescher1, F. Rösl2, L. Jansen3, A. Mayer4, P. Vaupel4, M. Dürst3, F. HoppeSeyler1
1German
Cancer Research Center (DKFZ), Molecular Therapy of Virus-Associated Cancers, Heidelberg, Germany
Cancer Research Center (DKFZ), Viral Transformation Mechanisms, Heidelberg, Germany
3University Hospital Jena, Department of Gynaecology, Jena, Germany
4University Medical Center Mainz, Department of Radiooncology and Radiotherapy, Mainz, Germany
2German
Introduction: Oncogenic HPVs are closely linked to major human malignancies, including cervical and head & neck cancer.
It is believed that HPV-positive cancer cells are under selection pressure to continuously express the viral E6/E7 oncogenes
("oncogene addiction"), that their intracellular p53 levels are reconstituted upon E6/E7 repression, and that E6/E7 inhibition
results in cellular senescence.
Objectives: These central dogmas of HPV-induced carcinogenesis are mainly based on functional studies performed under
standard cell culture conditions (21% O2). This neglects the fact that HPV-positive cancers often contain hypoxic regions
(<1.5% O2). Here, we investigated the HPV/host cell crosstalk under hypoxic conditions.
Material & Methods: The effects of hypoxia on viral oncogene expression, on p53 levels and on the senescence regulation
of HPV-positive cancer cells was assessed. Signal transduction pathways that determine the phenotype of hypoxic HPVpositive cancer cells were investigated. The response of hypoxic HPV-positive cancer cells towards chemotherapy was
analyzed. In vitro studies were accompanied by analyses of biopsies from cervical cancer patients.
Results: Hypoxia enables HPV-positive cancer cells to escape from the regulatory principles that act under normoxic
conditions: E6/E7 is efficiently repressed, yet, p53 levels do not increase. Moreover, E6/E7 repression under hypoxia does
not result in cellular senescence, due to hypoxia-associated impaired mTOR signaling via the inhibitory REDD1/TSC2 axis.
Instead, a reversible growth arrest is induced that can be overcome by reoxygenation. Impairment of mTOR signaling also
interfered with the senescence response of hypoxic HPV-positive cancer cells towards chemotherapy.
Conclusion: Collectively, our results indicate that hypoxic HPV-positive cancer cells induce a reversible state of dormancy,
with decreased viral antigen synthesis and increased therapeutic resistance, and may serve as reservoir for tumor
recurrence upon reoxygenation. This response is likely to affect the clinical behavior of HPV-positive cancers and should be
considered for the ongoing development of novel treatment strategies, such as immunotherapy or targeted E6/E7 inhibition.
Reference:
K. Hoppe-Seyler et al., PNAS, in press
Corresponding author:
Karin Hoppe-Seyler
[email protected]
Tumor Viruses
OP67
Identification of the tumor suppressor C/EBPα, a novel target of the HPV8 E6 protein, as a key regulator of the
stemness-regulating miR-203
A. Marthaler1, A. Fingerle1, E. Ebert2, Y. J. Kim2, C. Müller3, F. Grässer1, S. Majewski4, S. Smola1
1Saarland
University, Institute of Virology, Homburg, Germany
University, Institute of Pathology, Homburg, Germany
3Saarland University, Department of Dermatology, Homburg, Germany
4Medical University of Warsaw, Department of Dermatology and Venereology, Warsaw, Poland
2Saarland
Question: Genus beta-HPV infect cutaneous epithelium and their potential role in skin carcinogenesis has become a major
field of interest. A link between HPV5 or 8 infection and skin cancer was first demonstrated in patients suffering from
Epidermodysplasia verruciformis (EV), an inherited skin disease. Studies in transgenic mice have unraveled the E6 protein
as the major driver of HPV8-induced skin carcinogenesis, however, the underlying mechanism remained unclear. In this
study we show that HPV8 E6 regulates a key stemness pathway in keratinocytes.
Methods: Primary human keratinocytes and the skin keratinocyte cell line HaCaT were retrovirally engineered to express
HPV8 oncogenes. Cells were investigated in 3-dimensional organotypic and monolayer cultures for proliferation (BrdU- and
scratch assays) or differentiation using specific siRNAs as well as in EMSA and miR-203 reporter gene analysis. Functional
effects of HPV8 E6 (wild type and deletion mutant) were studied in Northern and Western Blot, qRT-PCR,
immunohistochemistry or by in-situ-hybridization of 3D-cultures and EV-biopsies.
Results: Our data show that the HPV8 E6 protein is a potent suppressor of the stemness-regulating miR-203. Thereby,
HPV8 E6 controls expression of the miR-203 target ΔNp63α, and in consequence proliferation and differentiation of
keratinocytes. We have identified CCAAT/enhancer-binding protein α (C/EBPα), a differentiation-inducing transcription factor
and suppressor of UV-induced skin carcinogenesis, as a novel p300-dependent target of the HPV8 E6 protein and as a
novel regulator of miR-203 expression. In vivo analyses of EV-lesions strongly support our findings.
Conclusion: In summary, our data provide evidence for a novel mechanism, how HPV8 can deregulate skin homeostasis in
a C/EBPα/miR203/p63-dependent manner potentially contributing to the enhanced susceptibility of HPV8-infected EVpatients for UV-induced skin carcinogenesis.
Corresponding author:
Anna Marthaler
[email protected]
Tumor Viruses
OP68
Peripheral and mature B cells are dispensable for Marek's disease virus pathogenesis
L. D. Bertzbach1, M. Laparidou2, B. Kaspers3, B. Schusser2, B. B. Kaufer1
1Free
University Berlin, Institute of Virology, Berlin, Germany
University Munich, Reproductive Biotechnology, Munich, Germany
3Ludwig Maximilian University Munich, Institute of Animal Psychology, Munich, Germany
2Technical
Introduction: B cells play a central role in the model of Marek's disease virus (MDV) pathogenesis. During primary infection,
MDV efficiently replicates in B cells and facilitates the transfer of the virus to T cells. MDV subsequently establishes latency
in infected T cells and induces transformation of CD4 T cells, resulting in lethal lymphomas.
Objectives: Despite many years of research, the exact role of different B cell subtypes in MDV pathogenesis remains poorly
understood. Recent advances in avian genetics have allowed the generation of genetically engineered chickens that lack
peripheral and mature B cells (JH-KO), but still harbor immature precursor B cells in the bursa.
Material & Methods: To determine the role of these B cells in MDV pathogenesis, we infected JH-KO or wild type chickens
with MDV RB1B and assessed the viral titers in the blood, Marek's disease and tumor incidence.
Results: Surprisingly, even in the absence of mature and peripheral B cells, Marek's disease and tumor incidence in JH-KO
chickens were comparable to wild type animals, suggesting that these B cells are dispensable for MDV pathogenesis. In
addition, viral loads in the blood and various organs of infected animals were not altered in JH-KO compared to wild type,
indicating that these mature and peripheral B cells are not required for virus replication and dissemination in vivo.
Conclusion: Taken together, our data shed light on the role of peripheral and mature B cells in disease incidence and
tumorigenesis, allowing us to further refine the current model of MDV pathogenesis.
Corresponding author:
Luca Danilo Bertzbach
[email protected]
Tumor Viruses
OP69
Epstein-Barr virus particles induce centrosome amplification and chromosomal instability
A. Shumilov1, M. H. Tsai1, H. J. Delecluse1
1German
Cancer Research Center (DKFZ), Pathogenesis of virus-associated tumors F100, Heidelberg, Germany
Introduction: The Epstein-Barr virus (EBV) is a human γ-herpesvirus that infects the majority of the world population [1].
The virus has a preferential tropism for B lymphocytes and it has been associated with several malignancies of lymphoid and
epithelial origin1. These include Burkitts or Hodgkins lymphomas as well as nasopharyngeal (NPC) and gastric carcinomas
[1]. Several studies have identified EBV lytic replication, the process that generates virus progeny, as a strong risk factor for
some of these diseases [2,3].
Objectives: We studied the cell division machinery of EBV-infected cells and explored mechanisms that underlie the effect
of EBV lytic replication on chromosomal instability and EBV-induced oncogenesis.
Material & Methods: EBV virus, primary B cells, epithelial cell lines, animal models, IFA, whole genome painting
Results: We found that EBV infection of B-lymphocytes in vitro and in an animal model leads to an increased rate of
centrosome amplification, associated with chromosomal instability (CIN). This effect was due to the virion itself as it could be
reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect their
target cells. A protein that is abundantly present in the EBV virion induced centrosome amplification and viruses that lacked it
largely lost this property.
Conclusion: Our findings identify a new mechanism through which EBV particles induce CIN without establishing a chronic
infection, thereby conferring a risk for the development of tumors that do not necessarily carry the viral genome.
References:
[1] Rickinson, A. B. & Kieff, E. D. in Field Virology Vol. 2 (ed Howley PM Knipe DM, Griffin DE, Lamb RA, Martin MA,
Roizman B, Straus SE) 2655-2700 (Lippincott Williams & Wilkins, 2007).
[2] Chien, Y. C. et al. Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. N
Engl J Med 345, 1877-1882, doi:10.1056/NEJMoa011610 345/26/1877 [pii] (2001).
[3] Hsu, W. L. et al. Independent effect of EBV and cigarette smoking on nasopharyngeal carcinoma: a 20-year follow-up
study on 9,622 males without family history in Taiwan. Cancer Epidemiol Biomarkers Prev 18, 1218-1226, doi:10559965.EPI-08-1175 [pii] 10.1158/1055-9965.EPI-08-1175 (2009).
Corresponding author:
Anatoliy Shumilov
[email protected]
Tumor Viruses
OP70
Characterization of the interaction of the host factor Kap1/TRIM28 and the Merkel cell polyomavirus encoded
oncoproteins
S. Siebels1, M. Czech-Sioli1, J. Theiss1, M. Spohn2, C. Schmidt1, A. Grundhoff2, N. Fischer1
1University
2Heinrich
Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
Merkel cell polyomavirus (MCPyV) is the only human polyomavirus associated with tumorigenesis. Similar to other
polyomaviruses MCPyV expresses a Large Tumor Antigen (LT) that together with Small Tumor Antigen (sT) contributes to
cellular transformation and is of critical importance for the initiation of the viral DNA replication. However, there are significant
differences of MCPyV early region compared to other polyomaviruses: (1) MCPyV LT encodes for unique linker regions of so
far unknown function flanking the Retinoblastoma protein (Rb) binding site, (2) MCPyV LT does not bind p53 and (3)
although truncating mutations resulting in a shortened LT protein in the tumor cells are a hallmark of MCPyV induced
tumorigenesis, in vitro experiments indicate sT as a major driver of cellular transformation.
Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our knowledge
how MCPyV ensures its own replication and how the virus contributes to cellular transformation.
We identified the KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in co-transcriptional regulation,
as a novel protein interaction partner of both T-Antigens. We confirmed these interactions by co-immunoprecipitation studie.
We demonstrate that the N-terminal RBCC domain of Kap1 interacts with the N-terminus of both T-Antigens. Applying our in
vitro MCPyV replication assay, we reveal that Kap1 knockdown or knockout results in a significant increase in viral DNA
replication highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Interestingly, different
to other DNA viruses viral gene expression is unaffected in the absence of Kap1. Enhanced viral DNA replication in the
absence of Kap1 is not a consequence of increased binding affinity of LT to the viral origin of replication. To investigate the
molecular mechanism underlying the observed changes we performed genome wide transcriptome analysis in cells devoid of
Kap1 in the presence/ absence of viral early gene expression.
We demonstrate that in the absence of Kap1 LT significantly changes cellular gene expression compared to cells expressing
Kap1. We will discuss the observed changes and how these might contribute to Merkle Cell Cancer pathogenesis.
Corresponding author:
Svenja Siebels
[email protected]
Tumor Viruses
OP71
BRD2/4-mediated chromatin association of LANA is important for latent replication of KSHV
M. Weidner-Glunde1, R. Lotke1, T. Günther2, J. Rückert1, A. Grundhoff2, T. F. Schulz1
1Hanover
2Heinrich
Medical School, Institute of Virology, Hanover, Germany
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
Kaposis sarcoma associated herpesvirus (KSHV) can establish a lifelong latent infection. It persists by replicating its
circularized genome in synchrony with the cellular DNA and attaching it to chromosomes during mitosis. One of the few
proteins expressed during latency is latency-associated nuclear antigen (LANA). It colocalizes with viral genomes in
characteristic nuclear speckles, mediates replication and tethering of viral episomes to mitotic chromosomes. LANA recruits
many cellular proteins, among others members of the BRD/BET family (Brd2 and Brd4), which act as chromatin adapters
and regulate transcription. BET proteins have been implicated in the attachment of LANA to chromatin through LANAs Cterminal chromatin binding domain (CBD), with which they interact. BET proteins play a critical role in the life cycles of many
persistent viruses. They regulate transcription mediated by KSHV LANA, Murine herpesvirus 68 ORF73 and Human
Papillomavirus E2 proteins. They participate in the integration site selection near transcriptional starts sites (TSS) for g
retroviruses. Additionally, BRD4 tethers the complex of Bovine Papillomavirus E2 protein and circular viral DNA to mitotic
chromosomes.
We observed higher solubility of LANA in latently KSHV-infected cells, which were treated with I-BET, an inhibitor that
displaces BET proteins from acetylated histones. ChIP seq analysis demonstrated that the increase in solubility is due to
release of LANA from the chromatin. Our ChIP seq results showed that LANA is preferentially associated with TSS. ChIP seq
for BRD2/4 performed in the same cells, showed high overlap with LANA binding sites. This suggests involvement of BET
proteins in the attachment of LANA to cellular chromatin. Using a short term replication assay, we showed that I-BET
treatment impairs latent KSHV replication. However, it did not affect the formation of LANA speckles, indicating that BETmediated chromatin association is not required for this process.
Therefore, BET proteins contribute to the preferential localisation of LANA, in the vicinity of TSS and thereby help to localise
latent episomes in chromatin regions that are favourable for their replication. This may provide a basis for therapeutic
interventions aimed at the elimination of latent viral genomes.
Corresponding author:
Magdalena Weidner-Glunde
[email protected]
Antiviral Therapy and Resistance
OP72
Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) represent regulators of cytomegalovirus
replication and putative targets for anti-herpesviral drugs
C. Hutterer1, J. Milbradt1, S. Hamilton2, M. Zaja3,4, J. Leban3, C. Henry3,4, M. Steingruber1, E. Sonntag1, I. Zeittraeger1, H.
Bahsi1, T. Stamminger1, W. Rawlinson2, S. Strobl3,4, M. Marschall1
1Friedrich
2Serology
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
and Virology Division, SEALS Microbiology Prince of Wales Hospital Randwick NSW 2013 and SOMS and BABS, University of NSW,, Sydney,,
Australia
34SC Discovery GmbH, Martinsried, Germany
4BioNTech Small Molecules GmbH, Martinsried, Germany
Introduction: Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in
immunocompromised individuals and neonates. The success of val-/ganciclovir therapy is hampered by low drug
compatibility and induction of viral resistance. A novel strategy is based on the exploitation of cell-directed signaling, e.g.
pathways with a known relevance for carcinogenesis and tumor drug development. Dual-specificity tyrosine phosphorylationregulated kinases (DYRKs) constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell
proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins,
including regulators of the cell cycle, thus particularly DYRK1B can induce cell cycle arrest, a critical step for the regulation of
HCMV replication.
Objectives: In this study, we developed an antiviral principle based on targeting DYRKs.
Results: We provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK
inhibitors. Using established replication assays for laboratory and clinically relevant strains of HCMV, a stringent
concentration-dependent inhibition of viral replication was determined. Mean inhibitory concentrations (EC50) of 0.71 ±
0.019 µM, Harmine; 0.63 ± 0.23 µM, AZ-191; 0.98 ± 0.08, SC84227; 0.60 ± 0.02, SC97202; and 6.26 ± 1.64, SC97208,
respectively, were measured with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of
the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover,
rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a
similarly high sensitivity to these compounds.
Conclusion: Summarized, we conclude that DYRK signaling represents a promising target pathway for the development of
novel anti-herpesviral strategies.
Corresponding author:
Manfred Marschall
[email protected]
Antiviral Therapy and Resistance
OP73
Tetrahalogenated benzimidazole D-ribonucleoside are active against RCMV
I. Woskobojnik1, A. Dittmer1, J. C. Drach2, L. B. Townsend3, S. Voigt4,5, E. Bogner1
1Charité
Medical School, Institute of Medical Virology, Berlin, Germany
of Ann Arbor, College of Pharmacy, Ann Arbor, United States
3University of Ann Arbor, Department of Chemistry, Ann Arbor, United States
4Robert Koch Institute, FG12 - Measles, Mumps, Rubella, and viruses affecting immunocompromised patients, Berlin, Germany
5Charité Medical School, Department of Pediatrics, Division of Oncology and Hematology, Berlin, Germany
2University
Benzimidazole D-ribonucleosides are potent and selective inhibitors of CMV infection that have been shown to target the
viral terminase, the enzyme complex responsible for viral DNA cleavage in single unit-length genomes and subsequent DNA
packaging into procapsids. Antiviral activity of compounds Cl4RB and BTCRB against RCMV was quantified by
measurement of plaque formation. Both compounds had an inhibitory effect against RCMV-E. Electron microscopy revealed
that in RCMV-E infected cells in the presence of the compound only a few virions were formed. In an initial study of the mode
of action, effects of the cleavage of concatemers was analysed by pulsed-field gel electrophoresis (PFGE). The results
showed that DNA concatemers synthesized failed to be processed in the presence of the compounds. To characterize the
behaviour of the antiviral compounds in a more physiological environment, a 3D cell culture model in which cells are
embedded in an extracellular matrix was established using rat-tail collagen I. Yield Assays showed a comparable viral growth
in the 3D vs. 2D cell culture, as well as inhibition in the presence of 10 µM Cl4RB or BTCRB for RCMV-E/GFP. These
results demonstrated that the tetrahalogenated benzimidazole D-ribonucleosides are effective against RCMV by preventing
cleavage of concatemeric DNA and nuclear egress of mature capsids.
Corresponding author:
Elke Bogner
[email protected]
Antiviral Therapy and Resistance
OP74
Virucidal activity of WHO-recommended formulations against enveloped viruses including Zika, Ebola and emerging
Coronaviruses
A. Siddharta1, S. Pfaender2, N. Jane Vielle2, R. Dijkman2, M. Friesland1, B. Becker3, J. Yang4, M. Engelmann1, D. Todt1, M.
Windisch4, F. Brill3, J. Steinmann3,5, S. Becker6, M. Alves2, T. Pietschmann1, M. Eickmann6, V. Thiel2, E. Steinmann1
1Twincore
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
of Bern, Institute of Virology and Immunology, Vetsuisse Faculty, Bern, Switzerland
3Dr. Brill und Partner, Institute of Hygiene and Microbiology, Bremen, Germany
4Institut Pasteur Korea, Applied Molecular Virology, Seoul, South Korea
5University Duisburg-Essen, University Hospital Essen, Institute of Medical Microbiology, Essen, Germany
6Philipps University Marburg, Institute of Virology, Marburg, Germany
2University
Introduction: To control viral transmission, hand hygiene and surface disinfection are important prevention methods and
remain the key efforts to reduce the spread of infection. The World Health Organization (WHO) published two alcohol-based
formulations to be used in healthcare settings and outbreak-associated infections, but inactivation efficacies of these
products have not been determined against (re-) emerging viruses.
Objectives: In this study, we evaluated the virucidal activity of these WHO products in a comparative analysis against
different enveloped viruses.
Material & Methods: Virucidal activity studies were performed with a quantitative suspension test with 30 seconds exposure
time. One part by volume of test virus suspension and one part by volume of the organic load were mixed with eight parts by
volume of one of the two WHO formulations at different concentrations
Results: Zika virus (ZIKV), Ebola virus (EBOV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East
Respiratory Syndrome coronavirus (MERS-CoV), bovine coronavirus (BCoV), hepatitis C virus (HCV), influenzavirus (H1N1)
and Modified Vaccinia virus Ankara (MVA) served as test viruses. All viruses tested were highly susceptible to the WHO
formulations. WHO formulation II, which is based on isopropanol, demonstrated a better virucidal effect compared to the
ethanol-containing WHO formulation I. Coronaviruses and ZIKV showed the highest susceptibility to both WHO formulations.
Higher product concentrations were required for complete inactivation of EBOV and HCV, whereas H1N1 and MVA
displayed the highest stability.
Conclusion: In conclusion, WHO-recommended alcohol-based formulations were validated with different enveloped viruses.
A strong virucidal effect against emerging pathogens including ZIKV, EBOV, SARS-CoV and MERS-CoV could be
demonstrated implicating the usability of these WHO formulations in healthcare and outbreak-associated viral infections.
Corresponding author:
Eike Steinmann
[email protected]
Antiviral Therapy and Resistance
OP75
Labyrinthopeptin A1 and A2 efficiently inhibit cell entry of primary human respiratory syncytial virus isolates
S. Blockus1, S. Haid1, S. M. Wiechert1, M. Wetzke2, H. Prochnow3, R. Dijkman4, B. Wiegmann5, M. A. Rameix-Welti 6, J. F.
Eléouet7, P. Duprex8, V. Thiel4, G. Hansen2, M. Brönstrup3, T. Pietschmann1
1Twincore
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
Medical School, Paediatric Pneumology, Allergy and Neonatology, Hanover, Germany
3Helmholtz Centre for Infection Research, Chemical Biology, Braunschweig, Germany
4University of Bern, Institute of Virology and Immunology, Vetsuisse Faculty, Bern, Switzerland
5Hanover Medical School, Cardiothoracic, Transplantation and Vascular Surgery, Hanover, Germany
6Hôpital Ambroise Paré, INSERM U1173 Infection - Inflammation, Boulogne-Billancourt, France
7INRA, Molecular Virology and Immunology, Jouy-en-Josas, France
8Boston University School of Medicine, Microbiology, Boston, United States
2Hanover
Introduction: The human respiratory syncytial virus (hRSV) is an enveloped, non-segmented, negative-strand RNA virus of
the genus Pneumovirus and the family of Paramyxoviridae. It is the most common cause of lower respiratory tract disease in
infants and it is a major risk for severe infections in the elderly or immunocompromised patients. Overall hRSV leads to
approximately 64 million infections and 199.000 in-hospital deaths worldwide. Due to the lack of effective treatment options
there is a need for new antiviral drugs.
Objective: In this study we aimed to identify small molecules with promising antiviral activity against hRSV and to investigate
the mode-of-action of selected compounds.
Methods: A cell-based high-throughput screening system, based on firefly-luciferase luminescence was developed and used
to screen a compound library. The IC50 and CC50 of promising molecules were determined using an indirect reporter gene
assay and a direct FACS-based assay. Time-of-drug-addition and lentiviral hRSV pseudotype assays were performed to
explore the mode of action of lead compounds. Finally, infection competition assays and virus-drug dilution assays were
conducted.
Results: Labyrinthopeptin A1 and A2, two lantibiotics and secondary metabolites of Actinomadura namibiensis emerged as
potent inhibitors of primary clinical hRSV strains. IC50 values were in the low micromolar range without cytotoxicity up to an
approximately 100-fold higher dosage. The antiviral effect could be shown in carcinoma derived HEp2 and A549 cells as
wells as in physiologically relevant air-liquid interphase cultures involving primary human lung epithelial cells. These
compounds inhibited laboratory and clinical strains of hRSV subtypes A and B. In addition, lentiviral hRSV pseudotypes with
or without resistance mutations against palivizumab or fusion inhibitors in clinical development were susceptible to
labyrinthopeptin A1 and A2. Time-of-drug-addition as well as the lentiviral pseudotype assays indicated that both compounds
inhibit cell entry of hRSV.
Conclusion: Labyrinthopeptin A1 and A2 exert broad antiviral activity against primary hRSV subtype A and B strains
including palivizumab and fusion inhibitor resistant viral variants. Thus, these molecules are candidates for further
development as antivirals for treatment of hRSV infections.
Corresponding author:
Sebastian Blockus
[email protected]
Antiviral Therapy and Resistance
OP76
HCV Resistance Profile Evolution in a GT1b, DAA-naïve Patient before, on and after Failing Triple DAA Therapy
E. Knops1, N. Schübel1, E. Heger1, M. Neumann-Fraune1, R. Kaiser1, S. Inden1, P. Kalaghatgi1, S. Sierra1
1University
of Cologne, Institute of Virology, Cologne, Germany
Background: New direct-acting-antiviral (DAA) therapies have high success rates, though they are still lower in cirrhotic
patients. The presence of resistance-associated-mutations (RAMs) can play a role in DAA therapy failure. Over 50% of GT1infected patients display baseline RAMs that may affect the susceptibility to DAAs [4].
Methods: A 52 year-old patient enrolled in the PEPSI Study was analysed [Fig. 1]. Fibrosis stage was determined by
Acoustic Radiation Force Impulse (ARFI) Imaging. Four blood samples were obtained: baseline, failure and two follow-ups.
Viral RNA was amplified by RT-PCR, sequenced with next generation sequencing (NGS) and interpreted with
geno2pheno[HCV]. Phylogenetic analysis was done using RAxML under a GTRCAT model of nucleotide substitution.
NS5A-to-NS5B amplicons from baseline and failure samples were cloned in E. coli to analyse the concomitant presence of
RAMs in these genes within individual viral strains.
Results: Upon detection of F4 fibrosis, he underwent a 12 week-long therapy of Ombitasvir co-formulated with Paritaprevir
and Ritonavir, plus Dasabuvir and Ribavirin [Fig.1]. Baseline sample was analysed twice, once before therapy start and once
retrospectively after failure. Retrospective RT-PCR amplification with improved laboratory protocols followed by NGS
sequencing revealed RAMs in the NS5A and NS5B viral genes compromising two of the three DAAs used in the therapy.
After failure, new RAMs were detected in NS3, NS5A and NS5B genes. Molecular cloning in E. coli demonstrated that the
RAMs in NS5A and NS5B genes at baseline and after failure were concomitantly present within individual viral strains.
Sampling at weeks 10 and 30 after stopping therapy showed that all RAMs remained. Phylogenetic analysis showed that
baseline viruses did not re-emerge after 30 weeks in the absence of therapy pressure, but the resistant viruses persisted.
Conclusion: This is the first report showing presence of RAMs simultaneously in different genes within individual viral strains
from a DAA-naïve patient infected before the DAA era. These RAMs most likely led to therapy failure. In difficult-to-treat
patients, baseline sequencing can help in the selection of the optimal therapy.
Corresponding author:
Saleta Sierra
[email protected]
Figure 1
Antiviral Therapy and Resistance
OP77
Risk of HIV-1 low level viremia to treatment failure in the AREVIR cohort in Germany
N. Lübke1, A. Pironti2, E. Knops3, B. Jensen4, M. Oette5, S. Esser6, T. Lengauer2, J. Timm1, R. Kaiser3
1Heinrich
Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
Planck Institute of Informatics, Saarbrücken, Germany
3University of Cologne, Institute of Virology, Cologne, Germany
4Heinrich Heine University Dusseldorf, Department of Gastroenterology, Hepatology and Infectiology, Dusseldorf, Germany
5Augustinerinnen Hospital, Clinic for General Medicine, Gastroenterology, and Infectious Diseases, Cologne, Germany
6University Duisburg-Essen, University Hospital Essen, Clinic of Dermatology, Essen, Germany
2Max
Background: The German-Austrian guidelines for the treatment of HIV infection define therapeutic success as the reduction
of the HIV-1 viral load (VL) below 50 copies/ml. Low level viremia (LLV) is defined as repeated VL measurements between
50 and 200 copies/ml after initial therapeutic success. LLV has been previously associated to virological failure (VF). Here,
we provide an independent analysis of the association of LLV and other factors with VF.
Methods: The AREVIR database, comprising clinical and virological data of therapy-naïve and -experienced HIV-1-infected
patients in Germany, was queried for patients who attained confirmed therapeutic success under ART and who experienced
confirmed LLV thereafter. We constrained our query to therapies in which the VL was measured at least once every 24
weeks. We defined VF as a confirmed VL greater than 200 copies/ml following therapeutic success. p-values were calculated
with Fishers exact and Wilcoxon rank sum test.
Results: The database query resulted in 2,485 first-line and 3,657 further-line therapies. LLV occurred in 294 (4.8%) of these
therapies, specifically in 47 (1.9%) first-line and in 247 (6.8%) further-line therapies. The majority of patients showing LLV
were treated with PI-based therapies (165/294; 56%), followed by NNRTI-based regimens (76/294; 26%).
53 out of 294 (18%) patients experienced VF after LLV with a median VL at failure of 472 copies/ml (range 203-116590
cop/ml) after a mean LLV episode of 77.4 weeks (σ=68.0). The failure rate was increased in therapy-experienced patients
(48/247; 19.4%), as compared to therapy-naïve patients (5/47; 10.4%); (p=0.2129). There was no difference in VF between
PI-based and NNRTI-based therapies regardless of the backbone (33/165; 20% and 13/76; 17.1%, respectively; p=0.6049).
Among all drug classes, VF was never related to entry inhibitors, integrase inhibitors or the more recently approved
compounds DRV, TPV, and RPV (45/204 vs. 0/83, respectively; p<0.0001).
Conclusion: The prevalence of LLV in patients on suppressive ART is low (4.8%). Nevertheless, 18% of patients with LLV
experienced VF thereafter. The strongest predictor for VF after LLV was a treatment regimen exclusively containing drugs of
the older generations. Therefore, episodes of LLV in patients treated with drugs with high potency and a high barrier to
resistance are not predictive of VF.
Corresponding author:
Nadine Lübke
[email protected]
Evolution of Viruses – Viruses in Evolution
OP78
Molecular basis of cell culture adaptation of street rabies virus from fox and dog
S. Nemitz1, M. Christen1, F. Pfaff2, S. Finke1
1Friedrich
2Friedrich
Loeffler Institute, IMVZ, Greifswald - Isle of Riems, Germany
Loeffler Institute, IVD, Greifswald - Isle of Riems, Germany
A typical feature of fixed Rabies viruses (RABV) is improved virus production in cell cultures and a stable time course of
disease in animal models. In most cases adaptation to non-natural growth conditions increases at the expense of virulence,
given that required virus doses for disease induction are generally increased and often the ability to infect through peripheral,
intramuscular routes is lost.
To investigate whether specific mutations in non-fixed street viruses can be correlated with cell culture adaptation and
attenuation in vivo, recombinant clones of highly virulent street viruses of dog and fox origin (rRABV DogA, rRABV Fox) were
passaged on three different cell lines (murine neuroblastoma cells NA 42/13, hamster kidney cells (BHK clone BSR) and
canine epithelial cells (MDCK-II).
Deep-sequencing of the viruses after 10 passages revealed a remarkable stability of the genome, with most amino acid
exchanges in the glycoprotein G. Most interestingly, cell type specific amino acid substitutions occurred independently in fox
and dog derived viruses. Whereas virus passages on NA 42/13 cells led to previously described increase in G protein
glycosylation sites (N266D), on BSR cells an additional mutation at position 444 led to sequences comparable to vaccine
virus sequence patterns, present in BHK cell passaged RABV vaccine viruses. Passages on MDCK-II cells let to specific but
different amino acid replacements. The data suggests that only one or two mutations are required to improve interaction with
cellular pathways in a cell specific manner. Furthermore, mutations selected on BSR cells were correlated with increased
virus release. By confocal microscopy we found that non-adapted G protein accumulated in the ER, showing that cell culture
adaptation does not occur on entry but on assembly/release level with cell type specific molecular targets involved.
Corresponding author:
Sabine Nemitz
[email protected]
Evolution of Viruses – Viruses in Evolution
OP79
A novel alphaherpesvirus associated with fatal diseases in banded penguin chicks (Spheniscus humboldti and S.
demersus)
F. Pfaff1, C. Schulze2, P. König1, K. Franzke3, S. Bock2, A. Hlinak2, J. Kämmerling4, A. Ochs5, A. Schüle5, T. C. Mettenleiter6,
D. Höper1, M. Beer1
1Friedrich
Loeffler Institute, Institute of Diagnostic Virology, Greifswald - Isle of Riems, Germany
State Laboratory, Frankfurt (Oder), Germany
3Friedrich Loeffler Institute, Institute of Infectology, Greifswald - Isle of Riems, Germany
4Tierpark Cottbus, Cottbus, Germany
5Berlin Zoological Garden, Berlin, Germany
6Friedrich Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
2Berlin-Brandenburg
Penguin diphtheria, a high mortality disease complex observed in wild and captive juvenile penguins, is characterized by
diphtheroid stomatitis and necrotizing gastroenteritis. Although it was frequently discussed that the disease may be of
primarily viral cause, this has never been proven. In this study, we independently isolated and characterized a novel avian
alphaherpesvirus, preliminarily designated as spheniscid alphaherpesvirus 1 (SpAHV-1), from captive African and Humboldt
penguins (Spheniscus demersus and S. humboldti) suffering from diphtheroid-necrotizing oropharyngitis / laryngotracheitis
and enteritis, respectively. Pathology, PCR diagnostics and electron microscopy suggested an alphaherpesvirus-like
infectious agent. A metagenomic workflow based on high-throughput sequencing was used to identify the causative agent.
The obtained data were additionally used to determine the complete genome sequences of the first two SpAHV-1 isolates.
SpAHV-1 comprises a class D genome with a length of about 164.3 kbp, a G+C content of 45.6% and encodes 86 predicted
ORFs. Three predicted proteins have solely been reported for SpAHV-1 and exhibit no sequence homology with other
herpesviruses. The isolates from both penguin species share 96.6% overall protein identity, indicating a close evolutionary
relationship. Taxonomic association of SpAHV-1 to the genus Mardivirus was sustained by gene content analysis of 60
orthologous genes. A phylogenetic analysis of 52 herpesviral core genes from a subset of 27 selected alphaherpesviruses
further supported this classification. The detected unique pattern of predicted proteins and the rather remote position to other
mardiviruses, characterizes SpAHV-1 as interesting new herpesvirus species. In summary, this study provides detailed
information about a novel avian alphaherpesvirus that is most likely the causative agent of penguin-diphtheria-like fatal
diseases in banded penguins. The determined full-genome sequences may serve as basis for the development of diagnostic
tools, in order to investigate virus presence in similar cases of penguin diphtheria and shed more light on the evolution of
herpesviruses in general.
Corresponding author:
Florian Pfaff
[email protected]
Evolution of Viruses – Viruses in Evolution
OP80
Specific virulence determinants of H7 avian influenza viruses in chickens – The hemagglutinin multibasic cleavage
site and beyond
E. M. Abdelwhab1, J. Veits1, J. Stech1, T. C. Mettenleiter1
1Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
Avian influenza viruses (AIVs) contain one of 16 hemagglutinin (H1-16) and one of 9 neuraminidase (N1-9) subtypes which
are used for classification. Whereas the majority of AIVs exhibit low pathogenicity in infected birds, H5 or H7 subtypes can
naturally transform into highly pathogenic (HP) AIV by single mutations, recombination and/or reassortment after circulation
in gallinaceous poultry. Acquisition of a HA multibasic cleavage site (mCS) connecting the HA1 and HA2 polypeptides is a
characteristic for all HPAIVs; however the amino acid sequence of the mCS varies. Here, we summarize our research in the
last five years on virulence of H7 viruses in chickens. Overall, virulence determinants are virus-specific. A LP H7N1 virus
requires a specific mCS motif and three mutations in the HA2 polypeptide to shift to a fully pathogenic phenotype. These
HA2 mutations were unique to this strain; none of HP AIVs in the public databases possessed all three mutations.
Interestingly, two successive passages in chickens of the precursor LP H7N1 carrying this mCS motif but lacking the HA2
mutations, resulted in increased virulence without acquisition of any of these three mutations indicating presence of yet other
alterations leading to high pathogenicity. Further, we studied virulence of four H7N7 viruses in chickens after acquisition of
three different mCS. Two out of three mCS motifs were sufficient for immediate shift of three H7N7 viruses to HP
phenotypes; viruses carrying the third mCS motif were significantly less virulent. The fourth H7N7 virus exhibited high
virulence only after acquisition of gene segments from HP AIVs indicating a role for non-HA gene segment in virulence.
There was no correlation between virulence in chickens and replication in avian and mammalian cells in culture or HA
cleavability, but tropism was modulated by mCS and/or reassortment. In conclusion, although the mCS motif is an important
virulence marker for evolution of HP AIVs from LP progenitors additional virulence markers within and beyond the HA
contribute in a strain-specific manner. Driving forces for acquisition of such specific mCS or ancillary mutations remain to be
investigated.
Corresponding author:
Elsayed M. Abdelwhab
[email protected]
Evolution of Viruses – Viruses in Evolution
OP81
Phylogenetic and functional analyses propose an avian precursor of currently circulating mammalian bornaviruses
S. Runge1, J. Müller1, S. Kessler1, P. Staeheli1, D. Rubbenstroth1
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Gemany
Question: Bornaviruses (family Bornaviridae) are negative-sense single-stranded RNA viruses of the order
Mononegavirales. Members of this family were identified in mammals, reptiles and avian species of various orders. Some
bornaviruses are causative agents of fatal diseases in endangered birds, agricultural animals such as horses and sheep, and
even humans. Based on phylogenetic analyses, we hypothesized that the common ancestor of currently circulating avian
and mammalian bornaviruses is of avian origin.
Methods & Results: In order to confirm this hypothesis and to gain more information on the zoonotic potential of
bornaviruses, we studied the infection and replication characteristics of a broad range of avian and mammalian bornaviruses
in avian and mammalian cells. We found that most avian bornaviruses were able to efficiently infect avian cells only. Only
those avian viruses that are closely related to mammalian viruses displayed ability to infect mammalian cells to a certain
degree. In contrast, mammalian virus isolates infected mammalian and avian cells, which is in line with the hypothesis of an
avian ancestor. To understand the mechanisms underlying the apparent host cell restriction of most avian bornaviruses in
mammalian cells, we studied different steps in bornavirus replication, including membrane fusion and viral polymerase
activity. In a polymerase reconstitution assay, polymerases of mammalian bornaviruses showed strong activity in avian and
mammalian cells, whereas avian polymerases were active mainly in avian cells, mirroring the infection characteristics of
these viruses. In contrast, avian and mammalian bornaviruses did not differ markedly in their ability to induce membrane
fusion of various cell types.
Conclusion: In summary, phylogenetic and functional analyses of bornaviruses suggest that circulating mammalian
bornaviruses evolved from an avian precursor and have retained their ancestors´ ability to replicate in avian cells. Avian
bornaviruses did not or only inefficiently replicate in mammalian cells, indicating a low zoonotic potential. Furthermore, our
study showed that viral polymerase activity rather than cell membrane fusion may be a crucial host range determinant of
bornaviruses.
Corresponding author:
Solveig Runge
[email protected]
Evolution of Viruses – Viruses in Evolution
OP82
The genome of RNA viruses is highly constrained by the convergent interplay between protein evolution and RNA
structure
A. N. Lozada Chávez1, M. Fricke1, M. Marz1, I. Lozada Chávez2
1Friedrich
Schiller University Jena, Faculty of Mathematics and Computer Science, Jena, Germany
of Leipzig, Department of Computer Science, Leipzig, Germany
2University
Background: Genome complexity in RNA viruses is restricted. Yet, genomic function is driven by conserved protein
domains, structured RNAs and regulatory signals, while evolutionary exploration is achieved through mutation, overlapping
genes [1], and intrinsic protein disordered regions (IPDRs) [2]. Thus, a conflict among the needs to encode both a functional
protein and a structured RNA as well as to keep genomic novelty raises the question of how these overlapping codes are
evolving together within and across RNA viral genomes.
Results: We systematically analyzed 5000 complete genomes from 12 different RNA viruses to estimate three major
features along each genome: selection (at the codon, domain and gene levels), amino acid conservation, IPDRs, and RNA
secondary structures. We found that RNA viral genomes are mainly driven by purifying selection at the codon level, and that
most viral proteins exhibit several short IPDRs. However, structural genes are characterized by an enrichment of disordered
motifs and protein regions under positive selection; particularly, those accessory and regulatory proteins involved in
interactions with other viral and host factors. By contrast, non-structural genes are usually dominated by more protein regions
under negative selection and less disordered motifs. As in [3] and [4], we found a negative correlation between the extent of
base-pairing in RNA structures and amino acid variability located at the same genomic regions. Likewise, amino acid sites
encoded by structured RNAs exhibited stronger purifying selection than those not involved in structure-forming RNA. Finally,
we found that coupled genes with the relaxed action of selection within overlapping regions are characterized by short
sequences, more amino acid variability and IPDRs regions, as well as "flexible" RNA structures.
Conclusion: Our findings suggest that RNA structures and protein sequences in RNA viruses are likely forced to constraint
to each other rather than to evolve independently. This convergent interplay seems to promote the genome architecture into
associated region-specific divisions of labor.
References:
[1] PMID: 27209091
[2] PMID: 24823319
[3] PMID: 21135148
[4] PMID: 19661910
Corresponding author:
Alejandro Nabor Lozada Chávez
[email protected]
Evolution of Viruses – Viruses in Evolution
OP83
Virus-virophage-host interactions in the marine protozoan Cafeteria roenbergensis
M. Fischer1, T. Hackl2
1Max
Planck Institute of Medical Research, Heidelberg, Germany
Institute of Technology, Cambridge, United States
2Massachusetts
Viruses are the most abundant biological entities on this planet and have shaped the evolution of cellular life in profound
ways. Many different types of host-virus relationships have developed, ranging from parasitic to mutualistic modes. In this
talk, I will introduce a tripartite microbial system that consists of the marine heterotrophic nanoflagellate Cafeteria
roenbergensis, its lytic giant virus CroV, and the virophage mavirus. Virophages are small double-stranded DNA viruses that
exert a beneficial effect on their host cell population by inhibiting the propagation of giant viruses. The mavirus virophage is
particularly fascinating as it bears close genetic similarity to a widespread class of eukaryotic DNA transposons called
Mavericks/Polintons, suggesting that mavirus may also exist in a transposon-like state. We have shown experimentally that
mavirus readily integrates into the nuclear genome of its host C. roenbergensis. The resulting provirophage genome is stably
maintained by the host cell and its genes are transcriptionally silent under normal conditions. However, when a mavirusbearing host cell is infected by CroV, the mavirus genes are activated and infectious virophage particles are produced and
released from the cell via lysis. Although the virophage response is too late to rescue the initially CroV-infected cells, the
newly released reactivated mavirus particles can inhibit CroV replication in subsequent co-infections, which results in survival
of the host population. Initial screening of Cafeteria genomes for provirophages has revealed several endogenous maviruslike genomes, suggesting that virophage-mediated protection against giant virus infection may be widespread in this class of
protists.
Corresponding author:
Matthias Fischer
[email protected]
Figure 1
Vaccines
OP84
Tropism of attenuated RABV after oral administration in different reservoir species
V. te Kamp1, T. Nolden2, S. Nemitz1, C. Freuling1, T. Müller1, S. Ortmann3, C. Kaiser3, A. Vos3, P. Schuster3, S. Finke1
1Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
GmbH, Innsbruck, Austria
3IDT Biologika GmbH, Dessau-Rosslau, Germany
2ViraTherapeutics
Oral vaccination against RABV with live attenuated virus vaccines is highly effective in foxes and thus has led to successful
elimination of rabies from Western Europe fox populations. However, significantly higher minimum effective doses are
required in other carnivore reservoir species like raccoons, skunks and dogs.
In order to investigate replication of attenuated RABV after oral administration, foxes were orally vaccinated with a
recombinant, GFP expressing RABV with a genetic background of the oral vaccine strain SAD B19.
By confocal laser scan imaging of tissue samples palatine tonsils were observed as a main site of virus replication with
characteristic and locally restricted distribution of infected cells. Notably, also the time course of infection was surprisingly
restricted, indicating that the main triggers for the induction of adaptive immune reactions arise in the first four days after
infection with vaccine virus.
Assuming that palatine tonsils infection is a main correlate of immunity induction, we further investigated whether oral
immunization with the GFP-expressing RABV differs in raccoons, skunks and dogs. Imaging analysis of tonsils as well as
qRT-PCR screening including also other tissues of the oropharyngeal tract confirmed that those species that require higher
virus doses for oral immunization are less efficiently infected.
These data suggest that carnivore reservoir species substantially differ in the susceptibility to oral live vaccine infection and
that this may determine potency of an oral RABV live vaccine.
Corresponding author:
Verena te Kamp
[email protected]
Vaccines
OP85
Attenuation mechanisms of the Junin virus vaccine strain, candid #1
S. Paessler1
1University
of Texas Medical Branch, Pathology, Galveston, United States
Argentine hemorrhagic fever (AHF) is a severe, potentially lethal disease with an at-risk population of over 3 million
individuals in the pampas region of Argentina. The etiological agent of AHF is Junin virus (JUNV), a member of the
arenavirus family. While treatment options for AHF are limited, an effective live attenuated vaccine strain (Candid #1) was
developed through serial passaging in mouse brain tissue and is currently administered to at-risk individuals with a high
degree of success. We have previously demonstrated that the GPC of JUNV is the primary gene responsible for the
attenuation of Candid #1 in a guinea pig model of AHF that mimics the clinical illness. In contrast to the GPC of the highly
virulent Romero strain, we show that the GPC of the Candid #1 strain expresses higher amounts of pre-cleaved full length
G1/G2 and less cleaved G2 than the virulent wild-type GPC in cell culture. The most significant accumulation of pre-cleavage
G1/G2 is associated with an amino acid substitution in G1 (T168A) that eliminates an N-linked glycosylation motif that is
utilized by the protein. This reduction in cleavage by the SKI-1/S1P protease correlates with a significant reduction in Candid
#1 GPC expression on the cell surface compared to the wild-type Romero GPC, and an increase in the ER retention of
Candid #1 GPC. The retention of the protein in the ER is due primarily to the absence of the N-linked glycan at N166, and
trafficking of Candid #1 GPC to the cell surface can be partially restored by restoring the N-linked glycosylation motif at
N166-T168. We show that the retention of the Candid #1 GPC in the ER results in the aggregation of improperly processed
GPC through the formation of inter-chain disulfide bridges, and leads to degradation of the protein in lysosomal
compartments through autophagy.
Corresponding author:
Slobodan Paessler
[email protected]
Vaccines
OP86
Adeno-associated virus (AAV) vector as a vehicle for a broadly protective influenza vaccine
D. Demminger1, S. Norley2, T. Wolff1
1Robert
2Robert
Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
Koch Institute, FG18 - HIV and other Retroviruses, Berlin, Germany
Introduction: Influenza viruses represent a major threat to public health. The strain specific seasonal vaccine is insufficient
to efficiently protect against drifted or emerging viruses as demonstrated by occasional mismatches of the vaccine with
circulating strains. Furthermore, seasonal vaccination does not provide protection against new pandemic or emerging
zoonotic viruses. Additionally, its production process in eggs is time consuming. Hence, there is an exceptional need for the
production of broadly protective vaccines that induce long-lived protective immunity not only against the vaccine strain but
also drift-variants thereof, as well as against more distantly related known and unknown strains.
Objectives: The use of AAV-vectors as vehicles for hemagglutinin (HA) antigens that focus the antibody response towards
the antigenically conserved HA-stalk.
Material & Methods: AAV-vectors harboring chimeric, headless or wild-type HA derived from
A/California/07/2009(H1N1pdm2009)-HA were used to immunize C57BL/6-mice. Serum antibody responses were analyzed.
A second set of mice was immunized and eventually challenged with the homologous influenza virus and survival was
monitored.
Results: Vaccination of mice with wild-type HA induced limited but neutralizing sero-reactivity against H1 viruses only. In
contrast, sera of mice immunized with chimeric HA reacted against all tested viruses (H1N1pdm2009, seasonal H1N1,
H3N2), although these antibodies were not neutralizing in vitro. Initial challenge experiments showed that mice were partially
protected against the homologous H1N1pdm2009-virus after immunization with chimeric or headless HA.
Conclusion: We show that the immunization with AAV-Vectors expressing chimeric HAs induces a remarkably broad crossreactive anti-Influenza virus antibody response in mice which is most likely directed against the HA-stalk. This immune
response could confer partial protection against infection with the homologous virus strain. To further improve the strength
and breadth of the induced immunity against the HA stalk domain, optimized headless HA antigens are being evaluated at
the moment. Challenge trials with heterologous and heterosubtypic influenza strains are ongoing.
Corresponding author:
Daniel Demminger
[email protected]
Vaccines
OP87
Efficacy testing of a newly developed MVA-based vaccine against Ebola virus in a mouse model
A. Kupke1,2, A. Volz3,4, E. Dietzel1,2, J. Schmidt1,2, H. Shams-Eldin1, L. Sauerhering1, C. Herden5, M. Eickmann1,2, S.
Becker1,2, G. Sutter3,4
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Centre for Infection Research (DZIF), Marburg, Germany
3Ludwig Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
4German Centre for Infection Research (DZIF), Munich, Germany
5Justus Liebig University Giessen, Institute of Veterinary Pathology, Giessen, Germany
2German
Based on the recombinant Modified Vaccinia virus Ankara (MVA), new vaccine candidates comprising the EBOV
glycoprotein (GP) or nucleoprotein (NP) were developed and tested for efficacy in a mouse model. Type I interferon receptor
knockout (IFNAR -/-) mice were immunized with the different vaccine candidates in a prime-boost regimen and mice
vaccinated with either PBS (mock) or wild type MVA vector served as controls. As EBOV-specific antibodies were detectable
by ELISA and virus neutralization assay, mice were infected intranasally with EBOV and monitored for clinical signs up to 14
days post infection. Serum and organ samples were evaluated for viral load by quantitative real time RT-PCR. Moreover,
organ samples were examined histopathologically.
Mice vaccinated with MVA-GP and MVA-NP developed high titers of EBOV-specific antibodies which were partially virusneutralizing. In contrast to the mock-vaccinated mice, which succumbed to EBOV infection, all mice vaccinated with MVA-GP
or MVA-NP survived and showed decreased viral load in serum and organs. Furthermore, MVA-GP or MVA-NP vaccinated
mice showed reduced organ pathology including reduced liver necrosis.
In summary, the MVA-based vaccines against EBOV GP and NP induced a protective immunity in mice with no observable
severe adverse effects and are therefore efficient and safe candidates for clinical trials.
Corresponding author:
Alexandra Kupke
[email protected]
Vaccines
OP88
Priming with a potent HIV-1 DNA vaccine frames the quality of T cell and antibody responses prior to a poxvirus and
protein boost
B. Asbach1, J. Köstler2, K. Kibler3, B. Perdiguero4, M. Esteban4, B. L. Jacobs3, D. C. Montefiori5, C. C. LaBranche5, N. L.
Yates5, G. D. Tomaras5, G. Ferrari5, K. E. Foulds6, M. Roederer6, G. Landucci7, D. N. Forthal7, M. S. Seaman8, N. Hawkins9,
S. G. Self9, S. Phogat10, J. Tartaglia10, S. W. Barnett11, B. Burke11, A. D. Cristillo12, S. Ding13, J. L. Heeney14, G. Pantaleo15,
R. Wagner1,2
1Institute
of Medical Microbiology and Hygiene, Molecular Microbiology (Virology), Regensburg, Germany
Hospital Regensburg, Institute of Clinical Microbiology and Hygiene, Regensburg, Germany
3Arizona State University, Biodesign Institute, Tempe, United States
4Consejo Superior de Investigaciones Científicas, Centro Nacional de Biotecnología, Madrid, Spain
5Duke University Medical Center, Durham, United States
6National Institutes of Health, Vaccine Research Center, Bethesda, United States
7University of California, Irvine School of Medicine, Irvine, United States
8Beth Israel Deaconess Medical Center, Center for Virology and Vaccine Research, Boston, United States
9Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, United States
10Sanofi Pasteur, Swiftwater, United States
11Novartis Vaccines and Diagnostics, Inc., Cambridge, United States
12Advanced BioScience Laboratories, Inc., Rockville, United States
13EuroVacc Foundation, Lausanne, Switzerland
14University of Cambridge, Lab of Viral Zoonotics, Cambridge, United Kingdom
15Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland
2University
The use of heterologous immunization regimens together with the employment of various improved vector systems and
antigen designs has proven to lead to vigorous increases in immunogenicity when HIV-1 vaccine candidates are assessed in
non-human primates. In order to resolve interdepencies between different delivery modalities, we compared three different
poxvirus boost regimens after a DNA prime. Three groups of rhesus macaques were each immunized with the same DNA
vaccine encoding for Gag, PolNef, and gp140 at weeks 0, 4 and 8. At week 20, the groups were boosted either (i) by
administering the poxviral replication-competent NYVAC-KC-vector by scarification, or (ii) by i.m. injection, or (iii) by i.m.
injection of the replication-deficient NYVAC-vector, carrying the same antigens. Finally, macaques were boosted with
adjuvanted, recombinant gp120 protein at weeks 28, 32, and 49 in order to enhance humoral responses. The regimen
elicited very potent CD4+ and CD8+ T cell responses in a well-balanced manner, peaking 2 weeks after the NYVAC-boost. T
cell responses subsequently declined and were hardly influenced by subsequent protein boosts. T-cells were broadly
reactive and polyfunctional, with high fractions of cells secreting all three cytokines assessed. All animals exhibited antigenspecific humoral responses already after the poxvirus boost, that by trend slightly increased following protein administration.
Polyclonal reactivity of IgG antibodies was highest against C clade Env-proteins, yet with substantial cross-reactivity towards
other clades. Serum IgA responses were absent. Substantial ADCC activity, and very high ADCVI activity were observed in
sera obtained after the last protein boost.
The absence of major differences between the groups indicates that the potent priming induced by the DNA vaccine initially
framed the epitope specificity and polyfunctionality of the T cell responses in such a way that the subsequent poxvirus boost
only led to an increase in the response magnitudes without skewing the quality. This emphasizes the importance of selecting
the best mixture of vector systems in heterologous vaccination regimens.
Corresponding author:
Benedikt Asbach
[email protected]
Vaccines
OP89
Needle-free delivery of measles virus vaccine to the lower respiratory tract of non-human primates elicits optimal
immunity and protection
R. de Swart1, R. de Vries1, L. Rennick2, G. van Amerongen1, S. McQuaid3, J. Verburgh1, S. Yüksel1, A. de Jong1, K. Lemon2,
T. Nguyen1, M. Ludlow2, A. Osterhaus1, P. Duprex2
1Erasmus
Medical Center, Department of Viroscience, Rotterdam, Netherlands
University School of Medicine, Microbiology, Boston, United States
3Queen's University of Belfast, Centre for Cancer Research and Cell Biology, Belfast, United Kingdom
2Boston
Introduction: Measles vaccines developed fifty years ago have greatly reduced global childhood morbidity and mortality.
The standard route of measles virus (MV) vaccination is subcutaneous injection, whereas MV is an airborne pathogen.
Programs evaluating needle-free measles vaccination have been hampered by a lack of knowledge about the target cells of
licensed live-attenuated vaccine viruses. These vaccines typically contains a low antigenic dose, making replication in the
host essential for induction of protective immune responses.
Objectives: The aim of this study was to assess the in vivo tropism of live-attenuated MV and determine whether respiratory
MV vaccination should target the upper or lower respiratory tract.
Material & Methods: Four groups of twelve cynomolgus macaques were immunized with 104 TCID50 recombinant
Edmonston-Zagreb expressing enhanced green fluorescent protein. The virus was grown in MRC-5 cells and formulated with
stabilizers and excipients as used in the commercial MVEZ vaccine produced by the Serum Institute of India. Animals were
immunized by intra-muscular injection, intra-tracheal inoculation, intra-nasal instillation or aerosol inhalation. Six animals of
each group were euthanized at early time points, the other six were followed up to assess immunogenicity and protection
from challenge infection 14 months after vaccination.
Results: EGFP-positive MV-infected cells were detected locally in the muscle, nose, lungs and draining lymph nodes, while
systemic vaccine virus replication and viremia were virtually absent. Macrophages, dendritic cells and tissue-resident
lymphocytes were the predominant target cells. Animals vaccinated via the respiratory route had the highest specific serum
antibody and best protection levels when the virus was delivered to the lower respiratory tract.
Conclusion: This represents a unique study that sheds light onto how this highly efficacious live-attenuated MV vaccine can
be delivered by needle-free methods. We conclude that respiratory vaccination with live-attenuated measles virus requires
vaccine delivery to the lower respiratory tract.
Corresponding author:
Rik de Swart
[email protected]
Viral Pathogenesis and Persistance
OP90
Cytomegalovirus infection promotes the development of allergic airway disease
S. Reuter1, J. Maxeiner2, J. Podlech3, K. Freitag3, A. Heinz4, C. Belz4, R. Holtappels3
1Experimental
Asthma Research, Leibniz Centre for Medicine and Biosciences Borstel, Borstel, Germany
Medical Center Mainz, Asthma Core Facility of the Research Center of Immunotherapy (FZI), Mainz, Germany
3University Medical Center Mainz, Institute of Virology, Mainz, Germany
4University Medical Center Mainz, Department of Pulmonary Medicine, Mainz, Germany
2University
There is increasing evidence that respiratory viral infections early in life as well as allergic sensitization are important risk
factors for the development of asthma. Viral respiratory tract infections are also the main cause of asthma exacerbation in
children and adults. Yet, the underlying mechanisms are not clear. Cytomegalovirus (CMV) is a highly prevalent human
pathogen targeting the lungs as major organ side with interstitial pneumonia being the most dreaded manifestation in
immunocompromised hosts. However, in the status of immunocompetence, CD8 T cells control CMV infection and prevent
viral pathology. To investigate a possible interference of CMV and allergic airway disease we made use of a prevalent
murine model of allergic inflammatory disease of the lungs which is based on intratracheal (i.t.) sensitization and aerosol
challenge with ovalbumin (OVA) as otherwise harmless antigen. Indeed, we can show that mCMV infection in the
sensitization phase induces an OVA-specific inflammation of the airways. Pulmonary inflammation is characterized by
increased numbers of inflammatory cells in lung tissue and in the airways, as well as by the development of goblet cell
metaplasia. Furthermore, mCMV significantly enhances the influx of activated CD8 T cells into the lungs and strongly
augments the CD8 T-cell response against the dominant OVA-specific epitope SIINFEKL, a specificity that is barely
detectable after OVA application alone. mCMV modulates the migration and activation of OVA-positive dendritic cell
subpopulations in the draining tracheal lymph node which could explain the mCMV induced enhancement of the OVAantigen specific pulmonary inflammation. Thus, our data suggest that mCMV is a potential cofactor supporting the
development of an allergic inflammatory disease of the airways.
Corresponding author:
Rafaela Holtappels
[email protected]
Viral Pathogenesis and Persistance
OP91
Differential interactions of a virus suppressor of RNA silencing with miRNAs modulate plant gene expression and
antiviral measures early in infection
R. Pertermann1, S. Tamilarasan1, T. Gursinsky1, G. Gambino2, J. Schuck1, L. Schwien1, H. Lilie1, R. Golbik1, V. Pantaleo3, S.
E. Behrens1
1Martin
Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Halle/Saale, Germany
of Sustainable Plant Protection, Consiglio Nazionale delle Ricerche, Research Unit of Gruglias, Torino, Italy
3Institute of Sustainable Plant Protection, Consiglio Nazionale delle Ricerche, Research Unit of Bari, Bari, Italy
2Institute
RNA silencing is a central component of the plant's immune response. It is effectively counteracted by viral suppressor
proteins of RNA silencing, VSRs, which operate, for example, by high-affinity sequestration of antiviral small interfering RNAs
(siRNAs). The close similarity of siRNAs to cellular micro RNAs (miRNAs) triggers current research aimed at understanding if
and how VSRs may intervene with plant gene expression. Analyzing miRNA-VSR interactions first by biochemical means, we
found the Tombusvirus VSR p19 to bind different miRNAs at considerably different affinities and demonstrated specific
miRNA and protein features determining this characteristic. We then investigated the activities of the miRNAs 162, 168 and
403 in in vitro silencing and in planta infection experiments in the absence and presence of the VSR. MiRNAs 162, 168 and
403 regulate the expression of the Dicer-like protein DCL1, and of the Argonaute proteins AGO1 and AGO2, respectively,
which are all important constituents of antiviral RNA silencing. Interestingly, we observed that in the presence of p19 and
correlating with its miRNAs binding affinities, DCL1-, AGO1- and AGO2 mRNA silencing were differently modulated. This
was particularly relevant early in infection, where the concentration of virus-derived siRNAs is low. Further data revealed that
the non-related Cucumovirus 2b VSR shows a miRNA binding profile that closely resembles that of p19. Thus, our study
suggests differential binding of miRNAs by VSRs a broadly evolved viral tool that coordinately modulates cellular gene
expression during the initial infection phase. It further highlights the close interrelationship of plant miRNA- and pathogen
evolution.
Corresponding author:
Sven-Erik Behrens
[email protected]
Viral Pathogenesis and Persistance
OP92
The role of infection and depletion of lymphocyte subsets in the pathogenesis of measles-induced immune
suppression
B. Laksono1, C. Grosserichter-Wagener2, S. Langeveld1, R. de Vries1, R. Huizinga2, J. van Dongen2, P. Katsikis2, M.
Koopmans1, M. van Zelm2, R. de Swart1
1Erasmus
2Erasmus
Medical Center, Department of Viroscience, Rotterdam, Netherlands
Medical Center, Department of Immunology, Rotterdam, Netherlands
Introduction: Measles is characterised by a transient immune suppression that leads to an increased risk of opportunistic
infections, but paradoxically also induces strong measles virus (MV)-specific immune responses that last a lifetime. One of
the cellular receptors used by MV is CD150, which is expressed on various subsets of immune cells. In vivo MV infections of
non-human primates showed that MV preferentially infects CD150 + dendritic cells and lymphocytes. Memory T-cells proved
to be more susceptible than naive T-cells, which led us to propose immune amnesia as a mechanism of measles-induced
immune suppression. MV also infects human and macaque B-cells, resulting in lymphoid tissue follicular depletion. However,
susceptibility of different B-cell subsets has not been defined.
Objectives: We aim to compare the relative susceptibility of human lymphocyte subsets to MV infection and determine their
production levels of new infectious MV particles in vitro.
Material & Methods: Lymphocytes were obtained from human peripheral blood or tonsils for in vitro infection with
recombinant MV expressing EGFP. CD150 expression and EGFP levels were determined by flow cytometry. Peripheral
blood naive and memory lymphocytes were sorted with a multicolour cell sorter prior to infection.
Results: We show that CD150 expression level was lower on B-cells than on T-cells, but observed no differences between
memory and naive B-cell subsets. Surprisingly, all B-cell subsets were more susceptible to MV infection than memory Tcells. Furthermore, infection of sorted naive and memory lymphocyte subsets did not only confirm that B-cells are highly
susceptible to MV infection, but also showed that they produced more infectious virus than T-cells. Interestingly, memory Bcells expressed higher levels of EGFP, suggesting they can produce more infectious viral particles than naive cells.
Conclusion: Our findings suggest a crucial role for B-cells in the pathogenesis of measles: whereas MV-infected T-cells are
responsible for virus dissemination during viremia, B-cells may be the most important viral factories, amplifying the infection.
Based on these observations, we conclude that depletion of B-cells contributes to measles-induced immune suppression.
Corresponding author:
Brigitta Laksono
[email protected]
Viral Pathogenesis and Persistance
OP93
To cut or not to cut – RNA target selection of the KSHV host-shut-off factor SOX
C. Vogt1,2, M. M. Gaglia2,3, J. Bohne1, B. A. Glaunsinger2
1Hanover
Medical School, Institute of Virology, Hanover, Germany
of California, Berkeley, United States
3Tufts University School of Medicine, Boston, United States
2University
Introduction: Viruses explore many ways to induce host shut-off aiding viral replication while at the same time inhibiting
basic cellular functions. In KSHV one host shutoff mechanism is the massive degradation of mRNAs which starts in the
delayed early phase of viral replication and continues until the end of the viral life cycle. This shutoff is executed by the
KSHV SOX protein. SOX has a dual function. Its DNase activity is important to resolve branched structures during viral
genome replication and it exhibits an RNA endonuclease activity in the cytoplasm. This activity induces a general mRNA
decay. SOX targets a broad range of transcripts of both cellular as well as viral origin. There are several examples how
endonucleases can achieve their specificity: a common strategy is cleavage near a specific landmark feature like the 5' end
or stop codon. Previous findings (Gaglia et al. 2015) showed that in the case of SOX cleavage is not associated with such
features. But interestingly, SOX cleaves its target RNAs at specific positions.
Objectives: We set out to answer the question how KSHV SOX can target such a variety of different transcripts while on the
other hand it cleaves at specific sites. Our hypothesis is that SOX uses a combination of a degenerative sequence motif and
structural features.
Material and Methods: To gain insights in the structure of some of the best characterized cleavages sites of SOX we used
the so called "in-line probing", a RNA structural probing method based on the fact that RNA degrades differentially depending
on its structure.
Results: We took the best characterized cut sites for SOX and by using RNA structural probing methods compared the
structures of these sites and their surrounding sequences.
Here we show that the analyzed SOX cleavage sites are embedded in a secondary structure with both the cut site and a
preceding A-stretch, which is characteristic for a great portion of identified cut sites, in unpaired regions. These results are
also in agreement with previous in silico predictions (Gaglia et al. 2015).
Conclusion: The RNA structures of the cut sites we tested here display some common structural features. This finding
reinforces our hypothesis that SOX target recognition might be a combination of structural and sequence features.
Corresponding author:
Carolin Vogt
[email protected]
Viral Pathogenesis and Persistance
OP94
Antiviral immunity, galectins and the interaction of hantavirus with professional antigen presenting cells
M. Raftery1, F. Weege2, A. Karlas2, T. Meyer2, R. G. Ulrich3, D. H. Krüger1, B. Klempa1,4, G. Schönrich1
1Charité
Medical School, Berlin, Germany
Planck Institute of Infection Biology, Berlin, Germany
3Friedrich Loeffler Institute, Institute of Novel and Emerging Infectious Diseases, Greifswald - Isle of Riems, Germany
4Biomechanical Research Center, Slovak Academy of Science, Institute of Virology, Bratislava, Slovakia
2Max
Introduction: Hantaviruses (Bunyaviridae) are rodent-borne pathogens which within the natural host cause asymptomatic
and persistent infection. Transmission to humans occurs via the respiratory route and leads to one of two serious illnesses;
hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS). Both diseases are
characterized by increased vascular permeability and decrease in platelet counts.
Objectives: A humanized mouse model has demonstrated that in vivo immune responses involving CD8 cells contribute to
the development of this disease. However, why viral strains vary in lethality is still unknown. We tested if the varying lethality
of different viral strains could be linked to differences in the infection of immune cells.
Material & Methods: Data was generated from infection of immune cells in vitro. Analysis of viral replication was analysed
by titration, qRT-PCR, and immunohistochemistry. Identification of genes involved in hantavirus infection of immune cells
was perfomed by cell sorting and sequencing of a library of cellular knock-outs generated by CRISPR technology.
Results: Several immune cell types were identified to be permissive or otherwise react to hantavirus infection, resulting in
bystander activation of lymphocytes, release of neutrophil extracellular traps from neutrophils, infection of several types of
antigen presenting cell including novel cell types and consequent modulation of T cell function. Among hantavirusmodulating genes several Galectins, sugar-binding soluble molecules involved in the immune response, were identified and
analysed further. We also found that apathogenic viruses were non-infectious to professional antigen presenting cells by a
post entry mechanism. Candidate cellular genes restricting the replication of apathogenic and pathogenic strains were
identified and analysed.
Conclusion: We identify a series of cellular genes that may be responsible for the induction and progression of HFRS in
humans
Corresponding author:
Martin Raftery
[email protected]
Viral Pathogenesis and Persistance
OP95
Reverse genetics for a feline coronavirus field isolate to study the molecular pathogenesis of FIP
R. Ehmann1, C. Kristen-Burmann1, B. Bank-Wolf1, M. König1, T. Hain2, J. Ziebuhr3, G. Tekes1
1Justus
Liebig University Giessen, Institute of Virology, Giessen, Germany
Liebig University Giessen, Institute of Medical Microbiology, Giessen, Germany
3Justus Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
2Justus
Introduction: Feline coronaviruses (FCoVs) are widespread in the cat population with seropositivity of up to 90%. Based on
their pathogenic potential, FCoVs are classified into two biotypes: feline enteric coronavirus (FECV) causes inapparent
persistent infections, whereas feline infectious peritonitis virus (FIPV) is the causative agent of the fatal systemic disease
called feline infectious peritonitis (FIP). According to the current view, FIPV evolves from FECV by acquiring mutations. So
far, the genetic changes responsible for the biotype switch have not been identified.
Objectives: The identification of mutations responsible for FIP-development requires reverse genetics approaches suitable
to produce, characterize and manipulate genetically defined field viruses. However, the development of reverse genetic
systems for FCoV field viruses represents a major technical challenge because these viruses do not grow in standard cell
culture systems. All currently available reverse genetic systems are based on cell culture-adapted FCoVs and none of them
is suitable for studying mutations involved in FIP pathogenesis.
Material & Methods: In this study, the full-length sequence of an FECV field isolate was determined, and the entire FECV
genome was integrated as a cDNA into the vaccinia virus genome to produce recombinant FECVs.
Results & Conclusion: The experiments showed that we were able to overcome the technical challenges associated with
establishing reverse genetic systems for field viruses. We produced high-titer virus stocks derived from an FECV field isolate
using the newly established reverse genetics system. Efficient viral rescue was demonstrated by Western blotting using an M
protein-specific monoclonal antibody, which allowed us to detect the structural M protein in purified viral particles. Typical
coronavirus particles were detected by electron microscopy. Furthermore, RNA protection assay confirmed the presence of
FECV genomic RNA in recovered recombinant FECV particles. This system provides an excellent basis to identify mutations
that contribute to the development of FIP using both in vitro and in vivo model systems.
Corresponding author:
Rosina Ehmann
[email protected]
Host Cell Factors and Modulation
OP96
Genetic manipulation of the IRE1-dependent ER stress pathway reveals an unexpected role of XBP1u as a repressor
of cytomegalovirus replication
F. Hinte1, E. van Anken2, B. Tirosh3, W. Brune1
1Heinrich
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
Raffaele Scientific Institute, Milano, Italy
3Institute of Drug Research/ The Hebrew University, Jerusalem, Israel
2San
During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality
control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER
homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of
which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the evolutionary most conserved. Once activated, IRE1
splices the X-box binding protein 1 (XBP1) mRNA, leading to translation of the transcription factor XBP1s. In addition, IRE1
can recruit TRAF2 to activate JNK. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage.
However, the molecular mechanisms and the viral proteins responsible for UPR modulation remain poorly defined.
We have previously shown that murine cytomegalovirus (MCMV) briefly activates the IRE1-XBP1 signaling pathway
immediately after infection. Therefore, we wanted to investigate the impact of the IRE1-dependent signaling on murine
cytomegalovirus (MCMV) infection.
We show that genetic inactivation of IRE1 in murine embryonic fibroblasts results in reduced expression of viral proteins and
a strong inhibition of viral replication, suggesting that IRE1 signaling is important for the viral life cycle. Re-introduction of
IRE1 using a doxycycline-inducible system restored viral replication in a dose-dependent manner up to wildtype levels. By
contrast, genetic ablation of XBP1 or TRAF2 had only minor effects on viral productivity. XBP1 and IRE1 knockout cells differ
in that XBP1 ko cells express neither the spliced nor the unspliced XBP1 protein whereas IRE1 ko cells express only the
unspliced XBP1 (XBP1u). To test whether XBP1u represses MCMV replication, we expressed an "unspliceable" XBP1
mRNA in XBP1 ko cells. Indeed, MCMV replication was massively reduced under these conditions.
Based on these results we conclude that XBP1u (which lacks the transactivator domain of XBP1s) is not only a negative
regulator of XBP1s during ER stress but also a repressor for MCMV replication. Moreover, we propose that IRE1 is needed
during MCMV infection to relieve the XBP1u-mediated repression of viral replication.
Corresponding author:
Florian Hinte
[email protected]
Host Cell Factors and Modulation
OP97
Flavivirus infection uncouples translation suppression from cellular stress responses
H. Roth1, V. Magg1, F. Uch1, P. Mutz1, P. Klein1, K. Haneke2,3, V. Lohmann1, R. Bartenschlager4, O. T. Fackler5, N. Locker6,
G. Stoecklin2,3, A. Ruggieri7
1University
2University
of Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
of Heidelberg, Division of Biochemistry I, Center for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, Mannheim,
Germany
3University of Heidelberg, Center for Molecular Biology of University of Heidelberg (ZMBH), Heidelberg, Germany
4University of Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
5University of Heidelberg, Department of Infectious Diseases, Integrative Virology, Heidelberg, Germany
6University of Surrey, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, Guildford, United Kingdom
7University of Heidelberg, Molecular Virology, Heidelberg, Germany
Introduction: Viruses strictly depend on host cell translation for the production of new progeny. Since infected cells also
synthesize antiviral proteins to limit virus infection modulation of host cell translation represents a frequent strategy by which
viruses optimize their replication and spread.
Objectives: In this study, we aimed at defining the complex interaction of two major human pathogens, Dengue virus
(DENV) and Zika virus (ZIKV), with the host cell translation machinery in human cells.
Material & Methods: Modulation of host cell translation was studied by using polysome profiling and single-cell analyses of
de novo protein synthesis in human hepatoma cells infected with DENV and ZIKV. Activation of related stress response
pathwaysand stress granule (SG) formation were analyzed by Western Blot and immunofluorescence analyses, respectively.
Results: Our study reveals that flavivirus infection causes an unexpected and potent repression of host cell translation while
synthesis of viral proteins remains unaffected. DENV genome translation was previously reported to switch from capdependent to cap-independent genome translation when host cell translation is experimentally inhibited. Importantly, our
results confirm that this switch occurs in the context of a natural DENV infection and therefore represents a vital strategy for
flavivirus replication. Translation suppression is often tightly linked to the activation of the eIF2α-dependent stress response.
Our results indicate that during DENV and ZIKV infection translation repression was uncoupled from the disruption of SG
formation and eIF2α signaling. Additionally, none of the canonical pathways leading to translation suppression that are
usually hijacked by viruses were involved in DENV-induced translation repression. Instead, our results demonstrate that
DENV infection activates the p38-Mnk1 signaling pathway that regulates the phosphorylation of eIF4E and is important for
virus production.
Conclusion: Together, these results describe host cell translation repression as a novel and conserved strategy employed
by flaviviruses to ensure efficient replication in human cells, and define several molecular levels by which flaviviruses hijack
host cell translation and stress responses to favor virus production.
Corresponding author:
Hanna Roth
[email protected]
Host Cell Factors and Modulation
OP98
Inhibition of cytosolic phospholipase 2α impairs coronavirus replication by interfering with virus-induced replicative
organelle formation
C. Müller1,2, M. Hardt3, D. Schwudke4, B. W. Neuman5, S. Pleschka1,2, J. Ziebuhr1,2
1Justus
Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
Centre for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Germany
3Justus Liebig University Giessen, Imaging Unit, Giessen, Germany
4Research Center Borstel, Bioanalytical Chemistry, Borstel, Germany
5University Ave, Texakarna, United States
2German
Introduction: Similar to other (+)ssRNA viruses, coronaviruses induce membrane rearrangements in infected cells, resulting
in organelle-like "virus factories" that carry virus-encoded multi-subunit complexes that drive viral RNA synthesis. These
replicative organelles (ROs) contain high concentrations of viral and cellular proteins involved in viral replication/transcription
and may act to protect viral structures from host defense mechanisms. Based on these observations, it seems reasonable to
suggest that enzymes involved in cellular lipid metabolism have important roles in coronavirus replication.
Objective: To assess to role of cellular cytosolic phospholipase 2a (cPLA2α) activity in the replication of corona- and other
RNA viruses.
Material & Methods: Characterization of RO formation in virus-infected cells (TEM, CLSM) in the presence of smallmolecular-weight inhibitors of cPLA2α; determination of effects of reduced cPLA2α activity on viral replication (virus titration,
Northern and Western blot); Lipidome analysis of infected cells
Results: The data suggest that the cytosolic phospholipase 2α (cPLA2α) is one of the host cell factors that drive the
production of coronavirus-induced ROs. cPLA2α catalyzes the hydrolysis of membrane-associated phosphoglycerides at the
sn-2 position, thereby releasing a fatty acid and generating a lysophospholipid (LPL). We show that, in human coronvirus
229E-infected Huh7 cells, inhibition of cellular cPLA2α activity results in impaired viral protein and RNA accumulation as well
as reduced RTC and RO formation. Furthermore, inhibition of cPLA2α activity is shown to result in reduced LPL levels,
suggesting an involvement of LPL in producing the membraneous structures required for coronavirus replication. We also
found that inhibition of cPLA2α activity suppresses the replication of several other (+)ssRNA viruses known to induce
intracellular membrane rearrangements, such as HCV and Dengue virus (Menzel et al., 2012), MERS-CoV, and Semliki
forest virus, whereas poliovirus, human rhinovirus 1A (HRV1A), Influenza virus (IV) and vaccinia virus (VV) replication was
not affected.
Conclusion: Taken together, the data provide convincing evidence to suggest that cellular cPLA2α activity has important
roles at different steps of the replication cycle of +RNA viruses from various families.
Corresponding author:
Christin Müller
[email protected]
Host Cell Factors and Modulation
OP99
PI3K–Akt hyperactivation and metabolic changes upon alphavirus infection
B. Thaa1,2, L. Liu1, M. Mazzon3, C. Castro4, G. M. McInerney1
1Karolinska
Institutet, MTC, Stockholm, Sweden
of Leipzig, Institute of Virology, Leipzig, Germany
3University College London (UCL), MRC Laboratory for Molecular Cell Biology, London, United Kingdom
4University of Cambridge, Cambridge, United Kingdom
2University
Alphaviruses are a group of mosquito-transmitted ss(+)RNA viruses including relevant human pathogens such as
chikungunya virus (CHIKV) and Ross River virus (RRV), which cause high fever and debilitating joint pain. We demonstrated
previously that infection with the model alphavirus Semliki Forest virus (SFV) hyperactivates the pro-survival PI3K–Akt–
mTOR signalling pathway in cells, inducing internalisation of viral replication complexes from the plasma membrane. Now,
we have identified the molecular mechanism for this phenotype: An SFV mutant where a single tyrosine residue in the nonstructural protein nsP3 was replaced by phenylalanine (SFV-YF) failed to hyperactivate the pathway. Concomitantly,
replication complexes of SFV-wt but not SFV-YF were efficiently internalised from the plasma membrane. Coimmunoprecipitation experiments revealed that nsP3 of SFV-wt but not SFV-YF specifically binds to a factor of the pathway,
explaining the phenotype. In addition, we identified the same activation motif in the pathogenic RRV and eliminated virusinduced PI3K–Akt activation by a corresponding YF mutation.
Reprogramming of the cellular metabolism upon virus infection is increasingly recognised as a determinant of viral growth.
Since PI3K–Akt activation is often linked to augmented glucose metabolism, we analysed metabolic changes upon SFV
infection by NMR spectroscopy. Infection with SFV-wt reprogrammed glucose metabolism towards the synthesis of new
metabolic intermediates, providing building blocks for progeny virions. Inhibition of the pathway or infection with SFV-YF
prevented these changes and reduced viral titres. Thus, we suggest that sustained glucose metabolism due to virus-induced
PI3K–Akt activation maximises viral growth and may represent an attractive antiviral target.
Corresponding author:
Bastian Thaa
[email protected]
Host Cell Factors and Modulation
OP100
Role of the endogenous protease testisin in combination with a secreted Legionella pneumophila protease in
activation of influenza virus HA
A. Arendt1, B. Schmeck2, A. Flieger3, W. Garten1, H. D. Klenk1, E. Böttcher-Friebertshäuser1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
University Marburg, Institute of Lung Research, Marburg, Germany
3Robert Koch Institute, Bereich Wernigerode, Wernigerode, Germany
2Philipps
The major complication of influenza virus infection are co-infections with bacteria. Typical co-infecting bacteria are those
colonizing the nasopharynx like Staphylococcus aureus or Streptococcus pneumoniae. Although Legionella pneumophila
isn"t a classical co-infecting bacterium, some cases were reported during 2009 pandemic. The central model for co-infection
is that the viral infection primes the airway epithelium for bacterial attachment. Interestingly, some bacteria have been shown
to influence viral replication either by direct activation of the viral hemagglutinin precursor or indirectly by activation of host
HA-activating proteases.
Multicyclic replication of influenza virus is dependent on the presence of an appropriate host cell protease, as the HA
precursor molecule requires proteolytic cleavage to gain its fusion capacity. HeLa cells enable multicyclic replication of
influenza virus without the addition of trypsin, indicating that these cells express an endogenous HA-activating protease.
Addition of L. pneumophila liquid culture supernatant to the cell culture medium of influenza virus infected HeLa cells
promotes enhanced HA-activation and viral replication. In contrast, the bacterial supernatant does not support viral growth in
MDCK(H)-cells, which are deficient for any HA-activating protease. Moreover, the supernatant of a L. pneumophila strain
deficient for the metalloprotease ProA, does not influence HA-activation or viral replication in HeLa cells. Thus, it can be
concluded that an endogenous host cell protease present in HeLa cells is activated by the L. pneumophila supernatant in a
ProA dependent manner resulting in enhanced virus activation. siRNA mediated knockdown of the trypsin-like protease
testisin expressed endogenously in HeLa cells reduces viral spread and HA-cleavage in presence of L. pneumophila
supernatant.
Thus, we propose, that L. pneumophila ProA mediates activation of the endogenous protease testisin in HeLa cells, which in
turn leads to an enhanced influenza HA-activation and in consequence to an increased viral replication.
Corresponding author:
Annika Arendt
[email protected]
Host Cell Factors and Modulation
OP101
Hepatitis C Virus replication changes the host cell lipid profile and is sensitive to manipulations of the lipid
metabolism
S. Hofmann1, M. Krajewski2, C. Scherer1, V. Mordhorst1, V. Scholz2, M. Klabes1, M. Melling1, P. Truschow1, R. Reimer1, D.
Schwudke2, E. Herker1
1Heinrich
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
Center Borstel, Borstel, Germany
2Research
The Hepatitis C Virus (HCV) life cycle is tightly linked to the host cell lipid metabolism. To investigate changes in the
abundance of different lipid species we performed shotgun lipidomic studies of whole cell extracts and isolated lipid droplets
(LDs) of HCV-infected compared to uninfected hepatoma cells. Therefore, Huh7.5 cells were infected with an HCV reporter
virus (Jc1Flag-E2 NS5A/B-EGFP) and when infection rates were above 70% cells were either used directly as whole cell
extracts or for lipid droplet isolations. Lipids were extracted using the method of acidified Bligh & Dyer and analyzed by mass
spectrometry. Our results indicate that HCV infection induces an alteration of the ratio between neutral and membrane lipids
in whole cell extracts. In addition, we observed that HCV infection caused changes in the relative abundance of different lipid
species in whole cell extracts as well as in isolated LDs. HCV-infected cells had a higher relative abundance of
phosphatidylcholines (PC) and triglycerides (TG) with longer fatty acid chains than control cells. Moreover, the analysis of
free fatty acids revealed a significant increase of the levels of polyunsaturated fatty acids (PUFAs) caused by HCV infection.
Importantly, inhibition of the PUFA synthesis pathway with a high dose of a D6-desaturase inhibitor decreased virus
replication. In contrast, pretreatment with low inhibitor concentrations promoted viral uptake, most likely due to higher levels
of HCV entry factors at the cell surface. We additionally analyzed the impact of different fatty acids on viral RNA replication
and assembly and release of progeny virions and could detect striking differences between fatty acids depending on chain
length and saturation. Taken together our results suggest that HCV alters the lipid metabolism of the host cell to enhance its
replication.
Corresponding author:
Sarah Hofmann
[email protected]
Epidemiology and Public Health
OP102
Genetic history of HIV-1 subtype A reveals two independent introductions in Germany and increasing spread among
German MSM
K. Hanke1, N. R. Faria2, D. Kühnert3, K. Yousef4, A. Hauser1, K. Meixenberger1, A. Hofmann5, V. Bremer5, B. Bartmeyer5, O.
Pybus2, C. Kücherer1, M. von Kleist4, N. Bannert1
1Robert
Koch Institute, HIV and other Retroviruses, Berlin, Germany
of Oxford, Department of Zoology, Oxford, United Kingdom
3ETH Zurich, Department of Environmental Systems Science, Zurich, Switzerland
4Free University Berlin, Department of Mathematics and Computer Science, Berlin, Germany
5Robert Koch Institute, Division of HIV/AIDS, STI and Blood-borne Infections, Berlin, Germany
2University
Introduction: Molecular surveillance of HIV-1 diversity in Germany has revealed a recent increasing trend for subtype A
infections during 2013-2015, particularly in German men having sex with men (MSM). Subtype A was previously believed to
be mainly transmitted via heterosexual contacts and needle sharing.
Objectives: The aim of this analysis is to elucidate spatiotemporally the source and routes of subtype A spread across
geographic locations and distinct transmission groups in Germany.
Methods: 183 HIV-1 subtype A pol-sequences from patients diagnosed shortly after infection were collated with 560 closely
related publicly available subtype A sequences. Bayesian coalescent and birth-death analyses were performed to
reconstruct the population dynamics and the fluctuations in the epidemic spread of the German subtype A epidemic. Discrete
trait diffusion models were used to infer epidemiological linkage among geographic regions and transmission groups. Finally,
multi-type birth-death models were used to estimate changes in the effective reproduction number (Re) for each of the
transmission groups.
Results: Our phylogenetic reconstructions indicate two evolutionary distinct sources of the German subtype A epidemic: an
Eastern European variant (originating itself from Middle & Western Africa) and an Eastern African variant. A total of 13
transmission clusters of German origin were identified from which 6 show recent activity with at least two newly infected
individuals during the last 3 years. While older introductions originated mainly from Eastern Africa, since 2005 introductions
have occurred exclusively with the Eastern European clade. Moreover, we observed a change in the main route of subtype A
transmission: the beginning of the German epidemic (1985-1995) was dominated by heterosexual transmission, whereas
transmissions among MSM are strongly increasing since 2005.
Conclusion: We trace for the first time the genetic history of the German HIV-1 subtype A epidemic and its spread across
geographic regions and transmission groups. We further identify and characterize active German transmission clusters and
highlight key regions that are important targets for public health prevention strategies.
Corresponding author:
Kirsten Hanke
[email protected]
Epidemiology and Public Health
OP103
Impact of rainfall events on the concentrations of human adenoviruses, human noroviruses and bacteriophages in
urban surface waters
F. Kubek1, B. Süßenbach1, C. Arndt1, C. Mekonnen1, W. Seis2, R. Szewzyk1, H. C. Selinka1
1Federal
Environment Agency, FG II1.4 Microbiological Risks, Berlin, Germany
Wasser Berlin, Berlin, Germany
2Kompetenzzentrum
In highly populated areas, the presence of infectious human viruses in surface waters may intermittently constitute a public
health risk, especially when these waters serve multifunctional purposes. Whereas in clean flowing areas such waters may
be used for drinking water abstraction, ground water recharge, as bathing sites or for recreational purposes, other areas may
be used as water transportation pathways, drainage sites for rainfall run offs from roads, discharge areas for clear water or
combined sewer overflows. During heavy rainfall, various water quality zones may get temporarily mixed, depending on the
environmental conditions and virus loads.
The BMBF-funded project FLUSSHYGIENE investigates the entry and dynamics of hygienically relevant microorganisms and
pathogens in multifunctional water bodies and hydrologic circles, with focus on temporarily occurring hygienic loads.
Methods: In the first phase of this project, virological data were collected over several months in regular monitoring intervals
as wells as by event-directed campaigns (e.g. after rainfall events) as basis for future quantitative microbial risk
assessments. Concentrations of human adenoviruses and human noroviruses (GGII) were monitored by defined sampling
strategies and determined by quantitative Real Time PCR. Bacteriophages (coliphages) were quantified by plaque assays.
Results: Under dry weather conditions, concentrations of adenoviruses and noroviruses in the river Spree were mostly
under the detection limit (< 10exp1/100 ml). Coliphages were present in the range of 10exp1 – 10exp2/100 ml. A campaign
called flowing wave was operated in July 2016 in the river Spree after a heavy rainfall event exceeding 6 mm per 5 min. In a
time interval of about 24 h after rain fall, concentrations of human adenoviruses, human noroviruses and coliphages were
increased by about 3-4 log units compared to dry weather conditions, before decreasing to normal concentration values. A
second campaign, monitoring another rain event for almost 90 h led to similar increases in virus detection and confirmed
these results.
Conclusion: These data show that heavy rainfall events, leading to combined sewer overflows in urban areas, may severely
affect the water quality for an extended period of time. Therefore, it is important to consider inclusion of such data of eventrelated changes in river virus concentrations for preventive estimations of potential public health risks by quantitative
microbial risk assessment (QMRA).
Corresponding author:
Hans-Christoph Selinka
[email protected]
Epidemiology and Public Health
OP104
The Global Polio Eradication Program - a never ending story?
S. Böttcher1, F. Feil2, S. Diedrich1
1Robert
Koch Institute, National Reference Center for Poliomyelitis and Enteroviruses, Berlin, Germany
for Social Affairs, Health and Equal Opportunities of Lower Saxony, Hanover, Germany
2Ministry
The global effort to eradicate polio has been one of the largest public health initiatives in history. Poliomyelitis, a devastating
disease paralyzing more than 350.000 children each year in the late 80s, has disappeared from our minds during the last two
decades. This is to owe to the enormous efforts of the Global Polioeradication Initiative (GPEI) and its outstanding partners
WHO, Rotary International, CDC, UNICEF and the Bill & Melinda Gates Foundation. The success of the polio eradication
efforts is mainly based on three pillars: effective vaccines (oral/live vaccine, OPV, and inactivated vaccine, IPV), surveillance
and containment of polioviruses to minimize the risks for facility-associated reintroduction of virus into polio-free
communities. These aspects will be discussed:
(1) After declaration of eradication of wild poliovirus (WPV) type 2 in 2015, a worldwide switch from trivalent OPV to bivalent
OPV was achieved in April 2016. Furthermore, all OPV-only using countries introduced at least one IPV dose in their national
vaccination scheme.
(2) To interrupt transmission of polioviruses, WHO recommends testing stool samples from patients with acute flaccid
paralysis (AFP). Since polio free countries mainly in Europe and North America did not meet the WHO criteria for AFP
surveillance, alternative systems were established. In Germany, a nationwide laboratory network for enterovirus diagnostic
(LaNED) supervised by the National Reference Center for Poliomyelitis and Enteroviruses has been established to detect
polioviruses/enteroviruses in stool and CSF samples from patients with aseptic meningitis/encephalitis or acute flaccid
paralysis.
(3) By endorsing the WHO Polio Endgame strategic plan 2013-2018 together with the Global Action Plan (GAPIII) all
member states committed to implementing appropriate containment of Poliovirus type 2 strains in essential facilities (PEF) or
to destroy those materials. Facility-associated risks can be eliminated by a thorough nationwide search for, and destruction
of, all infectious and potentially infectious materials, including WPV, vaccine derived polioviruses (VDPV), and OPV viruses.
In Germany, the Protection against Infection Act (IfSG) will be changed to obligate the laboratories to fulfill the requirements
according to GAPIII.
Corresponding author:
Sindy Böttcher
[email protected]
Epidemiology and Public Health
OP105
Primary infection vs secondary infection of HCMV in pregnancy and impact for the outcome in newborns – Why the
incoming data from China do change the incomplete and even wrong view, based on published data from
Birmingham (Alabama) and Sao Paolo (Brazil)
G. Jahn1, J. Yang1, L. Ye1, J. Xu1
1University
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
Question: Primary infection by HCMV in pregnancy brings up bad outcome in about 15% of the living newborns. Data from
Alabama and Brazil (high prevalence) showed that the same range in secondary HCMV infection can lead to symptomatic
newborns. Secondary HCMV infection is highly dependent on lifestyle. Major transmission routes: close contact to children
less than three years and the number of sexual partners during pregnancy. This is the ground for the risk to acquire a new
HCMV gp-type.
Methods: All newborns of two different population groups living in Shanghai were screened by PCR from DBS at birth
between Nov. 2011 to Dec. 2012 in the Obstetrics and Gynecology Hospital of Fudan University and the Community Health
Center of Pujiang Town. Two different primers were taken for PCR. The follow-ups were done for clinical symptoms and
laboratory findings.
Results: All together 17 of 1780 investigated DBS (0.9%) were positive. 10 out of the 17 PCR positive newborns could be
clinically investigated in follow ups. None of the PCR positive newborn had clinical sign of HCMV infection. Just one single
case demonstrated brainstem auditory evoked potentials. The rate of HCMV newborn infection in CHC of Pujiang Town was
1,6% in contrast to 0,2% of the newborns seen at the Hospital of Fudan University. The population of Pujiang were
characterized by migrants with lower income and education levels in contrast to the population seen at Fudan University,
which had higher incomes and more education.
Conclusion: Our data fit with other data (CDC, Atlanta) from the Shandong Province (CDC) were 0,7% of HCMV infected
newborns were seen and all were asymptomatic. Similar data were published from Turkey. In China and Turkey HCMV is
highly prevalent (> 90% are positive). The outcome concerning HCMV disease in newborns is totally different between
China, Turkey vs Alabama and Sao Paolo. The reason for that is most likely the difference in lifestyle of the population. This
matter of fact is usually totally ignored in the scientific community; but it matters.
Corresponding author:
Gerhard Jahn
[email protected]
Epidemiology and Public Health
OP106
Steep rise of norovirus cases and emergence of a new recombinant strain GII.P16-GII.2 in Germany in season 20162017
S. Niendorf1, S. Jacobsen1, M. Faber1, T. Bock1
1Robert
Koch Institute, Berlin, Germany
Human noroviruses (NV) are causative of 18% of acute gastroenteritis (AGE) cases worldwide, and have a peak season
during the winter month (November to March) in the northern hemisphere. In 2016, the NV season started in Germany with
an unexpected high number of NV infections. In November 2016, >14,500 laboratory confirmed cases were reported to the
national public health authority compared to a median of 7,810 cases of same month of the previous five years. It has been
shown previously that the emergence of a new NV variant is regularly associated with an increase of reported NV infection.
In this study, we conducted genotyping analyses of actual circulating NV strains in order to assess whether new emerging
strains might be responsible for the current steep rise of norovirus infections.
We characterized 175 stool specimens from 69 norovirus outbreaks and 65 samples from sporadic cases between
September and December 2016, from 13 federal states of Germany.
We identified new emerging recombinant GII.P16_GII strains which in outbreaks and in sporadic cases of AGE. Using
SimPlot analyses, the recombination point was mapped to the ORF1/ORF2 junction region. The predominant strain GII.P16GII.2 was detected in 29 of 69 investigated outbreaks and can be considered as the etiologic agents in 31 of 65 cases of
sporadic AGE. This strain was detected in nine of 13 federal states of Germany tested so far. Additionally, a minor variant
GII.P16-GII.4 2012 was found in 10% of outbreaks and in 10% of sporadic cases. Other well-known circulating genotypes,
e.g. GII.P4 2009-GII.4 2012, GII.Pe-GII.4 2012, GII.P17-GII.17, were detected less frequently.
Further molecular and epidemiological investigations are needed to assess whether the emerging new recombinant NV
strain GII.P16-GII.2 can replace the most resident GII.Pe-GII.4 2012 strain and which impact the detected recombinants
have toward sizes of outbreaks, symptoms associated to NV infections and herd immunity of the population not only in
Germany and Europe.
Corresponding author:
Sandra Niendorf
[email protected]
Epidemiology and Public Health
OP107
A four year survey of Adenovirus circulation in patients with acute respiratory infections
A. Heim1, S. Buda2, B. Schweiger3
1Hanover
Medical School, Institute of Virology, Hanover, Germany
Koch Institute, FG Respiratory Transmissible Diseases, Berlin
3Robert Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
2Robert
Circulation of human adenoviruses (HAdV) associated with acute respiratory infections has not been studied systematically
for many years, whereas case reports and outbreak investigations on severe and fatal respiratory infections caused by a few
potentially highly virulent HAdV types (e.g. E4, B14, B21a) were published. However, without epidemiological data on the
circulation of these types any conclusion on their virulence is questionable.
Over a period of four years 1900 respiratory specimens of out-patients with acute respiratory infections were collected by the
"Arbeitsgemeinschaft Influenza" and tested by HAdV real time PCR for the "Netzwerkprojekt respiratorische Infektionen der
Nationalen Referenzzentren und Konsiliarlabore". Molecular typing was performed by imputed serology (sequencing of the
loop 2 of the major neutralization epitope).
293 of 1900 patients (15.4%) were positive for HAdV DNA and 104 of these (5.5 %) had high HAdV loads > 1x10 4 copies/ml,
which are associated with acute respiratory infections. By contrast, low HAdV loads are usually associated with HAdV
persistence subsequent to acute infections. Therefore, typing was only performed in case of patients with high virus loads.
Types of species HAdV-C predominated in three of four study years. In total HAdV-C types were detected in 75 of 104
patients, with type HAdV-C2 most frequently found in 42 patients, followed by type HAdV-C1 in 24 patients. Circulation of
species HAdV-B was only detected in a single year when 25 patients were positive for HAdV-B3 in comparison to 23 patients
positive for HAdV-C types. Circulation of species HAdV-E was noted in another year with only two patients positive for type
HAdV-E4. Potentially highly pathogenic types HAdV-B14 and HAdV-B21a were not detected at all.
Conclusion: Absence of type HAdV-B21a in this large out-patient sentinel group of acute respiratory infections supported
the hypothesis on its high virulence because HAdV-B21a was detected during this time period only in cases of severe
pneumonia. Results on the predominant circulation of types HAdV-C1 and HAdV-C2 confirmed results of previous studies
from the 1950s to 1980s.
Corresponding author:
Albert Heim
[email protected]
Zoonoses
OP108
A comparative analysis of Thogotoviruses to assess their zoonotic risk
J. Fuchs1, A. Dick2, O. Daumke2, G. Kochs1
1University
2Max
Freiburg, Institute of Virology, Medical Center, Faculty of Medicine, Freiburg, Germany
Delbrück Center for Molecular Medicine, Berlin, Germany
Question: Thogotoviruses form a unique subclass of orthomyxoviruses. They are tick-transmitted and primarily cause
disease in livestock. However, a recent fatal human case of Thogotovirus (THOV) infection in the US suggests that these
viruses have a zoonotic potential. We previously showed that Thogotoviruses are controlled by the interferon (IFN) induced
innate immune system – most potently by IFN-induced Mx proteins. These antiviral effectors bind the nucleoprotein (NP) of
THOV, thus efficiently inhibiting viral replication.
We hypothesize that acquiring Mx resistance is an important step for Thogotoviruses to cross the species barrier. To this
end, we analyzed the interplay of nine Thogoto isolates from different parts of the world with the host innate immune system.
Methods: We studied the Thogoto isolates in cell culture systems and in mouse models regarding their virulence, organ
tropism, disease progression and sensitivity to Mx proteins.
Results: We found major differences in the organ tropism and virulence of the viral isolates. Low viral doses were lethal for
Mx-negative mice, whereas mice harboring a functional murine Mx1 were fully protected from high dose infections. By
infecting mice transgenic for human MxA we observed that one particular isolate - Jos virus - was replicating to normal titers
whereas the prototype THOV strain was dramatically inhibited. We subsequently cloned Jos virus NP and analyzed it in a
polymerase reconstitution assay confirming our observations in the mouse model. Sequence analyses and 3D modeling
revealed potential residues in NP that may determine the differences in sensitivity to human and mouse Mx proteins.
Conclusion: Our comparative analysis of phylogenetically distinct Thogotoviruses showed a dramatic protective effect of
murine Mx1 as well as human MxA. However, we identified Jos virus that surprisingly escaped MxA restriction, indicating a
possible zoonotic potential. Future analysis of Jos virus will help to evaluate the zoonotic risk of various Thogotovirus
members and reveal their capacity to overcome species barriers by escape of the host innate immune system.
Corresponding author:
Jonas Fuchs
[email protected]
Zoonoses
OP109
Global assessment of protein de novo synthesis in response to infection with avian and human influenza A virus
B. Bogdanow1, K. Eichelbaum1, A. Sadewasser2, K. Paki2, M. Flöttmann3, X. Wang4, J. Hou4, W. Chen4, E. Klipp3, L.
Wiebusch5, T. Wolff2, M. Selbach1
1Max
Delbrück Center for Molecular Medicine, Proteome Dynamics, Berlin, Germany
Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
3Humboldt University Berlin, Department of Biology, Berlin, Germany
4Max Delbrück Center for Molecular Medicine, Berlin, Germany
5Charité Medical School, Laboratory of Pediatric Molekular Biology, Berlin, Germany
2Robert
While influenza A virus (IAV) encodes and carries the enzymatic machinery for genome replication and transcription, it is
inherently dependent on the host machinery for mRNA translation. IAV assures the selective translation of viral mRNA over
host transcripts by inducing the shut-down of host protein synthesis while maintaining host translational competence.
To detect protein de novo synthesis at a global level upon infection we employed a combined SILAC and metabolic pulse
labeling approach with the methionine analogous azidohomoalanine (AHA). With the help of this technique, we aimed to
understand the molecular barriers to a non-permissive infection setting. Therefore, we monitored protein synthesis profiles in
A549 cells that were either infected with a permissive seasonal IAV isolate (H3N2/A/Panama) or with an avian, nonpermissive isolate (H3N2/A/Mallard).
We observed the expected overall shut-down in host protein synthesis for both strains, which was more pronounced at later
intervals. Cluster analyses revealed subsets of host proteins that escaped the shut-down, including proteins involved in
antiviral response, immune response and translation. We further observed that genes related to the interferon response were
subject of strain-specific regulation. Synthesis of viral proteins was potently induced and showed striking strain differences
for the M1, HA and NA proteins. mRNA levels obtained via RNA-seq indicate that the differences in protein production
between the two strains could largely be explained by differences at the mRNA level. Furthermore, we observed differences
in splicing of the M1 mRNA between the two strains. Experiments using eukaryotic expression vectors containing the coding
sequence of the M segment suggest that 73 nt around the 3' splice site are responsible for strain-specific control of M1
mRNA splicing.
Ongoing functional studies aim to elicit the contribution of strain-specific control of M1 splicing to the differential infection
outcomes of the two strains. In conclusion, our approach demonstrates the impact of IAV infection on the host translational
machinery at proteomics scale.
Corresponding author:
Boris Bogdanow
[email protected]
Zoonoses
OP110
SARS coronavirus may have originated from frequent recombination events between SL-CoVs in a single
horseshoe bat habitat
B. Hu1, X. L. Yang1, X. Y. Ge1, W. Zhang1, B. Li1, D. S. Luo1, Y. Z. Zhang2, M. N. Wang1, P. Daszak3, L. Wang4, J. Cui1, Z. L.
Shi1
1Chinese
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
Institute of Endemic Diseases Control and Prevention, Dali, China
3EcoHealth Alliance, New York City, United States
4Duke-NUS Medical School, Programme in Emerging Infectious Diseases, Singapore, Singapore
2Yunnan
Horseshoe bats are recognized as the natural reservoirs of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV),
as an increasing number of SARS-like coronavirus (SL-CoV) have been detected in this bat family since 2005. However,
gaps remain between currently known bat SL-CoVs and the direct progenitor of SARS-CoV. We have conducted a 5-year
surveillance of SL-CoV in a cave inhabited by horseshoe bats in Yunnan, China. Our results revealed that genetically diverse
bat SL-CoVs were circulating in this single location, including different novel strains with high sequence similarity to SARSCoV in the highly variable N-terminal domain (NTD) and receptor-binding domain (RBD) of S protein and the ORF8 region,
respectively. Meanwhile, compared with other SL-CoVs, strains identified from this cave exhibited higher sequence similarity
to SARS-CoV in the non-structural proteins. Evidence supported that frequent recombination events have occurred at
multiple genomic sites between different bat SL-CoVs within this cave and may have given birth to the direct ancestor of
SARS-CoV. These findings offered important new insight into understanding the origin of SARS-CoV and addressed the
potentially ongoing threat posed by bat SL-CoVs.
Corresponding author:
Zheng-Li Shi
[email protected]
Zoonoses
OP111
Hantavirus infection in human gut and hamster animal model
P. T. Witkowski1, C. C. Perley2, R. L. Brocato2, J. W. Hooper2, C. Jürgensen3, J. Schulzke4, D. H. Krüger1, R. Bücker4
1Charité
Medical School, Institute of Virology, Berlin, Germany
States Army Medical Research Institute of Infectious Diseases (USAMRIID), Virology Division, Ft. Detrick, United States
3Charité Medical School, Division of Hepatology and Gastroenterology, Berlin, Germany
4Charité Medical School, Institute of Clinical Physiology, Berlin, Germany
2United
Question: Hantaviruses are zoonotic agents that cause hemorrhagic fever with renal and/or cardiopulmonary
manifestations, reaching fatality rates of up to 50%. A large proportion of hantavirus patients also suffer from gastrointestinal
complications of unclear cause. Puumala hantavirus (PUUV), the predominant endemic hantavirus in Europe, is associated
with mild forms of hemorrhagic fever with renal syndrome. PUUV is transmitted to humans by exposure to aerosolized
excreta from infected rodents. In this study we demonstrate that PUUV can infect the organism via the alimentary tract.
Methods: We investigated susceptibility of the human small intestinal epithelium for PUUV infection, based on the polarized
Caco-2 cell culture system, an established model for intestinal barrier function, and analyzed the resistance of virus particles
to gastric juice. In vivo we infected Syrian hamsters with PUUV via the intragastric route and tested seroconversion and
protective immunity against subsequent lethal hantavirus challenge.
Results: PUUV retains infectivity in gastric juice for at least some time in pH >3 and is able to infect polarized human Caco-2
monolayers. This small intestinal cell model exhibited viral association with endosomal antigen EEA-1, followed by virus
replication and loss of epithelial barrier function with concomitant basolateral (serosal) occurrence of viruses. Cellular
disturbance and depletion of the tight junction protein ZO-1 appeared after prolonged hantavirus infection. Subsequent cell
rounding and detachment was observed, leading to paracellular leakage. Moreover, experimental PUUV infection of Syrian
hamsters by the intragastric route led to seroconversion and protection against subsequent challenge by lethal Andes virus
in a dose-dependent manner, confirming that PUUV retains infectivity when administered directly to the gut.
Conclusion: We have evidence that the oral route of transmission, potentially by contaminated food, is plausible for PUUV
infection. The results of our work denote a new aspect of hantavirus pathogenesis to be included in epidemiological
considerations.
DHK and RB contributed equally
Corresponding author:
Peter T. Witkowski
[email protected]
Zoonoses
OP112
HPAIV H5 evolution requires adaptation of the hemagglutinin by elevation of the fusion competence activation pH
U. Wessels1, E. S. M. Abdelwhab1, J. Veits1, S. Mamerow1, O. Stech1, D. Hoffmann2, M. Beer2, T. C. Mettenleiter1, J. Stech1
1Friedrich
2Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
Loeffler Institute, Institute of Diagnostic Virology, Greifswald - Isle of Riems, Germany
Introduction: Highly pathogenic avian influenza viruses (HPAIV) cause fatal disease in poultry leading to enormous losses
world-wide. Moreover, repeated zoonotic infections have raised serious concerns of a novel pandemic. Previously, we found
that, beside the essential polybasic hemagglutinin (HA) cleavage site (HACS), a lethal phenotype in chicken requires
additional virulence determinants in the HA and other genes as NA, PB2, NP, and M.
Objectives: In this study, we focused our follow-up search on the HA.
Material & Methods: Using the reverse genetics systems from the HPAIV A/Swan/Germany/R65/2006 (H5N1, R65) and the
low-pathogenic strain A/Teal/Germany/WV632/2005 (H5N1), we generated several HA reassortants, HA1/HA2 chimeras and
point mutants. This panel of viruses was subjected to investigate the HA fusion competence activation pH and their virulence
in chicken and mice.
Results: Remarkably, the R65 HA variants with exchanged HA1 and/or the point mutations S123R or I124T were found to
display a notable decrease of activation pH up to one magnitude. Furthermore, this pH decrease is paralleled by extreme
reduction of virulence down to 0% both in chicken and mice. To address the frequency of those two amino acid exchanges in
natural H5 HPAIV, we surveyed public sequence databases. Among 2899 full-length HA protein sequences, we found the
single exchanges HA1 123S or 124I at a very low frequency of 0.6% each, but in dual combination at the overwhelming
majority of 97.2%, indicating that the two amino acid exchanges together at the HA1/HA2 interface form an essential
virulence determinant.
Conclusion: The strong requirement for such a dual motif at the HA1/HA2 interface has formed a threshold for emergence
of contemporary H5N1 HPAIV. Moreover, because of their polybasic HACS requiring prevention of premature activation of
fusion competence in the acid trans-Golgi network, this dual motif is very likely a relevant virulence determinant both in avian
and mammalian hosts including humans. Overall, beside acquisition of a polybasic HACS, the evolution of HPAIV from lowpathogenic precursors requires adaptation of the HA by elevation of the fusion competence activation pH.
Corresponding author:
Zoonoses
OP113
Determinants of highly pathogenic H7N7 avian influenza virus pathogenicity and transmission
C. Dreier1, P. Resa-Infante1,2, A. Otte1,3, A. Preuß1, D. van Riel1,4, S. Bertram1,5, H. D. Klenk6, G. Gabriel1,5
1Heinrich
Pette Institute, Viral Zoonosis and Adaptation, Hamburg, Germany
Molecular Biology Laboratory, Grenoble, France
3Institute of Biochemistry, University of Kiel, Kiel, Germany
4Erasmus Medical Center, Department of Viroscience, Rotterdam, Netherlands
5University of Lübeck, Institute of Biology, Center for Structural and Cellular Biology in Medicine, Lübeck, Germany
6Philipps University of Marburg, Institute of Virology, Marburg, Germany
2European
Highly pathogenic H7N7 avian influenza viruses (HPAIV) have the ability to cause lethal infections in poultry and humans.
However, the molecular mechanisms involved in H7N7 HPAIV pathogenicity in mammals as well as those required for
transmission between mammalian species remain largely unknown. In this study, we analyzed the molecular determinants of
H7N7 HPAIV pathogenicity and transmission using a ferret-adapted H7N7 HPAIV variant.
To investigate the molecular determinants of H7N7 ferret adaptive signatures, a series of molecular assays have been
performed. Viral pathogenicity and transmission were studied using mouse and guinea pig models.
In this study, we have identified mutations in the HA gene of the ferret-adapted H7N7 that increase viral pathogenicity in
mice and facilitate contact transmission between guinea pigs.
To conclude, a ferret-adapted H7N7 HPAIV variant is able to mediate contact transmission in guinea pigs and significantly
increase pathogenicity in mice upon acquisition of adaptive changes in the HA gene.
Corresponding author:
Carola Dreier
[email protected]
Host Cell Factors and Modulation
OP114
Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration
V. Gonzalez-Motos1, C. Jürgens1, B. Ritter1, O. Larsen2, T. L. Rovis3, S. Jonjic3, T. Krey1, T. F. Schulz1, B. B. Kaufer4, U.
Kalinke5, A. E. Proudfoot6, M. M. Rosenkilde2, A. Viejo-Borbolla1
1Hanover
Medical School, Institute of Virology, Hanover, Germany
of Copenhagen, Copenhagen, Denmark
3University of Rijeka, Rijeka, Croatia
4Free University Berlin, Institute of Virology, Berlin, Germany
5Twincore GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
6NovImmune, Geneva, Switzerland
2University
Introduction: Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the
peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed
by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV infects leukocytes in tonsils and
other regional lymph nodes, modifies their activity and hijacks them for its spread to the skin and neurons. In spite of its
importance in pathogenesis, the mechanism of VZV dissemination remains poorly understood.
Objective: To determine whether VZV modulates leukocyte migration to facilitate virus spread.
Material & Methods: We expressed and purified the recombinant soluble ectodomain of VZV glycoprotein C (rSgC) –full
length and mutated constructs- and determined its chemokine binding properties by surface plasmon resonance. Transwell
experiments with rSgC, wild type and mutated chemokines, were used to determine the functional relevance of the
interactions. Recombinant VZV expressing GFP instead of gC or GFP fused to the C-terminus of gC were generated using
the bacterial artificial chromosome technique. We used western blot, direct fluorescence and immunofluorescence to
determine the location of gC during infection.
Results: VZV rSgC binds chemokines with high affinity and potentiates their activity, enhancing the migration of monocyte
and T cell lines and, more importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation
of chemokine activity occurs through the C-terminal part of gC ectodomain, containing two predicted immunoglobulin-like
domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the
chemokine receptor. We detected gC at the plasma membrane and in the supernatant of infected cells. We are currently
investigating the modulation of chemokine activity during infection.
Conclusion: Our data suggest that gC enhances the recruitment of leukocytes for their infection and thereby facilitates VZV
systemic dissemination in humans.
Corresponding author:
Victor Gonzalez-Motos
[email protected]
Figure 1
Host Cell Factors and Modulation
OP115
Macrophage-released TRAIL contributes to impaired alveolar fluid clearance after influenza A virus infection
C. Peteranderl1, B. Selvakumar1, L. Morales-Nebreda2, E. Lecuona2, T. Wolff3, R. Morty1, W. Seeger1, J. I. Sznajder2, G.
Mutlu2, S. Budinger2, S. Herold1
1Universities
Giessen & Marburg Lung Center, Department of Internal Medicine II, Giessen, Germany
University, Feinberg School of Medicine, Chicago, United States
3Robert Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
2Northwestern
Influenza A viruses (IAV) cause primary viral pneumonia resulting in acute respiratory distress syndrome (ARDS) associated
with severe alveolar edema formation. As impairment of edema resolution in ARDS patients is correlated with high mortality,
we investigated metabolism and function of Na,K-ATPase, a major regulator of fluid homeostasis, to define mechanisms
affecting alveolar fluid clearance (AFC) in IAV infection.
In vivo IAV infection of wildtype (wt) mice resulted in reduced AFC, edema formation and hypoxia that occurred in parallel
with a decrease in total and plasma membrane expressed Na,K-ATPase α1 subunit (NKAα1). NKAα1 was primarily
decreased in non-infected cells in a monoculture of alveolar epithelial cells (AEC) and in presence of co-cultured, infected
macrophages. We found paracrine signaling of type I interferons (IFN) and the macrophage-released, IFN-dependent
cytokine TRAIL (TNF-related apoptosis inducing ligand) to be sufficient to decrease NKAα1 in a CaMKKβ- and AMPKdependent way. Blockade of this pathway using specific chemical inhibitors, adenoviral overexpression or siRNA approaches
restored NKAα1 levels as well as vectorial water transport in ex vivo infected AEC. Additionally, trail -/- or ifnar-/- mice, mice
transduced with a dominant-negative form of AMPK or ccr2-/- mice lacking pulmonary macrophage recruitment showed
improved NKAα1 levels and AFC after IAV infection. In parallel, inhibition of Na,K-ATPase channel activity by ouabain
reduced the amount of IAV infected cells, implying a role for Na,K-ATPase in the IAV replication cycle.
Together, this work demonstrates that AFC is inhibited after IAV infection in non-infected neighboring cells by paracrine
IFN/TRAIL/DR5 signaling resulting in AMPK-mediated decrease of plasma membrane NKAα1. Targeting these pathways
may be a novel therapeutic strategy to improve AFC, oxygenation and finally outcome in patients with IAV-induced ARDS.
Corresponding author:
Christin Peteranderl
[email protected]
Host Cell Factors and Modulation
OP116
Inhibition of replication and pathogenesis of Coronaviruses by targeting viral 2'-O-Methyltransferase
S. Wang1, C. Zeng1, Y. Wang1, R. Pan1, Y. Chen1, D. Guo1
1Wuhan
University, State Key Laboratory of Virology, College of Life Sciences & School of Medicine, Wuhan, China
The 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate
in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs
and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune
recognition in host cells. Previous studies showed that the effective 2'-O-MTase activity of coronaviruses (CoVs) is
commonly relied on the interaction of nonstructural protein 16 (nsp16) and nsp10, forming nsp10/nsp16 complex. Recently,
the nsp10-derived peptide could broadly inhibit the 2'-O-MTase activities of betacoronavirus and gammacoronavirus in vitro
and the viral replication in MHV infection and SARS-CoV replicon models. Here, based on our current and previous studies,
we optimized the peptides or other novel inhibitors systematically using computer-aided drug design (CADD) and
biochemical assay screening. Interestingly, all the inhibitors exerted robust inhibitory effects in vivo in MHV-infected mice by
impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production
at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2'-O-MTase
activity can be an ideal antiviral target.
Corresponding author:
Deyin Guo
[email protected]
Host Cell Factors and Modulation
OP117
Marburg virus VP30 balances the XBP1-dependent unfolded protein response
C. Rohde1, S. Becker1, V. Krähling1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
One third of all cellular proteins are produced within the endoplasmatic reticulum (ER). Too many unfolded proteins impose
stress on the ER leading to the activation of several signaling cascades, generally referred to as unfolded protein response
(UPR). XBP1s (X-Box binding protein 1 spliced), one of the main transcription factors involved in UPR is dependent on
posttranscriptional splicing of the XBP1 mRNA by IRE1, a UPR sensor protein. XBP1s then binds to cis-acting elements e.g.
UPRE (UPR element) enhancing the expression of many genes, e.g. chaperones, in order to restore ER homeostasis. Under
non-UPR conditions the XBP1 mRNA is not spliced resulting in translation of XBP1u (XBP1 unspliced) instead of XBP1s.
XBP1u recruits its own mRNA to the ER membrane to support IRE1 splicing if ER stress arises.
UPR can support viral propagation since e.g. upregulation of chaperones might be beneficial for viral proteins, however,
terminal UPR leading to apoptosis may impair the viral replication process. The aim of our study was to examine how
Marburg virus (MARV) infection and single MARV proteins interact with UPR signaling pathways. To analyze if MARV
proteins regulate UPR signaling, we utilized luciferase assays to monitor the activity of the UPR-specific element UPRE.
Interestingly, while MARV infection did not activate UPR the recombinant expression of the viral glycoprotein GP induced
UPRE-dependent luciferase activity. Expression rates of GP during infection and transfection were the same. Using a tagged
variant of XBP1, we demonstrated at whole population and single cell level that XBP1s was activated by expression of GP.
In contrast, ATF6, another transcription factor relevant during UPR, was not activated. The apparently contradictory results of
MARV-infected versus GP-transfected cells could be reconciled by showing that MARV VP30 counteracted GP-activated
UPR. Remarkably, VP30 was also able to reduce UPR activated by thapsigargin. The function of VP30 toward balancing
UPR was further characterized by co-immunoprecipitation studies. Interestingly, VP30 directly interacted with XBP1u, not
XBP1s, which might influence the availability of the XBP1 mRNA during MARV infection. We discovered a tight regulation of
the XBP1-dependent UPR by MARV VP30 that is assumed to be needed for efficient MARV propagation.
Corresponding author:
Cornelius Rohde
[email protected]
Host Cell Factors and Modulation
OP118
Comparative loss of function screens reveal common pathways required by Paramyxoviridae and Pneumoviridae
K. Pfeffermann1, D. Anderson2, S. Y. Kim3, B. Sawatsky1, P. Duprex4, M. A. Garcia-Blanco2,3,5, V. von Messling1,2,6
1Paul
Ehrlich Institute, Veterinary Medicine, Langen, Germany
Medical School, Programme in Emerging Infectious Diseases, Singapore, Singapore
3Duke University, Department of Molecular Genetics and Microbiology, Durham, United States
4Boston University School of Medicine, Microbiology, Boston, United States
5University of Texas Medical Branch, Department of Biochemistry and Molecular Biology, Galveston, United States
6University of Quebec, INRS-Institut Armand-Frappier, Laval, Canada
2Duke-NUS
Introduction: Paramyxoviruses and pneumoviruses use the respiratory tract as a point of entry and share considerable
structural and functional homologies. Even though these virus families include many pathogens with great relevance for
human and animal health, host factors involved in their life cycle remain elusive.
Objectives: To identify common cellular host factors involved in the Paramyxo- and Pneumviridae life cycle as a basis for
new insights in the biology of these viruses and the development of rationally designed therapeutics.
Material & Methods: Comparative genome-wide siRNA screens with wild type measles, mumps and respiratory syncytial
viruses were conducted in A549 cells, a human lung adenocarcinoma cell line, that shares many key characteristics with
respiratory epithelial cells. One of the top hits, the ATP-binding cassette (ABC) transporter ABCE1, was chosen for further
characterization and its role in viral replication and translation was analyzed by viral growth kinetics and Western blot
analysis. Furthermore, its impact on viral as well as global cellular translation was elucidated using metabolic or catalyzed
fluorescent labeling of proteins.
Results: Comparative analysis of the three screens yielded 42 common proviral host factors, six of which, ABCE1, DOK7,
EIF3A, PRR15, RPS9 and USX1 were confirmed by subsequent validation screens. In addition to coatomer complex I, a
known dependency factor of negative strand RNA viruses in general, proteasomal degradation, RNA processing and
translation were the top pathways required by all three viruses. ABCE1 knockdown strongly inhibited the production of MeV
proteins, while only modestly affecting global protein synthesis. This not only confirms its supportive role in cellular protein
synthesis but also demonstrates that ABCE1 is absolutely required for efficient viral protein translation.
Conclusion: This study highlights the importance of ABCE1 as Paramyxo- and Pneumoviridae translation factor.
Furthermore, the data set will be an invaluable resource for the development of broad-spectrum antivirals not only against
the three viruses included but also against related new and emerging pathogens.
Corresponding author:
Kristin Pfeffermann
[email protected]
Host Cell Factors and Modulation
OP119
Restriction of HIV-1 infectivity by SERINC5 is not mediated by alterations of the lipid composition of viral particles
B. Trautz1, H. Wiedemann2, V. Pierini1, R. Wombacher1, B. Stolp1, A. J. Chase1, M. Pizzato3, B. Brügger2, O. T. Fackler1
1University
Hospital Heidelberg, Department of Infectious Diseases, Integrative Virology, Heidelberg, Germany
of Heidelberg, Biochemistry Center (BZH), Heidelberg, Germany
3University of Trento, Centre for Integrative Biology, Trento, Italy
2University
Introduction: HIV-1 Nef enhances virus replication and contributes to immune evasion in vivo by incompletely defined
molecular mechanisms. SERINC5 is a host cell factor that inhibits HIV-1 infectivity and is antagonized by Nef. This
antagonism is thought to occur via retargeting of the restriction factor from the plasma membrane to endosomal
compartments leading to the exclusion of SERINC5 from HIV-1 particles. The physiological functions of SERINC proteins are
poorly characterized with the one mechanistic study available suggesting a role in serine membrane incorporation for
phospholipid biosynthesis. The molecular basis of SERINC5-mediated inhibition of HIV-1 particle infectivity is unknown.
Objectives: We sought to address which of the proposed Nef effects on SERINC5 are essential for enhancement of HIV-1
particle infectivity and to test whether SERINC5 restricts virion infectivity via alteration of the lipid composition of virus
producing cells and/or HIV-1 particles.
Material & Methods: Using antagonism-deficient Nef mutants, we analyzed which of the proposed effects on SERINC5
(retargeting, surface downregulation, virion exclusion) are involved in counteraction. We used mass spectrometry to quantify
the lipidome of virus-producing cells and HIV-1 particles
Results: Characterization of Nef mutants revealed that retargeting and reduction of cell surface density of SERINC5 is
dispensable and insufficient, respectively for antagonism by Nef. While virion-incorporation of SERINC5 in the absence of
Nef was strictly associated with reduced particle infectivity, Nef enhanced HIV particle infectivity despite significant virion
incorporation of the restriction factor at high levels of SERINC5 expression. SERINC5 did not significantly alter the steady
state lipid composition of virus producing cells or HIV particles and also did not affect sphingolipid biosynthesis
Conclusion: Our results reveal that in addition to virion exclusion, Nef employs a yet-unknown mechanism to inactivate
virion-associated pools of SERINC5. From our lipid analysis we conclude that restriction of HIV-1 particle infectivity by
SERINC5 is not mediated by alterations of the steady state lipid composition of viral particles and that the restriction factor is
not involved in sphingolipid biosynthesis.
Corresponding author:
Birthe Trautz
[email protected]
Clinical Virology
OP120
Hepatitis E virus infection is common in neuralgic amyotrophy patients and associated with pathological CSF
M. Fritz1, B. Berger1, D. Endres2, O. Stich1, M. Panning3
1Albert
Ludwigs University, University Hospital Freiburg, Department of Neurology and Neurophysiology, Freiburg, Germany
Ludwigs University, University Hospital Freiburg, Department of Psychiatry & Psychotherapy, Freiburg, Germany
3Albert Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
2Albert
Background: The role of Hepatitis E virus (HEV) infection as a pathogen for neuroimmunological diseases is still
underestimated. There is growing evidence that, also in immunocompetent patients, an acute HEV infection can be
associated with peripheral nerve inflammation, most notably neuralgic amyotrophy (NA), an acute brachial plexus disorder
with pain and multifocal paresis. Here, we have analysed the largest cohort of patients with NA from Germany to date for
acute or resolved HEV infection.
Methods: In our study, 35 patients with neuralgic amyotrophy and a control group of 37 patients with VZV
meningoencephalitis/radiculitis were retrospectively tested for HEV infection. All patients were treated in the University Clinic
Freiburg from 2000-2016. We analyzed for serum anti-HEV IgM and IgG as well as HEV RNA in serum and CSF samples.
Results: A total of 49% of NA patients had a positive HEV serology. Four of these patients (11%) had an acute infection
(anti-HEV IgM positive). In the acute infection cases, HEV-PCR in serum was positive if the sample was examined < 2 weeks
after symptom onset (75% of acute cases). In one PCR-positive case we also detected HEV RNA in the CSF. In HEV PCRpositive cases all were of genotype 3. All four acute cases had elevated liver enzymes. HEV-positive NA was associated with
an elevated white blood cell count in the CSF (35% vs 6%, p<0.05) and a higher median age (57 vs 48 years, p<0.05), there
was no difference in clinical features. In a control group of patients with a VZV infection there were significantly less cases
with a positive HEV serology (22%, p<0.05) and there was only one anti-HEV IgM positive patient. None of the control
patients tested HEV RNA positive.
Conclusion: In a cohort of NA patients in south-west Germany we found that 49% had a positive HEV serology. Since the
prevalence of a HEV infection was significantly lower in the control group, this seems to be a specific effect. The association
with pathological CSF as well as the detection of HEV RNA in the CSF in one case argues for a causal relation of this
association. Therefore, we recommend testing for HEV infection in NA patients and evaluation of the outcome. The evidence
for treatment needs to be determined in future studies.
Corresponding author:
Marcus Panning
[email protected]
Clinical Virology
OP121
Effectiveness of Rotavirus vaccination in preventing rotavirus-related hospitalisations of under-five children,
Germany
C. Pietsch1, V. Schuster2, U. G. Liebert1
1University
2University
of Leipzig, Institute of Virology, Leipzig, Germany
of Leipzig, Department of Paediatrics, Leipzig, Germany
Background: Rotavirus immunisation was recommended by the local government of Saxony in 2008. Since 2013
vaccination is recommended nationwide. Vaccine coverage in infants increased from 36% (2008) to 83% (2014) in Saxony.
Methods: To assess the impact of vaccination on rotavirus-related hospitalisations 9,062 stool samples of under-5 children
with acute gastroenteritis were screened for rotavirus during 14 consecutive seasons and assigned to a pre-vaccine of 5 and
a post-vaccine cohort of 9 seasons, respectively. In the latter vaccination status was recorded and rotavirus genotypes were
determined.
Results: A total of 1,866 rotavirus infections were detected. Thereof 836 related to the post-vaccine cohort. The incidence of
rotavirus-related hospitalisations decreased by 67%. More than a quarter of avoided cases were attributed to herd effects. A
biennial pattern of rotavirus activity became apparent. The direct, indirect, total and overall vaccine effectiveness in all under5 children was 85%, 47%, 92% and 67%, respectively. Break-through infections of rotavirus-vaccinated infants were rare and
evenly distributed among those immunized with Rotarix and RotaTeq. However, direct vaccine effectiveness was reduced in
2012/2013, when many vaccinated children were infected by G2P[4] rotaviruses. During the study uncommon genotypes
such as P[6], G8 and G12 genotypes remained rare.
Conclusion: Rotavirus vaccines are highly effective. Break-through infections were rare and no selection of genotypes was
observed. The reduction of infections in unvaccinated children proves the benefit from herd protection. The bi-annual pattern
of seasonal morbidity is conceivably due to an accumulation of unvaccinated individuals, who initially escaped infection
because of herd protection.
Corresponding author:
Corinna Pietsch
[email protected]
Clinical Virology
OP122
CMV particles mediate acute myeloid leukaemia cell death via soluble factors irrespective of viral replication, gene
expression, and entry
L. Rink1, V. T. K. Le-Trilling1, S. Voigt2,3, M. Trilling1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Koch Institute, FG12 - Measles, Mumps, Rubella, and viruses affecting immunocompromised patients, Berlin, Germany
3Charité Medical School, Department of Pediatrics, Division of Oncology and Hematology, Berlin, Germany
2Robert
Acute myeloid leukemia (AML) is caused by malign transformations of cells from the myeloid lineage leading to their
uncontrolled proliferation and subsequent malfunction of hematopoiesis. Bone marrow ablation and subsequent
reconstitution e.g. by hematopoietic stem cell transplantation (HSCT) is a frequently used treatment option with curative
potential. Although HSCT recipients exhibit exaggerated vulnerability towards several infectious agents due to the ablation of
the immune system and immunosuppressive therapy, a surprising correlation between early (<d100) human cytomegalovirus
(HCMV) reactivation and reduced AML relapse has been documented. Based on the absence of this correlation in case of
lymphocytic malignancies as well as HSV, VZV, and EBV infections, we infer that an association between HCMV, its tropism,
and reduced AML relapse might exist.
Studying this correlation, we observed that very low multiplicities of HCMV infection cause overt death of several AML cell
lines. Boiling or sterile filtration of the HCMV inoculum abrogated the effect, but it was still evident upon treatment with pancaspase or necroptosis inhibitors. Furthermore, the effect was also observed in presence of Ganciclovir and upon contact
with UV-irradiated HCMV virions. Consistent with a dependency on the pentameric entry complex, HCMV strain TB40/E - but
not strain AD169 - entered AML cells as determined by pIE1-pp72 expression. However, both strains equally inhibited the
proliferation of several AML cell lines and induced AML cell death. We concluded that HCMV particles elicit this detrimental
effect on AML cells irrespective of viral replication, gene expression and entry. Consistent with the results of low MOI
infections, sterile supernatants from HCMV-experienced AML cells killed fresh AML cells. Based on the finding that such
supernatants activate NF-kB- and GAS reporter cells, we currently study if HCMV particles stimulate AML cells to induce a
suicidal innate immune response.
Corresponding author:
Lydia Rink
[email protected]
Clinical Virology
OP123
Are human parainfluenza viruses important pathogens in children with influenza-like-illness?
B. Schweiger1,2, B. Biere1, J. Reiche1,2
1Robert
2Robert
Koch Institute, Division of Influenza and Other Respiratory Viruses, National Reference Centre for Influenza, Berlin, Germany
Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
Introduction: Human parainfluenza viruses (PIV) are important pathogens of viral lower respiratory tract disease, and they
are second only to respiratory syncytial virus (RSV) as a cause of hospitalizations among young children. Symptoms include
common cold with fever, croup, bronchiolitis, and pneumonia. So far, there is limited information about the prevalence of PIV
in children with signs and symptoms of acute respiratory infection (ARI) or influenza-like illness (ILI) in pediatric outpatient
facilities.
Objectives: To study the importance of PIV in children with ILI, specimens from patients with respiratory symptoms were
investigated for PIV types 1 to 4 within the well-established German national outpatient influenza sentinel surveillance.
Patients & Methods: Within the sentinel, nasal or throat swabs from patients with ILI and/or ARI were regularly tested for
respiratory viruses including influenza viruses A and B, rhinovirus (RV), RSV, adenovirus (AdV), and metapneumovirus
(HMPV) (https://influenza.rki.de/Wochenberichte.aspx). In this study, specimens from children younger than four years were
retrospectively screened for PIV type 1 to 4 with a specific multiplex real-time PCR in a consecutive 4-year period. PIV
positive samples were sequenced by conventional RT-PCR.
Results: Between October 2012 and September 2016, about 3600 specimens were analyzed for six respiratory viruses. On
average, 23% of children with ILI were positive for influenza viruses. Besides influenza viruses, pathogens RSV (21%), RV
(21%), and AdV (16%) were found to considerably contribute to ILI. PIV were detected in 5% of the children. They
predominantly circulated before and after the influenza wave in late autumn/early winter and spring. PIV types 1 to 4 were
detected in every season in varying incidences. Moreover, sequencing facilitates first phylogenetic characterization of PIV in
Germany.
Conclusion: In children, ILI is caused by other viral pathogens than influenza viruses to a considerable amount. However,
children with ILI were rarely infected with PIV, and therefore, PIV play no significant role in children with ILI.
Corresponding author:
Janine Reiche
[email protected]
Clinical Virology
OP124
What is required for a HPV test to be suitable for the upcoming organized cervical cancer screening programme in
germany?
T. Iftner1, S. Brucker2, K. Neis3, A. Staebler4, M. Henes2, J. Haedicke-Jarboui1, G. Boehmer5, P. Sasieni6
1University
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
Hospital Tübingen, Department of Gynecology and Obstetrics, Tübingen, Germany
3Frauenärzte am Staden, Saarbrücken, Germany
4University Hospital Tübingen, Department of Pathology and Neuropathology, Tübingen, Germany
5Amedes Laboratory, Bad Münder, Germany
6Queen Mary University of Londo, Centre for Cancer Prevention, London, United Kingdom
2University
On Sept. 15th 2016 the "Gemeinsame Bundesausschuss (G-BA)" issued a directive for the upcoming organized Cervical
Cancer Screening Program in Germany. Women starting at the age of 35 years will be offered instead of the annual PAP
smear (cytology) a combined testing consisting of an HPV test in combination with the PAP smear every three years. Women
aged 20-35 will still have annual PAP smears.Requirements for a HPV DNA test in an organized population based screening
setting are as follows – the test shall be able to detect only the 13 HPV types classified by the IARC/WHO as carcinogenic to
humans. The clinical sensitivity and specificity of a HPV test for the detection of cervical pre-cancer (CIN2 or higher; CIN2+)
must not be lower than 90% and 98% of already established and in RCTs used HPV test systems, like the HC2 (QIAGEN),
respectively. Ideally all tests should have an FDA approval as the CE mark of a HPV tests does currently not request tests to
fulfill the requirements for highly critical pathogens in class C of the IVD regulations of the EC (International Medical Device
Regulators Forum), which should, however, be a pre-requirement for a mass-screening test.We performed in the past years
three HPV DNA and RNA test comparisons (Abbott: realtime high risk HPV test, Hologic: CERVISTA, APTIMA) in crosssectional studies (N= approx.10.000 each) based on routine screening populations and currently perform one new RNA test
comparison (APTIMA; N=9451) in a long-term prospective (5 years) routine screening cohort.The results of these
comparisons show that besides a high degree of automatization, inter- and intra-laboratory reproducibility and non-inferiority
in the clinical performance to established gold standard tests other features need to be considered. Tests like the CERVISTA
revealed in our comparative cross-sectional studies a twice as high positivity rate of women with normal cytology as the HC2
test leading to an increased referal rate. The Abbott realtime high risk HPV test missed cases of HPV31-positive CIN3. In
contrast the APTIMA RNA-based test consistently shows comparable clinical sensitivity to the HC2 in combination with
always higher clinical specificity. This would reduce the number of follow up procedures by 23% in a screening program by
keeping a high sensitivity for precancer. The results of these studies and a list of tests suitable for a screening program in
Germany using a 3-year screening interval will be presented.
Corresponding author:
Thomas Iftner
[email protected]
Clinical Virology
OP125
Molecular characterization of influenza A viruses circulating in Germany during 2014-2015 to 2016-2017
J. Naumann1, M. Wedde1, T. Wolff1, B. Schweiger1
1Robert
Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
Introduction: Influenza viruses cause global epidemics every year and can cause severe and fatal infections. In the context
of influenza surveillance of mild, severe and fatal cases, influenza A(H1N1)pdm09 and A(H3N2) viruses were characterized
and genes coding for external and internal proteins were analyzed.
Objectives: Assessment of molecular features of influenza A viruses (IAV) during 2014-2015 to 2016-2017.
Material & Methods: Viral specimens were collected during the season 2014-2015 to 2016-2017. RNA was extracted from
original material. After multisegment reverse transcription-PCR the amplified hemagglutinin (HA) genes were analyzed by
cycle sequencing (Applied Biosystems). For a subset of specimens whole genomes were sequenced (Miseq, Illumina).
Results: Concerning the prevalence of IAV, the HA-analysis revealed that A(H1N1)pdm09 viruses dominated in the season
2015-2016. Viruses of the clade 6B.1 were most prevalent and represent the new vaccine strain A/Michigan/45/2015
recommended for the southern hemisphere in 2017. At the beginning of the season 2016-2017 A(H3N2) viruses circulated
exclusively and most of the viruses belonged to the clade 3C.2a1 (with the reference strain A/Bolzano/7/2016).
The whole genomes of A(H1N1)pdm09 viruses that circulated during 2015-2016 were analyzed retrospectively using a
subset of PCR-positive specimens (three mild, one ambulant pneumonia, six severe, one fatal case). We analyzed fixed and
newly emerged amino acid (aa) substitutions of the internal genes and found that most of fixed aa substitutions that
accumulated since 2009 were located on the polymerase PB2, NS1 and, interestingly, on the disrupted PB1-F2 reading
frames. Phylogenetic analysis of the external and internal genes revealed that 50% of the viruses from severe cases cluster
together in their PB1, PA, HA, NA, M1 and NEP genes. However, none of these identified genetic groups was characterized
by a clade specific aa substitution. For one severe and one fatal case we found polymorphisms on HA-222 (D/G/N), an
indicator of viral pathogenicity.
Conclusion: Whole genome sequencing of A(H1N1)pdm09 viruses showed a different prevalence of aa substitutions along
the external and internal genes and offers a promising tool to get more detailed insights in the evolution of seasonal IAV.
Corresponding author:
Juliane Naumann
[email protected]
Abstracts of posters
Virus Receptors and Entry
P01
Porcine amino peptidase N domain VII has critical role in binding and entry of porcine epidemic diarrhea virus
H. J. Shin1
1Chungnam
National University, College of Veterinary Medicine, Taejon, South Korea
Porcine epidemic diarrhea virus (PEDV) infects swine intestinal cells causing enteric disease. Research has shown that the
entry into these cells is through porcine aminopeptidase N (pAPN) receptor. To gain insights into mechanisms of PEDVpAPN interactions, the present study aimed at identifying the domain that is critical for PEDV binding. To this end, NIH3T3
cell lines constitutively expressing pAPN or pAPN mutants were generated. The mutants were; domain VII deletion mutant
and domains IV-VI deletion mutant. In the latter, domain VII was linked to the transmembrane segment through domain III.
Results showed PEDV infection was restricted to pAPN and pAPN domain VII expressing NIH3T3 cells. Further, reducing
PEDV titre 10 fold resulted in 37.8% decrease in foci indicating positive correlation. A time course test at 12, 24, 36, 48 and
60 hr showed that foci increased 6 fold in the overall time range. Also, PEDV harvested from pAPN or domain VII expressing
NIH3T3 cells was induced indirect plaques in Vero cells confirming successful entry and replication. Collectively, our results
demonstrate that PEDV recognizes pAPN and that the main interactive point is lodged within domain VII of the pAPN. These
findings are important for therapeutic development as well as creating a platform for future studies on PEDV.
Corresponding author:
Hyun-Jin Shin
[email protected]
Figure 1
Figure 2
Virus Receptors and Entry
P02
Binding of murine norovirus to histo-blood group antigen impacts infection in cell culture
H. Wegener1,2, A. Mallagaray3, T. Schöne3, J. Lockhauserbäumer4, C. Uetrecht4, T. Peters3, S. Taube1
1University
of Lübeck, Institute of Virology and Cell Biology, Lübeck, Germany
of Lübeck, Graduate School for Computing in Medicine and Life Sciences, Lübeck, Germany
3University of Lübeck, Institute of Virology and Cell Biology, Lübeck, Germany
4Heinrich Pette Institute, Dynamics of Viral Structures, Hamburg, Germany
2University
Human Norovirus (HuNoV) infections are the worldwide leading cause of viral gastroenteritis. Successful infection of HuNoV
is linked to the presence of specific histo-blood group antigens (HBGAs) in the host. Binding to HBGAs is mediated by the
protruding (P) domain of the major capsid protein and the choice of a specific HBGA varies between genogroups and
individual strains. Additional carbohydrates, such as gangliosides and milk oligosaccharides have recently been shown to
interact with the HuNoV capsid. The precise mechanism, how binding to distinct carbohydrates determines the fate of
infection is still unclear, particularly because of the inability to efficiently culture HuNoV.
Although advances have been made in the establishment of a HuNoV cell culture system murine noroviruses (MNV) still
prove to be advantageous for studying infection by offering a robust cell culture system, a reverse genetic system and a
natural small animal host. For MNV it was shown that they also bind gangliosides, which impacts attachment and
dissemination in the host. However, a possible role for HBGA has not been reported.
To address this, we investigated HBGA binding of a MNV using saturation transfer difference nuclear magnetic resonance
(STD NMR) spectroscopy and native mass spectrometry (MS). Our data show that MNV binds to fucose and B antigen as
well gangliosides in a very similar manner as HuNoV. Pre-treatment of MNV with soluble fucose or B antigen but not GM3 or
galactose increased infectivity in cell culture.
We therefore conclude that the HBGA binding patterns of MNV and HuNoV are similar and that the role of HBGAs in
norovirus binding and infection can be investigated using MNV as model.
Corresponding author:
Henrik Wegener
[email protected]
Virus Receptors and Entry
P03
Flavivirus E protein stem interactions in virus entry
I. Medits1, F. X. Heinz1, K. Stiasny1
1
Medical University of Vienna, Department of Virology, Vienna, Austria
Introduction: Flaviviruses enter cells by receptor-mediated endocytosis. After virus uptake, the viral envelope protein E
mediates fusion of the viral and the endosomal membrane, triggered by the slightly acidic pH in endosomes. The current
fusion model is based on atomic structures of truncated forms of the E protein in their dimeric pre- and trimeric post-fusion
conformation. These structures lack the two transmembrane-domains and the so-called stem-region. The stem connects the
ectodomain and the membrane anchor and is hypothesized to be essential for fusion by zippering along the trimer core
during the conformational changes of E necessary for fusion.
Objectives: Since stem interactions are essential for fusion and hypothesized to provide part of the energy required for this
process, we wanted to gain information on these interactions as well as their role in the fusion process by a mutagenesis
approach using tick-borne encephalitis virus (TBEV), a major human pathogenic flavivirus.
Material & Methods: We introduced modifications (point mutations, deletions) into the stem of recombinant E proteins as
well as an infectious clone of TBEV and analyzed their effect on E protein trimerization, trimer stability and infectivity.
Results and Conclusion: We identified important interaction sites between the stem and the trimer core involved in the
stabilization of the post-fusion conformation. In addition, replacing conserved residues in the stem resulted in a strong
reduction in the production of infectious particles. Passaging experiments allowed the identification of resuscitating mutations
which led to a recovery of the viability of mutant viral particles to wildtype levels. Currently, we investigate whether the
observed phenotypes are caused by defects in virus entry, assembly or both processes.
Corresponding author:
Iris Medits
[email protected]
Virus Receptors and Entry
P04
Identification of candidate domains in the canine distemper virus attachment protein crucial for infection
M. Herren1, M. Wyss1, P. Plattet1
1University
of Bern, Division of Experimental Clinical Research, Bern, Switzerland
The so called morbillivirus fusion machinery consists of the Hemagglutinin glycoprotein (H) that is physiologically active as a
dimer of dimers, and of the Fusion protein (F) that is found to be active as a homo-trimer. Those transmembrane proteins
have a membrane proximal stalk domain and a membrane distal head domain. In order to trigger the process fusing host cell
and virus membrane, F has to undergo drastic irreversible conformational changes, to anchor itself into the host lipid bilayer
and later to fuse the membranes. F is bound to H in a regulatory manner before receptor engagement to ensure that F
triggering occurs only when close enough to the host cell.
The H protein head and stalk are interconnected by a putative flexible region in each monomer that probably allows the
heads to move relative to the stalk upon receptor engagement. In addition, the H flexible domain may also control folding of
functional H structures prior to receptor binding. To test this hypothesis, we generated mutants of the putative flexible H
region and determined their surface expression, their fusion triggering activity, their capability to interact with F, as well as
their receptor binding efficacy by functional assays.
Strikingly, although mutations conserving the hydrophobic properties at position I146 had no impact on any Hs bioactivities
(cell surface expression, F-binding, receptor-binding and F-triggering), charged and polar residues selectively inhibited the Ftriggering function.
Based on a tertiary structure prediction model, we are now investigating if hydrophobic residues at position 146 (conserved
among Morbilliviruses) encompassed within the flexible domain, are productively involved in the H stalk internal
reorganization as part of the F triggering signaling.
Shedding insight into amino acid function in the putative flexible H region might not only help understanding the mechanisms
involved in head movements necessary for viral cell entry, but may also support the stabilization of H-tetramers in prereceptor-binding states in order to facilitate crystallization which in turn could act as basis for drug designs.
Corresponding author:
Michael Herren
[email protected]
Virus Receptors and Entry
P05
Identification of functional determinants in the Chikungunya Virus E2 Protein
C. Weber1, E. Berberich1, C. von Rhein1, L. Henss1, E. Hildt1,2, B. Schnierle1
1Paul
Ehrlich Institute, Virology, Langen, Germany
Centre for Infection Research (DZIF), Braunschweig, Germany
2German
Background: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent
arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions, including Europe
and the United States of America. In the past, CHIKV has mainly affected developing countries, but has recently caused
large outbreaks in the Caribbean and Latin America. No treatment or licensed CHIKV vaccine exists.
Methodology/Principal Findings: Here, we have identified determinants in the CHIKV cell-attachment protein E2 that
facilitate cell binding. The extracellular part of the E2 gene is subdivided into the three domains, A, B, and C. These domains
were expressed in E. coli and as Fc-fusion proteins generated from HEK293T cells and used for cell-binding assays.
Domains A and B bound to all cells tested, independently of their permissiveness to CHIKV infection. Domain C did not bind
to cells at all. Furthermore, CHIKV cell entry was promoted by cell-surface glycosaminoglycans (GAGs) and domain B
interacted exclusively with GAG-expressing cells. Domain A also bound, although only moderately, to GAG-deficient cells.
Soluble GAGs were able to inhibit CHIKV infection up to 90%; however, they enhanced the transduction rate of CHIKV Env
pseudotyped vectors in GAG-negative cells.
Conclusion/Significance: These data imply that CHIKV uses at least two mechanisms to enter cells, one GAG-dependent,
via initial attachment through domain B, and the other GAG-independent, via attachment of domain A. These data give
indications that CHIKV uses multiple mechanisms to enter cells and shows the potential of GAGs as lead structures for
developing antiviral drugs.
Corresponding author:
Barbara Schnierle
[email protected]
Virus Receptors and Entry
P06
Establishment of replication-deficient VSVeGFP∆G viruses pseudotyped with Nipah virus glycoproteins
M. Elvert1, O. Dolnik1, L. Sauerhering1, A. Maisner1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Introduction: The Nipah virus Malaysian strain (NiV) is a zoonotic paramyxovirus that originates from fruit bats. During the
first outbreak in 1998, it caused an acute respiratory infection in pigs with a prominent virus replication in the respiratory
epithelium and efficient virus shedding in airway secretions. Humans infected by NiV-positive pigs mostly developed severe
encephalitis with high mortality rates. Less than 30% of the patients also showed flu-like symptoms, suggesting that there are
species-specific differences in the infection of human and porcine airway epithelial cells.
Objectives: As NiV is a BSL-4 classified virus, we aimed to establish replication-deficient VSV pseudotypes carrying the two
NiV glycoproteins F and G (VSV-NiVgp) to study NiV-mediated entry and cell tropism under BSL-1/2 conditions.
Methods: For VSV-NiVgp pseudotype generation, we used a recombinant VSVΔG genome encoding a GFP, and a modified
standard rescue protocol. In this, BHK cells were infected with MVA-T7 and were then transfected with T7-driven plasmids
encoding the VSVeGFPΔG genome and the three helper proteins (VSV-N, -P, -L). In addition, NiV-F and -G were
coexpressed. Undiluted purified cell supernatants containing VSVeGFPΔG virions with incorporated NiV glycoproteins were
then used for the infection of new cell cultures expressing the NiV glycoproteins. Pseudotypes from these cell supernatants
were purified and stock viruses were titrated on Vero cells by counting GFP-positive cells.
Results and Conclusion: In standard VSVΔG rescue protocols, VSV-G is coexpressed during the rescue. VSV-G carrying
pseudotypes are then used to infect cell cultures expressing the foreign glycoproteins. To get rid of the VSV-G containing
pseudotypes, these need to be neutralized by antibodies. As we exclusively used the NiV glycoproteins in our pseudotype
rescue protocol, this critical step of antibody neutralization could be avoided. With this modified experimental setup, we could
generate VSV-G free VSV-NiVgp pseudotype stocks with titers about 106/ml. Studies to analyze viral entry and cell tropism
of the VSV-NiVgp pseudotypes in differentiated respiratory epithelial cells from pigs and humans were recently started.
Corresponding author:
Mareike Elvert
[email protected]
Virus Receptors and Entry
P07
Native mass spectrometry analysis of norovirus - glycan mimetics interaction
H. Yan1, K. Bücher2, L. Hartmann2, R. Creutznacher3, A. Mallagaray3, T. Peters3, C. Uetrecht1,4
1Heinrich
Pette Institute, Dynamics of Viral Structures, Hamburg, Germany
Heine University Dusseldorf, Institute of Organic and Macromolecular Chemistry, Dusseldorf, Germany
3University of Lübeck, Institute of Virology and Cell Biology and Institute of Chemistry, Lübeck, Germany
4European XFEL GmbH, Hamburg, Germany
2Heinrich
Introduction: Human noroviruses (HuNoV) are non-enveloped RNA viruses belonging to the Caliciviridae family and being
the main cause of viral gastroenteritis. From crystallographic structures, it is apparent that the norovirus capsid protein
protruding (P) domain binds to glycans on the cell to mediate cell attachment [3]. Owing to the soft electrospray ionization
technique, native mass spectrometry (MS) enables characterization of non-covalent protein-glycan ligand interactions in a
single experiment without the need for titrations [6]. Whereas binding affinity to individual glycans is low, multivalency on the
cell surface supposedly results on strong avidity. To investigate the impact of multivalency on viral cell entry, binding of
multivalent glycan mimetics, linear oligomer chains with monosaccharides attached [7], to P domains is analyzed by native
MS.
Experiments: Different combinations of glycan mimetics and HuNoV strains were tested in native MS. Multivalent glycan
mimetics resembling the high density of glycans on cell surfaces were used for binding to P domain to characterize binding
affinity and avidity. The results are compared to binding of natural monovalent glycan ligands to elucidate how multivalency
impacts cell attachment.
Results: The results will elucidate binding profiles and dissociation constants of distinct noroviruses to glycan mimetics. This
will also explore whether glycan mimetics have potential as antivirals. Preliminary data show similar affinity for the first
binding event from single sugar to multi-valent glycan mimetics indicating that further adaptions are required to study avidity.
An introduction to native MS for non-covalent interaction study and latest results for the next generations of glycan mimetics
will be presented.
Keywords: Native mass spectrometry, human noroviruses, glycan-mimetics, P domain, binding interactions.
References:
[1] Mallagaray A, et al., (2015). Angew. Chem. 54:12014 –12019.
[2] El Hawiet A, et al., (2012). Anal. Chem. 84:50–58.
[3] Cao S, et al., (2007). J. Virol. 81:5949–5957.
[4] Prasad BV V, et al., (1999). Science 286:287–290.
[5] van den Heuvel RH and Heck AJ, (2004). Curr. Op. Chem. Biol. 8(5): 519 – 526.
[6] Uetrecht, C, & Heck, AJ, (2011). Angew. Chem. 50: 8248-8262.
[7] Ponader, D., (2012) Biomacromolecules. 13 (6): 845–18.
Corresponding author:
Hao Yan
[email protected]
Virus Receptors and Entry
P08
The autophagic pathway in Uukuniemi virus infection
A. Hoffmann1, H. Fleckenstein1, M. Simon1, S. Blobner1, V. Lang2, N. Brady3, P. Y. Lozach1
1University
Hospital Heidelberg, Infectious Diseases, Heidelberg, Germany
Cancer Research Center, Heidelberg, Germany
3Johns Hopkins University, Baltimore, United States
2German
Bunyaviridae is the largest family of RNA viruses, infecting a wide range of hosts including plants, vertebrates and
invertebrates worldwide. Many members cause severe disease with high mortality rates in life stock and humans such as
hepatitis, encephalitis and hemorrhagic fever. Currently no specific treatment is approved for human use. With a global
increase in outbreaks bunyaviruses represent a growing threat to public health and agricultural productivity.
Perferably, bunyavirus infection is prevented by targeting early virus-host cell interactions prior to genome release.
Bunyaviruses are late-penetrating viruses, a group of viruses that rely on late endosomal compartments for productive
penetration. To reach those late endosomal compartments Uukuniemi virus (UUKV), a prototype bunyavirus, relies on Rab5+
early endosomes while Rab7, a small GTPase critical for maturation of late endosomes, is not required. A genome wide
siRNA screen performed in our lab identified VAMP3 as a novel host cellular factor required for late penetration of UUKV.
Among other functions, VAMP3 was proposed to mediate initiation of autophagy and fusion between multivesicular bodies
and autophagosomes, thereby bridging the autophagosomal and enodcytic pathway. Interestingly autophagy related genes
WIPI1, Rab1b and FIP200 were also found in the list of potential hits from the siRNA screen. Therefore we hypothesize an
involvement of autophagy in UUKV infection.
To investigate the role of autophagy in UUKV infection, siRNAs and autophagy-deficient cell lines are used in FACS-based
infection assays and an autophagy-indicating cell line is used in microscopy-based assays.
Thus far we could demonstrate that silencing of Rab1b and key autophagy proteins Atg5 and Atg7 blocks UUKV infection.
Rab11a, proposed to regulate fusion between multivesicular bodies and autophagosoms, is also required for UUKV infection.
In addition fluorescently labeled UUKV particles localize to LC3-II positive vesicles, suggesting that UUVK enters
autophagosomes at some stage during the viral entry process.
In the context of previous findings, autophagosomes could display an alternative pathway for UUKV to reach acidic
endosomal compartments. Together, our results will provide a handle to develop specific treatments against emerging
pathogenic bunyaviruses.
Corresponding author:
Anja Hoffmann
[email protected]
Virus Receptors and Entry
P09
Monitoring Measles Virus transmission through a 3D tissue model of human airway mucosa
S. Derakhshani1, M. Steinke2, E. Avota1, S. Schneider-Schaulies1
1Julius
Maximilians University Würzburg, Würzburg, Germany
Hospital Würzburg, Würzburg, Germany
2University
Introduction: Parameters important in Measles Virus (MV) transmission from dendritic cells (DCs) to acceptor epithelial cells
such as cytoskeletal components or receptor clustering were determined in 2D systems. However, it remains unclear
whether and to what extent these also apply in a more complex enviroment such as the respiratory tract epithelium.
Objectives: Molecular mechanisms, structures and subcellular components essential for MV transmission from infected DCs
to epithelial cells will be analyzed in a 3D test system of the airway mucosa with human tracheobronchial epithelial cells
(hTEC), fibroblasts and, later on, an endothelial cell layer seeded on a clinically implemented biological scaffold with high in
vitro/in vivo correlation.
Material and Method: For 3D tissue model generation, fibroblasts are seeded apically on a scaffold derived from
decellularized porcine jejunal segment, which only consist the small intestinale submucosa (SIS- )[1]. hTEC were
subsequently added and the whole model was cultured under airlift condition.
DCs generated from monocytes were infected with recombinant MV (IC323-eGFP) for 24 h and were applied on the
basolateral surface of the collagen scaffold (by flipping over the model for 2 h), and spread of the infection was monitored by
flourescense microscopy.
Results: Barrier integrity of the models (70 – 90%) was confirmed 3 weeks of airlift conditions by FITC-dextran assay. After
24 h incubation with infected DCs, fluorescence microscopy was performed to monitor the DCs migration through the model
from basolateral to the apical compartment of the 3D tissue model. The GFP+ cells were identified at within the upper layer
of the model representing either transmigrated DCs or infected epithelial cells. A deteiled analysis of the spatial cellular
composition of the infected models is currently performed by immunocytochemistry.
Conclusion: Establishing this 3D model involving immune cells together with MV could give us a better insight for studying
the components involved in cellular MV transmission to target cells. In the current study we showed propagation of MV
through the airway 3D model creating a suitable condition for studying receptors, membrane domains and components
involved in the transmission process.
References:
[1] Steinke M et al. An engineered 3D human airway mucosa model based on an SIS scaffold (2014)
10.1016/j.biomaterials.2014.05.031.
Corresponding author:
Shaghayegh Derakhshani
[email protected]
Virus Receptors and Entry
P10
Coding and non-coding SCARB1 variants modulate hepatitis C virus replication cycle as well as the clinical features
of hepatitis C
S. Westhaus1,2,3, M. Deest2,4,3, A. T. X. Nguyen2,4,3, F. Stanke5,6, D. Heckl7, R. Costa2,4,3, A. Schambach8, M. P. Manns2,3, T.
Berg9, F. Vondran3,10, C. Sarrazin11,12,13, S. Ciesek1,2,3, T. von Hahn2,4,3
1University
Duisburg-Essen, University Hospital Essen, Insitute for Virology, Essen, Germany
Medical School, Department of Gastroenterology, Hepatology und Endocrinology, Hanover, Germany
3German Center for Infection Research (DZIF), Hanover, Germany
4Hanover Medical School, Institute of Molecular Biology, Hanover, Germany
5Hanover Medical School, Clinic for Pediatric Pneumology and Neonatology, Hanover, Germany
6German Center for Lung Research (DLZ), Partner sSite BREATH, Hanover, Germany
7Hanover Medical School, Department for Pediatric Hematology and Oncology, Hanover, Germany
8Hanover Medical School, Department Experimental Hematology, Hanover, Germany
9University of Leipzig, Department of Gastroenterology and Rheumatology , Leipzig, Germany
10Hanover Medical School, Department of Visceral and Transplant Surgery, Hanover, Germany
11University Hospital Frankfurt, Medical Clinic I, Frankfurt a.M., Germany
12St. Josefs-Hospital, Medizinische Klinik II, Wiesbaden, Germany
13German Centre for Infection Research (DZIF), Frankfurt a.M., Germany
2Hanover
Question: Scavenger receptor class B type I (SR-BI) is multiligand receptor that has a central role in lipid homeostasis of the
liver and is one essential hepatitis C virus (HCV) entry factor. SR-BI is encoded by the Scarb1 gene and a number of single
nucleotide polymorphisms (SNPs) associated with clinical phenotypes affecting serum lipid levels has been described. But
their impact on HCV replication cycle is incomplete characterised.
Methods: In this study the impact of both coding and non-coding SCARB1 variants on HCV infection was aimed to be
analysed. The generation of a SR-BI negative hepatoma cell line by CRISPR/Cas method (7.5/TS4-B2) allowed us to test
five known coding non-synonymous SNPs for their ability to bind HCV and mediate viral entry. In a genetic association study
four common synonymous SNPs reported to be associated with a clinical phenotype were analysed using a well
characterized cohort of chronically HCV infected patients. Furthermore, tissue from liver resections was collected and SR-BI
expression was analysed by immunoblot and correlated to SCARB1 genotype.
Results: Reduced HCV infectivity was observed in 7.5/TS4-B2 cells expressing the non-synonymous SNPs S112F and
T175A while the remaining coding variants (G2S, V135I, P297S) showed comparable infectivity to wildtype SR-BI. Moreover,
binding of soluble E2 was markedly reduced in cells expressing S112F and T175A. Although SR-BI protein expression was
below of other overexpressed variants it was above endogenous levels in parental Huh-7.5 and cell surface expression for all
SNPs was detectable. Genetic association studies for the four non-coding variants showed that homozygosity for the G allele
of SNP rs3782287 was association with significantly lower viral load. Furthermore, a sub-significant trend towards lower SRBI expression in liver tissue in GG homozygotes and viral load was observed. Haplotype analysis confirmed these findings
and identified haplotype blocks that predict treatment response and virus load.
Conclusion: We found that both coding and non-coding genetic variations of Scarb1 can affect HCV replication cycle:
common synonymous SNPs affect viral and rare coding variants may be associated with cellular resistance to HCV cell
entry. These findings underscore the relevance of SR-BI as an HCV receptor in the human host and may help to better
understand inter-individual variation in clinical course of HCV infection.
Corresponding author:
Sandra Westhaus
[email protected]
Virus Receptors and Entry
P11
Zika virus entry into permissive cells
K. Kosowicz1, K. Pyrć1
1Jagiellonian
University, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, Poland
Introduction: Zika virus (ZIKV), a mosquitoborne flavivirus known to infect humans for over 50 years, has recently come in
the spotlight due to massive outbreaks in Micronesia, South Pacific Islands and South America. It has been estimated that
since the first reported case in South America in May 2015 the total number of infections in humans in this continent could
have reached 1 300 000. The rapid spread and neuropathologic complications, associated with ZIKV infections, indicate a
need for research on the biology of infection followed by the development of a specific therapy.
Objectives: The main goal of this study was to elucidate the ZIKV entry route into permissive cells. Thorough understanding
of the process enables the identification of possible infection inhibitors. This concerns both the well-known agents specific for
the indicated cellular uptake route as well as development of novel inhibitors of ZIKV.
Material & Methods: All experiments were conducted on simian fibroblast-like kidney cell line Vero, permissive for ZIKV.
Interaction between ZIKV H/PF/2013 strain and cellular proteins was studied by confocal microscopy. The results were
confirmed by the use of specific inibitors of endocytic pathways and siRNA. Virus replication was scored by means of viral
RNA yield using RT-qPCR.
Results: We first showed using specific inhibitors that ZIKV requires internalization and endosome acidification prior to
infectious entry. Using confocal microscopy and small interfering RNAs we have observed both virus co-localization with
clathrin and its accumulation on the cell surface in the clathrin-depleted cells. The infection rate was greatly reduced by
amantadine and pitstop 1, two inhibtors of clathrin-mediated endocytosis.
Conclusion: Here, we report that ZIKV enters susceptible cells via clathrin-dependent endocytosis, which is a common way
exploited by several other viruses from this genus including West Nile virus, Kunjin virus, dengue virus and yellow fever virus.
The internalization process is heavily dependent on low pH in the endosomal compartment.
Figure 1: NH4Cl inhibition of ZIKV entry into Vero cells. The virus is stained with Alexa 488 dye in green, actin with Atto 633
in red and nuclei with DAPI. Panel A represents PBS-pretreated ZIKV-infected control cells, Panel B NH4Cl-pretreated ZIKVinfected cells.
Corresponding author:
Katarzyna Kosowicz
[email protected]
Figure 1
Figure 2
Virus Receptors and Entry
P12
Identification of potential receptor binding sites on glycoprotein O of the human cytomegalovirus (HCMV) by charge
cluster to alanine scanning
C. Stegmann1, F. Rothemund2, B. Adler3, C. Sinzger1
1Ulm
University Medical Center, Institute of Virology, Ulm, Germany
University Medical Center, Institute of Medical Microbiology and Hygiene, Ulm, Germany
3Ludwig Maximilian University Munich and Max von Pettenkofer Institute of Virology, Munich, Germany
2Ulm
The HCMV glycoprotein complex gH/gL/gO is required for infection with cell free virions. It was recently shown that gO
interacts with the platelet derived growth factor receptor alpha (PDGFR-alpha) on the surface of fibroblasts, and this
interaction is necessary for efficient infection. gO is highly polymorphic, with only 50 % conservation among HCMV strains.
We hypothesized that the interaction with PDGFR-alpha is mediated by conserved peptide sites on the surface of the
protein. To identify such interaction sites we targeted all conserved charge clusters of gO by a series of 21 mutations on the
background of a Gaussia luciferase-expressing reporter virus. The mutant viruses were reconstituted in fibroblasts and the
kinetics of virus spread was monitored by measuring luciferase secretion over time. To test whether mutants could still form
the gH/gL/gO complex, lysates of infected cells were analyzed by western blotting. To screen for potential interaction sites
we tested which of the mutations reduced sensitivity to inhibition by soluble PDGFR-alpha. All 21 mutants could be
reconstituted. Two mutants were severely impaired with 50 % reduced spreading rates, which was explained by a defect in
formation of the gH/gL/gO complex. Eleven mutants were moderately affected with 20-30 % slower virus growth, while seven
mutants were phenotypically indistinguishable from wildtype and one mutation led to 20 % enhanced virus spread. Regarding
inhibition of infection by a soluble PDGFR-alpha-Fc chimera, at least two of the moderately affected mutants displayed partial
resistance, indicating that binding of the soluble receptor is impaired. Remarkably, these two charge clusters are both located
within the N-terminus of the protein. In conclusion, our data obtained with a mutational scanning approach suggest
conserved charge clusters within the highly polymorphic N-terminus of gO as a potential site of interaction with the cellular
HCMV receptor PDGFR-alpha.
Corresponding author:
Cora Stegmann
[email protected]
Virus Receptors and Entry
P13
Characterization of SH-SY5Y, a non-lymphocytic cell line resistant to filovirus cell entry
F. J. Zapatero Belinchon1,2, E. Dietzel3, O. Dolnik3, K. Döhner4, R. Marques da Costa1,5,2, M. P. Manns2, S. Ciesek5, B.
Sodeik4, S. Becker3, T. von Hahn1,2
1Hanover
Medical School, Institute of Molecular Biology, Hanover, Germany
Medical School, Department of Gastroenterology, Hepatology und Endocrinology, Hanover, Germany
3Phillips University Marburg, Institute of Virology, Marburg, Germany
4Hanover Medical School, Institute of Virology, Hanover, Germany
5University Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
2Hanover
Ebola is a prototypical dead-end zoonotic infection. Nonetheless during the 2014-2016 West Africa outbreak it exhibited the
potential to cause large lengthy outbreaks and adapt to the human host. Filoviruses are widely regarded to be pantropic in
that they are able to infect a broad range of tissues with the exception being lymphocytes and lymphocyte-derived cell lines
that are resistant to filovirus cell entry.
We have Identified, through a filovirus glycoprotein pseudotype susceptibility cell screening, a novel non-lymphocytic
neuroblastoma cell line, SH-SY5Y, that is refractory not only to GP-driven transduction, but also to infection by chimeric
rVSV-EBOV-GP and authentic filoviruses. We then set out to analyze a panel of filovirus resistant and permissive cell lines
with the aim to understand the observed block to filovirus cell entry. Cluster analysis of gene array data exhibited no
correlation between reported filovirus entry factors and the tested cell lines' susceptibility to filoviral entry. With regard to
expression and functionality of known intracellular factors involved in filovirus entry (NPC1, TPC1/2, cathpesin B & L) SHSY5Y were not different from highly permissive cell lines used for comparison. Likewise, expression of reported cell surface
entry factors (TIM1, FOLR, Axl, ITGA5, ITGB1) was comparable between SH5Y and some highly permissive cell lines.
SH-SY5Y, a neuroblastoma cell line often used for neuronal function and differentiation studies, is refractory to filoviral cell
entry. We propose that it is by either lacking a currently unknown critical entry factor or by expressing a dominant restriction
factor with antifiloviral activity. Currently, heterokaryon formation experiments to differentiate between these possibilities are
ongoing.
Corresponding author:
Francisco Jose Zapatero Belinchon
[email protected]
Virus Receptors and Entry
P14
The cytoplasmic domain of the G protein of Kumasi virus, a novel member of the genus Henipavirus, determines
host species-specific functional activity of Kumasi virus surface glycoproteins
K. Voigt1, M. Hoffmann2, M. Müller3, J. F. Drexler3, C. Drosten3, G. Herrler1, N. Krüger1
1University
of Veterinary Medicine Hanover, Institute of Virology, Hanover, Germany
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
3University of Bonn Medical Centre, Institute of Virology, Bonn, Germany
2German
Hendra (HeV) and Nipah virus (NiV), two zoonotic henipaviruses, can lead to severe and fatal infections in humans and other
mammals, whereas infected bats do not show any symptoms. Previously it was assumed that the occurrence of
henipaviruses is restricted to regions of South East Asia (NiV) and Australia (HeV). In 2009, viral RNA of a novel henipavirus
(BatPV/Eid_hel/GH-M74a/GHA/2009 (Kumasi virus)) has been detected in the spleen of an African flying fox of the species
Eidolon helvum.[1] So far, the zoonotic potential of the Kumasi virus is unknown.
We recently showed that functional activity of the Kumasi virus fusion (F) and attachment glycoprotein (G), indicated by cellto cell fusion, was observed in bat cells but not in cells of other mammalian species. Moreover, an inefficient transport of
Kumasi virus G to the cell surface of non-bat cells has been linked to this restricted fusion activity. [2, 3]
In the present study, we aimed to resolve molecular factors that limit functional activity of Kumasi virus surface glycoproteins
to bat cells and might be determinants of the zoonotic potential of henipaviruses.
Here, we generated truncated versions of Kumasi virus G in which amino acid residues of the cytoplasmic domain have been
deleted. To analyse functional activity and host specificity, those proteins were subjected to fusion assays.
In contrast to the parental Kumasi virus G, the truncated protein was able to induce cell-to-cell fusion not only in bat cells but
also in cell lines of other mammalian species. However, the expression pattern of the truncated G protein did not differ from
the full-length G protein, indicating that neither expression level nor localization are responsible for this effect.
Our results suggest that the truncation in the cytoplasmic domain of Kumasi virus G enables a more efficient F and G
interaction, which is required for the triggering of the non-fusogenic F protein to transit into the fusion-active form.
References:
[1] Drexler et al., 2012.
[2] Krüger et al., 2013.
[3] Krüger et al., 2014.
Corresponding author:
Kathleen Voigt
[email protected]
Virus Receptors and Entry
P15
Analysis of herpes simplex virus type 1 glycoprotein L and D chimeras
A. Hofemeier1, S. Imdahl1, N. Kirschnick1, M. L. Romberg1, W. Hafezi1, J. Kühn1
1University
of Münster, Institute of Virology, Münster, Germany
Membrane fusion by herpes simplex virus type 1 (HSV-1) requires four essential glycoproteins: glycoprotein D (gD), L (gL), H
(gH) and B (gB). Binding of gD to one of its receptors, such as nectin-1 or herpesvirus entry mediator (HVEM, TNFRSF14),
results in the activation of the gH/gL heterodimer via the so-called profusion domain of gD. The signal from gH/gL is
transmitted to gB, which finally executes fusion. At present, the precise mechanism of membrane fusion induced by HSV-1 is
not fully understood.
Aim of the present study was to characterize triggering of membrane fusion by interaction of gD and gH/gL using chimeric
constructs consisting of gL and various domains of gD. These constructs were co-transfected with gB and gH expression
plasmids. The fusion activity was measured in stable Vero reporter cells expressing SEAP upon cell-cell fusion or CHO K1
cells transiently expressing SEAP reporter constructs and HSV-1 entry receptors, respectively.
A chimeric protein consisting of full length gL and the mature form of gD termed gLgD mediated cell fusion in Vero and CHO
cells at levels comparable to wild-type gL and gD. In contrast, a secreted chimera (gLsecgD) lacking the transmembraneand cytodomain of gD, was unable to induce cell fusion in Vero cells. However, gLsecgD still showed fusion ability in CHO
cells (approx. 50% of wt control). Furthermore, using the conformation sensitive gH antibody LP11, gLgD as well as gLsecgD
were shown to induce correct gH folding and cell surface expression. Interestingly, the gLsecgD260 chimera, truncated at
position 260 of gD, which lacks the profusion domain of gD, still showed residual yet significant fusion activity in CHO cells
overexpressing HVEM. Cell fusion induced by gLsecgD260 was not observed in CHO cells overexpressing nectin-1.
In conclusion, the divergent functions of gL and gD can be combined in a single multidomain protein. A chimeric construct
consisting of full length gL and the mature form of gD is able to functionally replace gL and gD in membrane fusion. Chimeric
constructs containing truncated forms of the gD portion, however, reveal remarkable, so far undescribed differences to wt gD
function. In the context of gLgD chimeras, the profusion domain of gD as well as membrane anchoring are no longer
essential for activation of gH and initiation of membrane fusion.
Corresponding author:
Joachim Kühn
[email protected]
Virus Receptors and Entry
P16
Entry determinants of the human endogenous retrovirus HERV-K(HML-2)
J. Wamara1,2, H. Al-Shehabi1, A. Richter1, N. Bannert1
1Robert
2Berlin
Koch Institute, Division for HIV and other Retroviruses, Department of Infectious Diseases, Berlin, Germany
University of Technology, Department of Applied and Molecular Microbiology, Institute of Biotechnology, Berlin, Germany
Introduction: Endogenous retroviruses are present in all vertebrates including humans and are commonly associated with
neurological disorders like ALS or multiple sclerosis and cancer. They endogenized into the genome of their hosts' cells
millions of years ago by infecting germline cells thus being able to be passed to the progeny. Although most of them have
been inactivated by post-insertional mutations, some proviruses of the HERV-K(HML-2) family remain largely intact and viral
particles are produced.
Objectives: We aimed to shed light on determinants of the viral entry of HML-2. Along these lines we have previously shown
that a D139A and Δ144-152 mutation in the Env protein allows regular protein expression and particle incorporation but
prevents entry.
Material & Methods: CrFK and SK-Mel 28 (NCI-60 panel) serve as HML-2 susceptible cells. The latter was kindly provided
by M. Schindler. Hep2 were used as HML-2-resistant cells. Virus-like particles (VLP) were produced in HEK 293Tcells and
concentrated by ultracentrifugation. Infections were carried out with normalized virus.
Mutations were made with sequence specific primers and the mutagenesis-PCR reaction was performed with PCR or
Mutagenesis Kits (Thermo Fisher or Agilent) according manufacturer's protocols.
Results: Here we report that two nearby N-glycosylation sites are necessary for the formation of the primary virus-receptor
complex that is essential for viral entry. Indeed a single mutation of the residues, N128A and N153A, respectively, was
sufficient to suppress the infectivity of VLP carrying these env mutants. Similarly, a mutation of the glutamic acid repeat
146EEE148 into 146AAA148 prevents an infection of susceptible cells with the corresponding VLP indicating a functional and/or
structural significance of the short single amino acid hydrophilic stretch.
Conclusion: These data are in agreement with the well-known contribution of specific amino acid residues and repeats to
protein structure and functions. It appears that the analyzed hydrophilic residues in this study may stabilized the structure of
the HML-2-Env at domain level and provide a specific conformation, which in turn allows the interaction of the virus with
target cell surface protein(s). The analyzed region is likely to function as a major receptor binding site (RBS) of the envelope
protein of HERV-K(HML-2).
Corresponding author:
Jula Wamara
[email protected]
Virus Receptors and Entry
P17
Functional relevance of the N-terminal domain of pseudorabies virus envelope glycoprotein H and its interactions
with glycoprotein L
M. Vallbracht1, S. Rehwaldt1, B. G. Klupp1, T. C. Mettenleiter1, W. Fuchs1
1Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
Membrane fusion is crucial for infectious entry and spread of herpesviruses. This process is mediated by the conserved viral
fusion glycoprotein (g)B, the presumably gB activating conserved heterodimeric gH/gL complex and receptor binding proteins
like alphaherpesvirus gD. In contrast to most viral fusion proteins, gB is not able to mediate membrane fusion in the absence
of the gH/gL complex.
Interestingly, while all three proteins are essential for penetration of the alphaherpesvirus pseudorabies virus (PrV), limited
direct cell-to-cell spread occurs in the absence of gL. After extended cell culture passage of gL-negative PrV, phenotypic
revertants were isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH
ectodomain.
The aim of the present study was to investigate the functional relevance of this structurally yet uncharacterized part of PrV
gH. To this end, we introduced an in-frame deletion of 66 codons at the 5 end of the plasmid-cloned PrV gH gene (gH32/98)
and characterized the fusion activity in transient transfection-fusion assays. Additionally, we inserted the engineered gH gene
into the PrV genome, in the presence or absence of wild-type or modified versions of gL, gD and gB. Penetration, plaque
formation, and infectious titers of PrV progeny were investigated.
The results show that deletion of the gL binding domain did not affect expression and processing of gH, but abrogated its
function in in vitro fusion assays. The corresponding PrV recombinant (pPrV-gH32/98K) was incapable of entry and spread.
Interestingly, in vitro fusion activity of mutated gH32/98 was restored when co-expressed with a hyperfusogenic variant of gB
(gBB4.1), obtained from the passaged gL-deletion mutant PrV-DgLPassB4.1. Moreover, the entry and spread defects of pPrVgH32/98K were partly compensated by gBB4.1 in cis, as well as in trans independent of gL.
Our results indicate that gL and the gL-binding domain of PrV gH are not strictly required for membrane fusion during virus
entry and spread, and that compensatory mutations affecting gB restore a fully functional fusion machinery.
Corresponding author:
Melina Vallbracht
[email protected]
Virus Receptors and Entry
P18
Epigallocatechin-3-gallate protects macaques from intrarectal simian/human immunodeficiency virus infection
J. B. Liu1, J. L. Li2, H. Liu1, X. Wang2, Q. H. Xiao1, Q. Y. Xian1, Y. Wang1, X. D. Li1, K. Zhuang1, L. Zhou1, R. H. Zhou1, T. C.
Ma1, Y. Persidsky2, W. Z. Ho2,1
1Wuhan
2Temple
University rsity School of Basic Medical Sciences, Animal Biosafety Level III Laboratory at Center for Animal Experiment, Wuhan, China
Univ., Pathology, Philadelphia, United States
Question: Epigallocatechin-3-gallate (EGCG), a natural and major ingredient of green tea, has been shown by a number of
in vitro studies to potently inhibit HIV-1 infection/replication in PBMC and macrophages. EGCG can also remodel seminal
amyloid fibrils and effectively suppress semen-mediated enhancement of HIV-1 replication, suggesting the potential use of
EGCG for preventing HIV-1 sexual transmission. In the present study, we evaluated the protective effect of EGCG on rectal
SHIV infection in rhesus macaques.
Methods: Sixteen male rhesus macaques were biweekly intrarectally administered either 2 ml of 5 mM EGCG (n=8) or PBS
(n=8) prior to the rectal exposures to 10 TCID50 of SHIVSF162P3N, an extensively used strain in macaque model of sexual
transmission. The EGCG or PBS treatment and viral inoculation were stopped when all the control animals became infected.
The plasma SHIV RNA over the course of study was longitudinally assessed. SHIV RNA and proviral DNA were also
measured in multiple gastrointestinal tissues and lymph nodes biopsied at week 20 and necropsied at the endpoint.
Results: Intrarectal administration of EGCG could protect seven of eight rhesus macaques from repetitive, intrarectal
challenges with low-dose SHIVSF162P3N. All protected animals showed no evidence of systemic and mucosal SHIV
infection as demonstrated by the absence of viral RNA, DNA and antibodies. In contrast, all control animals became infected
after SHIV challenges (a median of 2.5 times, range of 1-8 times, P=0.0009). The critical mechanisms of the EGCGmediated prevention of intrarectal SHIV transmission are its competition with gp120 for CD4 receptor binding and the
suppression of macrophage infiltration and activation in the rectal mucosa.
Conclusion: These data support further clinical evaluation and development of EGCG as a novel, safe and cost-effective
microbicide for preventing sexual transmission of HIV-1.
Corresponding author:
Wen-Zhe Ho
[email protected]
Virus Receptors and Entry
P19
Emerging bat hepatitis B viruses – evidence for co-factors restricting hepadnaviral entry and species-specificity
A. König1, A. Rasche2, A. Geipel1, S. Müller3, J. Geyer3, J. F. Drexler2, D. Glebe1
1Justus
Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
of Bonn Medical Centre, Institute of Virology, Bonn, Germany
3Justus Liebig University Giessen, Institute of Pharmacology and Toxicology, Giessen, Germany
2University
Background and Question. Infections with human hepatitis B virus (HBV) are still a major global threat, despite the availability
of antiviral therapy and a protective vaccine. HBV and related hepadnaviruses are believed to be host- and organ-specific,
due to their interaction with the liver-specific bile acid transporter (hNTCP) of hepatocytes. NTCP serves as the high-affinity
receptor for binding and entry of HBV due to its specific interaction with the N-terminal part of the preS1-domain of large viral
surface protein. However, we recently demonstrated that bat hepadnavirus (BatHBV) species TBHBV from Uroderma
bilobatum, (ub) infects human hepatocytes via hNTCP in vitro, in contrast to RBHBV and HBHBV from bat species
Hipposideros ruber (hr), and Rhinolophus alcyone (ra), respectively. Methods. To investigate the potential interaction of
BatHBV with human and corresponding host specific bat NTCPs we generated HepG2 cell lines stably transfected with h-,
ub-, ra-, or hrNTCP. All cell lines were tested for functional expression of NTCP. Host-dependent binding-specificity of
different BatHBVs to their host-derived NTCPs was analyzed via preS1-peptides binding of HBV, TBHBV, RBHBV and
HBHBV, respectively. Results. NTCPs from each host showed the highest binding capacity for the preS1-peptide derived
from its corresponding hepadnavirus, i.e. hNTCP from human liver showed highest affinity for HBV preS1-peptides.
Surprisingly, infection experiments using HDV particles pseudotyped (ppHDV) with the surface proteins of HBV, or BatHBVs
demonstrated that ppHDV/RBHBV and ppHDV/HBHBV were not able to infect via its host-specific NTCP efficiently. But,
ppHDV/HBV was able to infect efficiently hNTCP-expressing cells, and could surprisingly also use African bat derived
NTCPs (ra/hrNTCP) for infection. A similar result was observed for ppHDV/TBHBV. In conclusion, host-specific NTCPs
showed the highest binding affinity for preS1-peptides derived from its corresponding hepadnavirus, but this did not lead to
an enhanced susceptibility at least in human hepatocytes. These results indicate that additional restricting factor(s) might be
essential for early steps of hepadnaviral infection. These unknown factors could determine host specificity by interaction with
NTCP and/or the virus itself.
Corresponding author:
Dieter Glebe
[email protected]
Virus Receptors and Entry
P20
Receptor-targeted AAV – Improving cell selectivity and identification of cell surface receptors mediating cell entry
J. Hartmann1, F. Höpfner1, J. E. Carette2, H. Büning3, C. J. Buchholz1
1Paul
Ehrlich Institute, Molecular Biotechnology and Gene Therapy, Langen, Germany
University School of Medicine, Department of Microbiology and Immunology, Stanford, United States
3Hanover Medical School, Institute of Experimental Hematology, Hanover, Germany
2Stanford
Introduction: Adeno associated viral (AAV) vectors are increasingly used for gene transfer in basic research and clinical
settings with promising success. Cell entry of AAV is mediated by binding of its capsid to primary and secondary cell surface
receptors like heparan-sulfate proteoglycan (HSPG), integrin α5β1 or the recently identified KIAA0319L (AAVR) (Pillay et al.,
2016; Nature 530, 108-112). Recently, we described retargeting of AAV-2 by mutating the HSPG binding site in the capsid
proteins and simultaneously fusing a designed ankyrin repeat protein (DARPin) with high affinity for Her2/neu to the VP2
capsid protein. EpCAM, CD4 and GluA4 have been targeted with a high selectivity as well (Münch et al., 2015; Nat Comm 6,
6246). However, it is so far unclear if these particles use the target receptor for entry or only as primary attachment site.
Objectives: To gain a better understanding of the receptors involved in cell attachment and entry as well as off-target
transduction of receptor-targeted AAVs, integrin α5β1 and AAVR receptor usage were investigated
Material & Methods: GluA4-targeted AAV-2 vectors were mutated for integrin α5β1 binding and were assessed for entry
into GluA4-positive and -negative cells. The contribution of AAVR was analyzed using AAVR knockout cell lines expressing
GluA4.
Results: There was no reduction in gene transfer activity detectable after mutation of the integrin α5β1 binding site in GluA4AAV vector. Off-target transduction on GluA4-negative cells was, however, reduced 4-fold. The introduced capsid mutations
did not impact transgene packaging, but slightly reduced thermostability. AAVR-negative cell lines were resistant against wild
type AAV2. Interestingly, this was also valid for GluA4-AAV even when these cells expressed high levels of GluA4 which
mediated strong binding of vector particles.
Conclusion: Integrin α5β1 is not required for transduction of GluA4-targeted AAV, but seems to be responsible for
unspecific off-target transduction. On the other hand, AAVR is crucial for AAV entry and cannot be bypassed by altering the
receptor usage, at least not for GluA4.
Corresponding author:
Jessica Hartmann
[email protected]
Virus Receptors and Entry
P21
Arenavirus trVLPs as tools to study antibody neutralization
A. Leske1, K. Schnepel1,2, E. C. Dunham3, K. Shifflett3, A. Watt3, T. Hoenen3,4, A. Groseth1,3
1Friedrich
Loeffler Institute, Junior Research Group Arenavirus Biology, Greifswald - Isle of Riems, Germany
Moritz Arndt University Greifswald, Institute of Biochemistry, Greifswald, Germany
3National Institutes of Health, Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, Hamilton, MT,
United States
4Friedrich Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
2Ernst
Arenaviruses are responsible for several viral hemorrhagic fevers endemic to Africa and South America, and their causative
agents are classified as biosafety level (BSL) 4 agents. Unlike for other BSL4 agents, for the New World arenavirus Junín
virus (JUNV) a highly effective vaccination as well as a post-exposure treatment based on passive transfer of convalescent
serum are available. The role of neutralizing antibodies as the key protective determinant in JUNV infection is supported both
by the use of neutralizing antibody (nAb) response as a marker for protection following vaccination and the direct correlation
between the success of passive antibody therapy and the levels of nAbs administered.
Unfortunately, similar resources for the management of other New World arenavirus infections (i.e. those due to Machupo or
Guanarito virus) are not available. As such it was of interest to determine the extent to which nAbs against JUNV would also
be capable of targeting other related New World arenaviruses. However, the classification of many of these viruses as BSL4
agents poses significant challenges. Our goal was to use a recently established JUNV transcription and replicationcompetent virus-like particle (trVLP) system to quantify neutralization of a range of arenavirus glycoproteins (GPs) under
BSL1 conditions.
To this end JUNV trVLPs, which are composed of minigenomes (genome analogues in which the viral genes are replaced
with one or more reporter genes, e.g. a luciferase gene), the viral polymerase complex proteins (NP and L) and the viral
proteins responsible for budding and entry (Z and GP), were generated. The GP gene was then replaced with that of various
other JUNV species, as well as related Clade B arenaviruses (e.g. Tacaribe, Machupo, Guanarito, Sabiá). The resulting
trVLPs were incubated with serum samples containing anti-JUNV nAb and the reduction in infectivity was quantified.
Unfortunately, while nAbs against the vaccine strain are unsurprisingly active against a range of other JUNV strains, it
appears that nAbs against JUNV may have limited efficacy against other New World arenaviruses. In the future, this platform
will provide a rapid and easy means to help identify the targets of antibody neutralization.
Corresponding author:
Allison Groseth
[email protected]
Virus Receptors and Entry
P22
Phenotypic characterization of substitutions in the hemagglutinin of the 1968 pandemic influenza virus
J. West1, J. Baumann1, J. Röder1, J. Jansen1, J. Doedt1, N. Mounogou Kouassi1, H. D. Klenk1, M. Matrosovich1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Introduction: The hemagglutinin (HA) of the H3N2/1968 pandemic influenza virus was derived from an unknown ancestor
most similar to viruses isolated from ducks in Asia. Eight amino acid substitutions separated the HA of the human pandemic
viruses from HAs ofthe closest avian viruses (F-2L in the signal peptide; R62I, D81N, N92K, A144G N193S, Q226L and
G228S in the mature protein). Substitutions Q226L and G228S are well known to be essential for avian-to-human adaption;
they enabled virus binding to 6-linked sialic acid receptors in humans. Our previous studies suggested that some of the other
6 substitutions could be instrumental for the emergence of the pandemic virus, however, their phenotypic effects and specific
role in the adaptation remained unknown.
Objectives: We wanted to characterize the effects of 6 single-point substitutions in the HA mentioned above on receptor
binding and membrane fusion properties of the H3N2/1968 pandemic virus.
Methods: We generated 2:6 recombinant PR8-based influenza virus having HA and NA of A/Hong Kong/1/1968 (H3N2) and
its variants differing by amino acid substitutions in the HA. Virus binding to fetuin carrying either 3-linked or 6-linked sialic
acids wasdetermined in a solid-phase receptor-binding assay. Binding avidity ofthe viruses was compared based on their
ability to infect partially desialylated MDCK cells. To compare fusion activity of the viruses, we determined pH optimum of
acid-induced conformational transition of the HA and studied inhibition of viral infection in MDCK cells by the lysomotropic
agent ammonium chloride.
Results: None of 6 studied single-point amino acid substitutions in the HA showed significant effect on viral membrane
fusion activity. Two substitutions, D81N and N193S, significantly reduced binding avidity of the pandemic virus as compared
to the avian precursor.
Conclusion: Our data suggest that no significant changes in membrane fusion activity occurred during avian-to-human
adaptation of the avian HA precursor. By contrast, two amino acid substitutions, D81N and N193S, reduced viral binding
avidity suggestingtheir important role in the adaptation. Studies are in progress to characterize molecular mechanisms by
which these substitutions affect viral receptor binding activity and to assess their effects on viral fitness in humans.
Corresponding author:
Johanna West
[email protected]
Virus Receptors and Entry
P23
The role of LAMP1 binding and pH sensing by the spike complex of Lassa virus
H. Cohen Dvashi1, H. Israeli1, O. Shani1, A. Katz1, R. Diskin1
1Weizmann
Institue of Science, Structural Biology, Rehovot, Israel
Introduction: The primary cellular receptor of Lassa virus (LASV) glycoprotein complex is α-dystroglycan (α-DG) which is
recognized by the surface viral glycoprotein complex, and mediating cell entry through macropinocytosis. To effectively infect
cells, LASV needs to switch in endosomal compartment from a-dystroglycan to a protein termed LAMP1. We recently solved
the crystal structure of GP1 from LASV (Cohen-Dvashi et al., JVI, 2015). This structure revealed a unique cluster of three
histidine residues located on the surface of the protein that are highly conserved among the Old World Arenaviruses.
Objectives: to reveal the role of LASV GP1 histidine triad in the pH sensing and triggering of the spike complex and provide
a mechanistic role for binding LAMP1.
Material & Methods: we employed cell-cell fusion assays, virus-like particles infection assay and biochemical pull-down
assays, to study the effects of mutations in LASV GP1 histidine-triad.
Results: mutating each of the histidine residues to tyrosines reduced LASV GP1 binding to LAMP1 and impaired their
capability to infect cells in the context of the complete complex. Cell fusion assays showed that mutations in this histidine
triad do not hinder pH-induced membrane-fusion. Two of the histidine-triad mutated spikes (H93Y, H230Y) displayed
enhanced fusogenic activity. Mutating His230 to a positively charged arginine significantly lowered the pH needed for
triggering of the spike, indicating that it is a negative regulator of spike triggering.
Conclusion: our results indicate that LAMP1 binding is needed for efficient infection, but not required for membrane fusion
per se. We demonstrated that effective LAMP1 binding requires a positive charge at position 230 in the context of GP1, and
it is likely that LAMP1 donates a negatively charged group for this interaction. Our data suggest that the spikes are initially
primed but arrested during the gradual acidification in the endocytic pathway. After priming, triggering could then be achieved
by either binding to LAMP1 or by sufficiently low and non-physiologic pH.
References
Cohen-Dvashi H, Cohen N, Israeli H, Diskin R. 2015, J Virol
Cohen-Dvashi H, Israeli H, Shani O, Katz A, Diskin R. 2016, J Virol
Corresponding author:
Hadas Cohen Dvashi
[email protected]
Virus Receptors and Entry
P24
Entry of merkel cell polyomavirus into A549 cells
M. Becker1, M. Dominguez1, R. M. Schowalter2, C. B. Buck2, M. Schelhaas1
1University
2National
of Münster, Cell Biology of Virus Infection Unit, ZMBE, Münster, Germany
Cancer Institute, Tumor Virus Molecular Biology Section, Laboratory of Cellular Oncology, Bethesda, United States
Merkel Cell Polyomavirus (MCPyV) is a member of the polyomaviridae and was recently discovered in an aggressive form of
skin cancer known as Merkel cell carcinoma (MCC). Although reports indicate that MCPyV virions are shed from human skin,
the precise cellular tropism of the virus in healthy subjects remains unclear, in particular since MCPyV DNA has also been
detected in samples from a variety of other tissues. MCPyV is also different from other polyomaviruses in that initial
attachment requires sulfated polysaccharides such as heparan sulfates and/or chondroitin sulfates, before the virus engages
sialic acid residues as a co-receptor like other polyomaviruses. To explore the question, whether MCPyV differs significantly
in its entry process from other polyomaviruses, we analyzed the cell biological determinants of MCPyV vectors
(pseudoviruses) into A549 cells, a highly transducable lung carcinoma cell line, in comparison to Simian Virus 40, a well
studied polyomavirus, human papillomavirus type 16, and other control viruses.
Our results indicated that MCPyV enters cells via caveolar/raft mediated endocytosis but not macropinocytosis, clathrinmediated endocytosis or glycosphingolipid enriched carriers. The viruses internalized in small, tight fitting endocytic pits that
led the virus to endosomes and from there to the endoplasmic reticulum (ER). Trafficking required microtubular transport,
acidification of endosomes, and a functional redox environment such as other polyomaviruses. Based on the fact that only
minor amounts of viruses reached the ER and that the majority of viruses appeared to be stuck in endosomal compartments,
we suggest the trafficking from endosomes to the ER is a bottleneck of initial infection of A549 cells.
Corresponding author:
Miriam Becker
[email protected]
Virus Receptors and Entry
P25
Host cell tropism of Lassa virus in the human respiratory tract
H. Müller1, S. K. Fehling1, M. Matrosovich1, T. Strecker1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Lassa virus (LASV), a member of the family Arenaviridae, is a highly pathogenic hemorrhagic fever virus that can cause
severe systemic infections in humans. The natural reservoir of LASV is the natal multimammate mouse Mastomys
natalensis. Infection in humans mainly occurs via exposure to infectious rodent excreta through the respiratory or
gastrointestinal tracts. In this study we analyzed the interaction between LASV and respiratory epithelial cells to advance our
knowledge on the molecular mechanisms underlying LASV replication and host cell tropism in the respiratory tract.
Furthermore, we aimed to determine on which side LASV enters and leaves polarized bronchial epithelial cell layers in order
to understand the mechanism underlying LASV entry and dissemination in the human airway. To this end we established
well-differentiated primary cultures of human bronchial epithelial cells (HBEpC) grown under air-liquid interface conditions
that closely mimic the bronchial epithelium in vivo. Our major findings were: (i) HBEpCs are highly susceptible to LASV
infection. (ii) LASV can infect polarized bronchial epithelial cells via the apical or basolateral membrane, while progeny virus
particles are released predominantly from the apical surface. In vivo, such apical virus shedding from infected bronchial
epithelia might support virus transmission via airway secretions. (iii) Non-ciliated cells, specifically goblet cells and basal
cells, represent the initial target cells. In conclusion, our findings suggest that the bronchial epithelium supports efficient
LASV replication; however, host cell tropism of LASV is likely restricted and limited to non-ciliated cells.
Corresponding author:
Helena Müller
[email protected]
Innate Immunity
P26
Pre-activation with TLR7 agonist accelerates hepatitis B virus clearance in the mouse model
X. Huang1,2, G. Zhang2, J. Liu1, Y. Lin1, J. Hou2, X. Zhang2, M. Lu1
1University
Duisburg-Essen, University Hospital Essen, University Hospital Essen, Institute of Virology, Essen, Germany
of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
2Department
Background: Toll-like receptor (TLR) agonists are able to activate cellular signal pathways and induce the production of
cytokines. Currently, TLR7 agonist has been shown to be a potent antiviral in the animal models and patients with chronic
hepatitis B (CHB). Therefore, activation of TLR7 pathway may promote adaptive immune responses and contribute to HBV
control. Here we examined how TLR7 agonist modulates the host immune system and influence adaptive immunity if applied
in vivo.
Methods: C57BL/6 mice (male, 6-8weeks) received 20μg Imiquimod via tail vein injection. Non-parenchymal liver cells
(NPC) and splenocytes were isolated and analyzed for their phenotypes at different time points. At day 14 after injection,
mice were hydrodynamically injected with 10μg pAAV/HBV1.2 plasmid. Serum HBV markers were determined up to 3 weeks.
The frequencies and phenotypes of immune cell types in the liver and spleen were analyzed. The frequency and functionality
of HBV-specific CD8+ T cells in the liver and spleen were determined by dimer staining and intracellular cytokine staining
after stimulation with HBV peptides. Interferon-γ (IFN-γ) production of αCD3/CD28-stimulated splenocytes was detected by
specific ELISA.
Results: Imiquimod application itself did not induce significant change in the cell composition in the liver and spleen.
However, CD8 T cells from the spleen and NPCs showed a sustainably changed phenotype with up-regulated CD62L
expression within 4 weeks of Imiquimod injection. Compared with controls, the IFN-γ production of spleenocytes from
Imiquimod-treated mice were significantly increased with αCD3/CD28 stimualtion (p
Conclusion: Pretreatment with TLR7 agonist could modulate the host immune status and enhance the HBV-specific T cell
responses which facilitate HBV clearance in the mouse model. Thus, the TLR7 agonist may be used for immunotherapeutic
strategy to treat chronic HBV infection.
Corresponding author:
Xuan Huang
[email protected]
Innate Immunity
P27
APOBEC3-mediated coronavirus restriction through editing and no-editing mechanism
A. Milewska1,2, K. Pyc1,2
1Jagiellonian
2Malopolska
University, Microbiology, Kraków, Poland
Centre of Biotechnology, Kraków, Poland
The Apobec3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like type 3) family of cytidine deaminases inhibit
the mobility of diverse retrotransposons, retroviruses and other viruses. Although initial reports demonstrated that Apobec3
antiviral function is based predominantly on their mutational activity on DNA templates, further studies proved that these
enzymes exploit several mechanism, including editing and non-editing processes. An important finding in 2004 provided
evidence that Apobec3 deaminases are able to mutate HIV RNA genome, what expanded possible Apobec3 substrates to
viruses, which do not replicate through DNA intermediates [REF]. Up to date, no Apobec3-mediated deamination of viral
RNA genome has been shown as a restriction factor against RNA viruses. In the current work, we investigated the Apobec3
antiviral effect against human coronaviruses, using HCoV-NL63 as a model pathogen.
First, using a quantitative PCR we analyzed transcript levels of human Apobec3 genes in human airway epithelium cultures
after HCoV-NL63 infection. The analysis revealed an up-regulation of Apobec3A, -3C, -3DE and -3F in virus-inoculated
cultures. Next, we determined the influence of Apobec3 expression on HCoV-NL63 infection. We found that Apobec3C, -3F
and -3H significantly reduced HCoV-NL63 replication in naturally permissive LLC-Mk2 cells. Although no hypermutation of
viral RNA was observed after series of passages on Apobec3-expressing cells, we found that catalytically inactive
Apobec3C, -3F and -3H mutants were less effective in hampering HCoV-NL63 infection. Also, co-immunoprecipitation
studies showed that Apobec3 proteins exhibiting anticoronaviral activity are able to interact and bind HCoV-NL63
nucleoprotein. Taken together, our results showed that three of the Apobec3 proteins inhibit HCoV-NL63 infection in vitro.
We suggest that this antiviral activity is based on both deaminase dependent and independent mechanisms, such as
generating replication-deficient mutants and interfering in encapsidation of progeny virions.
References:
Bishop, K. N., Holmes, R. K., Sheehy, A. M., and Malim, M. H. (2004) APOBEC-mediated editing of viral RNA, Science 305,
645.
Corresponding author:
Aleksandra Milewska
[email protected]
Innate Immunity
P28
Expression pattern of individual IFN-α subtypes in chronic HIV infection
Y. Li1, B. Sun1, S. Esser2, H. Streeck2, R. Yang1, U. Dittmer3, K. Sutter3
1Chinese
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
Duisburg-Essen, University Hospital Essen, Clinic of Dermatology, Essen, Germany
3University Duisburg-Essen, University Hospital Essen, Virology, Essen, Germany
2University
Introduction: Interferon-α plays an important role in HIV pathogenesis. However, IFN-α is not a single protein but a
collective term for 12 individual IFN-α subtypes. Recent evidence suggests that the different subtypes exhibit very distinct
antiviral and immunomodulatory activities in HIV infection.
Objective: Here we determined the expression profiles of all IFN-α subtypes in HIV-positive patients and their association to
the induction of HIV restriction factors.
Patients & methods: We collected blood samples from ART-treated and treatment-naive HIV-positive patients and
determined the expression levels of different IFN-α subtypes and IFN-induced restriction factors by quantitative RT-PCR.
Results: HIV infection induced the expression of IFN-α RNA, but not IFN-b RNA, and the expression levels correlated with
plasma viral loads in individual patients. Expression levels of all IFN-α subtypes were increased due to HIV infection and
were reduced after successful ART. The IFN-α response was dominated by the most abundantly expressed subtypes 2, 4
and 21. HIV infection affected the magnitude of the IFN-α response, but not the overall pattern of the subtype expression,
except for IFN-α2, which was highly induced by HIV. Virus-induced IFN-α subtype responses correlated with the expression
of HIV restriction factors. In vitro stimulation of PBMCs revealed that the subtypes 6, 14, and 21 were most effective in
inducing expression of HIV restriction factors.
Conclusion: These results indicate that HIV infection induces a specific expression pattern of IFN-α subtypes. Since IFN-α
subtypes differ in their ability to induce expression of HIV restriction factors, this information is important for ongoing studies
on HIV therapy with IFN-α.
Corresponding author:
Kathrin Sutter
[email protected]
Innate Immunity
P29
Establishing robust induced pluripotent stem cell-derived model systems to study hepatitis C virus–host
interactions
A. Schoebel1, K. Roesch1, E. Herker1
1Heinrich
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
Knowledge of the activation and interplay between the hepatitis C virus (HCV) and the host is essential to understanding the
establishment of chronic HCV infection. Human hepatoma cell lines are widely used as an in vitro culture system to study
HCV replication, but numerous metabolic alterations and defective innate immunity hinder the study of virus-host
interactions. Here, we work on the establishment of a robust iPSC-derived hepatocyte model to study the interaction between
HCV and the host in a more physiological relevant manner with focus on the innate immune response to viral infection.
Following a published protocol, analysis of hepatic markers showed successful maturation of iPSCs. Upon infection, a higher
antiviral response is triggered in hepatocyte-like cells (iHLCs) derived from iPSCs than in hepatoma cells, as iHLCs show a
stronger induction of interferon-stimulated genes (ISGs) in presence of a lower viral load at day 1-3 post infection. Highest
fold-induction was detected for Viperin, an ISG known to play a role in HCV infection, as well as 2'-5'-oligoadenylate
synthase 2 (OAS2), suggesting a role for this ISG as well. Further, blocking the JAK-STAT-signaling pathway almost
completely diminished ISG-induction in iHLCs, correlating with a detectable increase in viral replication monitored in
Luciferase assays. ShRNA-mediated knockdown of pattern recognition receptors and key factors of type I interferon
response increased viral spreading in hepatoma cells. We also observed increased levels of HCV replication in iHLCs
expressing shRNAs against RIG-I and STAT2. However, knockdown-iHLCs were still able to clear viral infection, suggesting
the activation of alternative routes of innate immunity. Besides the disturbance of the antiviral response of the cells, HCV
particles derived from hepatoma cells show a higher density correlating with a lower specific infectivity due to the impaired
VLDL metabolism. Density gradient analysis of cell culture supernatant revealed that iHLCs secrete lipoproteins that are
comparable to VLDLs isolated from human serum samples, providing a possible platform for formation of virions comparable
to patient material. We conclude that iHLCs provide a suitable alternative for studying HCV in vitro with the advantage of an
intact innate immunity and metabolism.
Corresponding author:
Anja Schoebel
[email protected]
Innate Immunity
P30
Comparative study of human hepatoma cell lines and primary hepatocytes for hepatotropic virus innate immunity
studies
W. Nicolay1, J. Brüning1, S. Kahl1, F. Vondran2, T. Pietschmann1, G. Gerold1
1Twincore
2Medical
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
School Hanover, General, Visceral and Transplant Surgery, Hanover, Germany
The liver is the site of replication for numerous viruses including hepatitis C virus and hepatitis B virus, which both cause
chronic liver disease. Hepatocytes recognize invading viruses through innate immune sensors such as Toll-like receptors
(TLRs) and retinoic acid-inducible gene I (RIG-I) like receptors (RLRs). To study innate responses to hepatotropic viruses,
primary human hepatocytes (PHH) are challenging as cell material is limited and cells rapidly dedifferentiate ex vivo.
Therefore, we here aimed to characterize a panel of hepatoma cell lines and compare them to PHH in order to find optimal
surrogate systems for studying the innate immunity to hepatotropic agents in vitro.
Specifically, we compared the human hepatoma cell lines Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B and HepaRG to PHH
from four independent donors. We characterized the expression level of RNA sensors (TLR3, TLR7, TLR8, RIG-I and
MDA5), their adapter proteins (MAVS and Trif) and the interferon alpha receptor (IFNAR) using immunoblot, flow cytometry
and quantitative RT-PCR. We then challenged all cells with synthetic TLR and RLR agonists or vesicular stomatitis virus
(VSV) and monitored the induction of type I interferon (IFN-I) and IFN inducible genes (ISG).
We observed comparable expression levels of RNA sensors, their adaptors and IFNAR in all tested cells. Slightly reduced
RIG-I, MDA5 and MAVS expression levels were observed in Huh-6 and Huh-7 cells. All cells responded to IFN-I treatment by
inducing RIG-I and MDA5. When comparing innate responses to TLR3, TLR7/8 and RIG-I/MDA5 agonists, RIG-I/MDA5
activation induced the highest levels of IFN-I and ISGs in cell lines and PHH. PHH additionally responded to TLR3
stimulation. Huh-6 and Huh-7 cells displayed the poorest RIG-I/MDA5 responses of all tested hepatoma cell lines. Prestimulation of Huh-6 and Huh-7 cells with IFN-I could however rescue their deficiency in sensing cytosolic RNA agonists.
When infecting cell lines and PHH with VSV, we observed a differential pattern of IFN-I response with HepG2, HepG2-HFL
and Hep3B showing the highest levels of IFN-I induction.
In conclusion, we found that HepG2, HepG2-HFL and Hep3B cell lines best mimicked the innate immunity to RNA viruses in
PHH. This set of data might help design innate immunity studies of hepatotropic virus infection.
Corresponding author:
Gisa Gerold
[email protected]
Innate Immunity
P31
The chromatin remodeling factor SPOC1 – A novel player in the intrinsic defense against human Cytomegalovirus
A. Reichel1, M. Scherer1, A. C. Stilp1, N. Reuter1, S. Lukassen2, S. Schreiner3, A. Winterpacht2, T. Stamminger1
1Friedrich
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
Alexander University Erlangen Nuremberg, Human Genetics, Erlangen, Germany
3Technical University Munich and Helmholtz Centre Munich, Institute of Virology, Munich, Germany
2Friedrich
Introduction: SPOC1 (survival time-associated PHD finger protein in ovarian cancer 1) functions as a regulator of chromatin
structure and DNA damage response. It is able to bind H3K4me2/3 containing chromatin and promotes DNA condensation
by recruiting H3K9 methyltransferases, such as SETDB1. SPOC1 was demonstrated to act as a restriction factor for
Adenovirus (Ad) infection by repressing viral gene expression at the transcriptional level. To antagonize this repressive
function, Ad targets SPOC1 for proteasomal degradation immediately upon infection.
Objectives: In this study, we analyzed the role of SPOC1 for human cytomegalovirus (HCMV) infection.
Material & methods: Western blot analyses and quantitative real-time PCR experiments were performed with primary
human fibroblasts (HFF) and epithelial cells (ARPE-19) to analyze the fate of SPOC1 during HCMV replication. In order to
assess whether SPOC1 promotes or inhibits HCMV replication, we utilized SPOC1 overexpressing and SPOC1-depleted
HFF cells.
Results: Interestingly, and in contrast to Ad-infected cells, we observed a transient upregulation of SPOC1 during the early
phase of HCMV infection. This upregulation takes place both on transcript and protein level and was observed in various cell
types as well as with different HCMV strains. Furthermore, we discovered that SPOC1 is downregulated in a GSK3-β
dependent manner at later stages of infection. In this study, we demonstrate that overexpression of SPOC1 leads to a
decreased expression of viral early and late proteins, revealing a possible role in antiviral defense. Intriguingly, multistep
growth curve analyses displayed a severe replication defect on HFF cells overexpressing SPOC1. Moreover, these cells
showed both a decrease in viral replication and in virus particle release. Furthermore, we observed a substantial decrease in
the number of immediate-early protein 1 (IE1)-positive cells, indicating that SPOC1 acts on the initiation of viral IE gene
expression. In line with these findings, HFF cells depleted of SPOC1 displayed augmented initiation of viral gene expression.
Conclusion: Our findings provide evidence that SPOC1 functions as a restriction factor against HCMV infection by inducing
a silencing of viral IE gene expression.
Corresponding author:
Anna Reichel
[email protected]
Innate Immunity
P32
Infection of primary human hepatocytes with cell culture-derived Hepatitis B virus in vitro is accompanied by tolllike receptor 2 activation
R. Broering1, Z. Zhang1,2, M. Trippler1, C. Real1, M. Werner1, A. Paul3, G. Gerken1, J. Schlaak1,4, M. Lu2
1University
of Duisburg-Essen, University Hospital of Essen, Dept. of Gastroenterology and Hepatology, Essen, Germany
of Duisburg-Essen, Institute of Virology, Essen, Germany
3University of Duisburg-Essen, University Hospital of Essen, Dept. of General-, Visceral- and Transplantation Surgery, Essen, Germany
4Evangelisches Klinikum Niederrhein GmbH, Duisburg, Germany
2University
Background: Chronic viral hepatitis caused by Hepatitis B virus (HBV) infection is among the most frequent causes for liver
related morbidity and mortality. It has become clear that not only the adaptive but also the innate immune system is involved
in the pathogenesis of this infection. Here, we analyzed immune responses in primary human hepatocytes (PHH) infected
with HBV in vitro.
Methods: PHH were isolated after perfusion and digestion of liver tissue obtained after tumor resections or transplantations.
PHH were treated with cell culture-derived HBV particles (HepG2-117) or negative control particles and cultured for 1-10
days. Infection was monitored by the release of HBsAg and HBeAg, transcription of viral genes and immunohistochemistry
staining. Changes in gene expression were analyzed by microarray and quantitative RT-PCR. TLR ligands, western blot
analysis and neutralizing antibodies were used to characterize the initial immune response of PHH.
Results: PHH could be efficiently infected with cell culture-derived HBV particles, indicated by the secretion of viral antigens
and expression of viral mRNAs. The exposure of PHH to HBV particles induced a gene expression profile, comparable to
that induced by TLR2 ligand Pam3Cys, leading to the induction of inflammatory and chemoattractant cytokines. Treatment
with HBV as well as Pam3Cys activated ERK1, JNK and p38 mitogen-activated protein kinases as well as NFκB.
Furthermore, HBV-induced gene expression could be neutralized by TLR2-specific antibodies, whereas HBV infectivity was
not affected by these blocking antibodies. The expression of interferons or interferon-stimulated genes could be detected
neither initially nor at later time points after HBV infection. Interestingly, PHH isolated from HBV-infected patients revealed a
higher responsiveness to TLR2 stimulation compared to uninfected resection or transplantation controls, indicated by
elevated induction of cytokine gene expression.
Conclusion: The present data demonstrate that TLR2 may be involved in recognition of HBV during the infection process
and activate innate immune responses in PHH. Consistently, recent data suggest that TLR2 might be involved in antiviral
responses during hepadnaviral infection and play an important role in the pathogenesis of chronic HBV infection.
Corresponding author:
Ruth Broering
[email protected]
Innate Immunity
P33
Exploitation of PKR as a selector for the identification of immunostimulating influenza virus RNAs useful for
antiviral therapy
M. Budt1, C. Mache1, M. Zickler2, A. Beling2, T. Wolff1
1Robert
2Charité
Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
Medical School, Institute of Biochemistry, Berlin, Germany
Introduction: Current antivirals against influenza A viruses (IAV) are limited to few viral target structures and suffer from
rapid development of resistant virus mutants. Alternative treatment strategies could utilize the capability of the cellular
defense system to recognize and counteract an infection. Thus, sensors like the RNA-activated protein kinase R (PKR) or
RIG-I detect virus-specific RNA patterns, and mount an efficient immune response.
Objectives: Here, we employ PKR to identify novel immunostimulatory RNAs for the development of antiviral therapeutics.
Material & Methods: PKR was immunoprecipitated from IAV-infected cells and the associated RNA was first characterized
by deep sequencing. Individual RNAs were amplified by PCR, cloned and resynthesised by in vitro transcription. After
transfection of RNA into cells, PKR activation was determined by phospho-PKR immunoblot and RIG-I activation was
analyzed by luciferase assay. Viral replication was analyzed by plaque assay.
Results: PKR-bound RNA from IAV-infected cells displayed a predominance of large polymerase segments. The RNA pool
stimulated both PKR and RIG-I, indicating an overlap in target requirements for both sentinel pathways. Furthermore,
transfection of PKR-derived RNA conferred an antiviral protective effect to target cells, significantly reducing the replication of
oseltamivir-resistant IAV. Individual cloned RNAs were examined to identify the most potent stimulators and to delineate
responsible motives. While RNAs consisting of entire IAV segments were strong stimulators of the antiviral response, two
engineered RNAs lacking the dsRNA region were not. Similarly, dephosphorylation abolished the stimulatory capacity of the
RNA. Of note, PKR-derived IAV RNAs elicited a broadly protective effect that was also effective against a range of other
viruses like VSV and enteroviruses. Mechanistically, we observed an induction of interferon beta in RNA-transfected
fibroblasts. Consistently, cells recieving conditioned medium were protected a similar manner like the transfected cells,
arguing for a paracrine mode of action.
Conclusion: PKR is a useful selector for the identification of immunostimulating viral RNAs with potential for the
development of novel therapeutics.
Corresponding author:
Matthias Budt
[email protected]
Innate Immunity
P34
Mass-spectrometry based profiling of PKR-interaction partners identifies KSRP as a novel protein regulator of PKR
S. Sänger1, T. Wolff1
1Robert
Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
Introduction: The RNA-dependent protein kinase (PKR) is an interferon inducible, double-strand RNA-activated protein
kinase that plays a significant role in innate antiviral immunity. The activation of PKR by recognition of viral RNA or distinct
protein regulators involves dimerization, autophosphorylation and phosphorylation of numerous target factors. Downstream
effects of PKR activation include inhibition of translation and the induction of transcription factors that lead to production of
type I interferon.
Objectives: Due to its key role in antiviral immunity many viruses have evolved mechanisms to avoid PKR initiated effects.
We and others have previously described the influenza virus non-structural protein 1 (NS1) as an antagonist of PKR. We are
interested in further elucidating the role of cellular and viral factors in regulating PKR activation in the context of influenza
virus infection.
Material & Methods: We used a SILAC approach followed by LC-MS/MS analysis to identify precipitable interaction partners
of PKR upon influenza A wildtype or ΔNS1 virus infection and validated selected binding partners in independent
biochemical assays involving overexpression and knockdown studies, coprecipitation experiments and viral replication
analyses.
Results: We detected 47 cellular PKR binding proteins involved in various cellular pathways including RNA processing and
stress response. We validated the interaction between PKR and a subset of candidates in co-immunoprecipitation
experiments and could show that some of the identified proteins induced PKR phosphorylation upon overexpression.
Hereby, the KH-type-splicing regulatory protein (KSRP) was identified as a novel regulator of PKR. Overexpression and
knockdown experiments indicated that KSRP is able to support antiviral signaling by enhancing PKR activation in a process
that involves direct protein-protein-interaction.
Conclusion: Taken together, this study demonstrates the aptitude of quantitative mass spectrometry for elucidation of
cellular antiviral response pathways. Precipitation of PKR and binding partners revealed KSRP as a novel protein regulator of
PKR. The mechanism of KSRP mediated PKR activation seems to be complex and needs to be analyzed in greater detail in
ongoing studies.
Corresponding author:
Sandra Sänger
[email protected]
Innate Immunity
P35
Cholesterol and IFITM3 in influenza A virus entry
A. Kühnl1, A. Musiol1, C. Ehrhardt2, S. Ludwig2, T. Grewal3, V. Gerke1, U. Rescher1
1University
of Münster, Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation, Münster, Germany
of Münster, Institute of Molecular Virology, Centre for Molecular Biology of Inflammation, Münster, Germany
3University of Sydney, Faculty of Pharmacy A15, Sydney, Australia
2University
Interferon-induced transmembrane protein 3 (IFITM3) has recently been described as an important antiviral host factor, that
is highly efficient in blocking virus cell entry, for example of influenza A virus (IAV). Yet, the knowledge of its antiviral
mechanism remains incomplete. In particular the role of endo-/lysosomal cholesterol accumulation upon increased IFITM3
levels is unknown. Moreover its physiological relevance in case of interferon-driven immune responses remains elusive.
Here, we demonstrate that interferon stimulation affects cellular cholesterol distribution and that increasing cholesterol export
from the endo-/lysosomal compartment by NPC1 expression slightly rescued interferons inhibitory effects. Furthermore we
utilize expression of the Ca2+-regulated membrane-binding protein annexin A6 to induce comparable alterations on cellular
cholesterol distribution independent of IFITM3 and studied the impact on IAV cell entry. Infection was apparently diminished,
with fusion - at the stage of hemifusion or earlier - as the first step to be affected. We conclude that endo-/lysosomal
cholesterol accumulation implies a distinct antiviral function independent of any direct inhibitory effect of IFITM3. Furthermore
it is part of interferons antiviral capacity and therefore potentially participates in a crucial innate immune response.
Corresponding author:
Alexander Kühnl
[email protected]
Innate Immunity
P36
Effect of human APOBEC3C Cytidine deaminase on Herpes Simplex Virus 1 (HSV-1)
N. Kirschnick1, A. Hofemeier1, M. L. Romberg1, W. Hafezi1, J. Kühn1
1University
of Münster, Institute of Virology, Münster, Germany
Human APOBEC3 (A3) cytidine deaminases play an important role for innate immunity by restricting the replication of
retrotransposons and various exogenous RNA and DNA viruses such as retroviruses, hepadnaviruses, and
papillomaviruses. A3 proteins convert cytosines in single-stranded DNA to uracils, causing base substitutions and strand
breaks. Hyperediting of the genomes of the herpesviruses Epstein-Barr virus (EBV) and HSV-1 by APOBEC3C (A3C) was
detected in vitro and in vivo. In tissue culture, overexpression of A3C reduces replication of HSV-1 (Suspène R et al., J.Virol.
85, 2594-2601, 2011). The exact mechanism of action, however, has not been fully uncovered yet. Aim of this study was to
understand the effects of A3C on HSV-1 more precisely.
The inhibitory effect of A3C on HSV-1 replication in tissue culture could be confirmed. Overexpression of A3C in BHK cells
led to a moderate, yet significant reduction in virus titer. In addition, hyperediting of HSV-1 genomes was detected via
3DPCR. In comparison, a catalytic mutant of A3C (C97S) showed only a slight reduction in virus titer and no hyperediting.
Interestingly, transient overexpression of A3C strongly reduced the expression of various co-transfected reporter gene
constructs. The effect of A3C on reporter gene expression was most pronounced in CHO cells (90% reduction), and was
seen both with wild-type A3C and the cytidine-deaminase negative mutant of A3C (C97S). Quantitative RT-PCR showed,
that A3C reduced transcript levels of reporter genes.
In conclusion, our results indicate that A3C may restrict HSV-1 replication not only by genomic hyperediting but also by
reducing transcript levels apart from its deaminase function. To our knowledge, the negative effect of A3C on transcript levels
has not been shown so far. A mode of action could be that A3C binds to single-stranded DNA in the transcription bubble
thereby causing a "roadblock" in transcription.
Corresponding author:
Joachim Kühn
[email protected]
Innate Immunity
P37
A systematic approach to identify Interferon Stimulated Genes (ISGs) with antiviral potential against Ebola virus
R. P. Galao1, H. Wilson1, K. L. Schierhorn1, F. Debeljak1, S. Wilson2, C. M. Swanson1, S. Neil1
1King's
College London, London, United Kingdom
of Glasgow, Centre for Virus Research, Glasgow, United Kingdom
2University
Ebola Virus (EBOV) is an enveloped, negative-sense RNA virus of the Filoviridae family. This zoonotic virus is highly
pathogenic in primates and sporadically spreads to humans causing severe hemorrhagic fever, as highlighted by the latest
epidemic in West Africa. Given the hazardous nature of working with full-length filoviruses, we implemented a recently
described reverse genetics system that produces safe, transcription- and replication-competent virus-like particles (trVLP).
This system permits the study of EBOV"s cell biology, cellular entry, gene expression, assembly and innate immune evasion.
Expression of ISGs results from the production of type I-IFNs as a consequence of signaling cascades being triggered
through the activation of pattern recognition receptors by non-self-nucleic acids, such as those found in RNA viruses. Some
of these ISGs target specific steps of virus life cycle limiting its spread, replication and leading to the activation of adaptive
immune responses. EBOV is capable of counteracting host innate antiviral defenses, particularly by blocking the production
of type I-IFNs and the cellular responses to exogenously added type I-IFNs. Despite this, we observe that IFN treatment of
target cells inhibits trVLP production implying that many downstream target genes have antiviral activity against EBOV. We
have screened through a well-characterized library of approximately 400 human ISGs using the trVLP system and identified
around 30-40 candidates that may directly inhibit stages of EBOV replication including entry, gene expression, genome
replication and assembly.
The validation and characterization of candidates are now being addressed. Our recent findings present additional insights
into the mechanism associated with the innate immune response to EBOV infection, and further refine the understanding of
the cellular function of selected ISGs.
Corresponding author:
Stuart Neil
[email protected]
Innate Immunity
P38
Stress granule proteins inhibit Ebola virus replication in a transcription- and replication-competent virus-like
particle system
K. L. Schierhorn1, H. Wilson1, R. P. Galao1, F. Debeljak1, S. Neil1, C. M. Swanson1
1
King's College London, London, United Kingdom
Ebola virus (EBOV) is a highly pathogenic, nonsegmented, negative-strand RNA virus that replicates in the cytoplasm of
infected cells. The presence of viral RNA can trigger host cell antiviral mechanisms by activating RNA sensors like RIG-I and
PKR. Activation of RIG-I induces type I interferon expression (IFN) and PKR activation leads to translation inhibition resulting
in the formation of stress granules (SGs). SGs are non-membrane-bound cytosolic organelles that assemble when
translation is inhibited due to different stress conditions, such as viral infection. SGs are also amplification platforms for
sensing foreign RNAs and IFN induction. EBOV VP35 protein has been shown to inhibit PKR, RIG-I and visible stress
granule formation. However, whether SG proteins have antiviral effects on EBOV replication remains unclear. Using a
transcription- and replication-competent virus-like particle (trVLP) system, we performed an overexpression screen with
selected SG and RNA-binding proteins to determine whether they affected EBOV replication. We identified several SG
proteins that inhibit trVLP replication up to 96%. This inhibition cannot be overcome by high virus input. To eliminate proteins
with non-specific effects, we excluded candidates that inhibited co-transfected firefly luciferase expression. We are currently
investigating which step of the viral life cycle is inhibited for the top candidate proteins to identify their underlying mechanism
of action. Preliminary results suggest that some proteins may interfere with viral RNA transcription or replication whereas
others affect viral protein expression. In addition, we are investigating the impact of depleting SG proteins on trVLP
replication. In conclusion, our data suggest that specific SG proteins can inhibit EBOV replication.
Corresponding author:
Kristina L. Schierhorn
[email protected]
Innate Immunity
P39
The human cytomegalovirus ULb' region codes for multilayered modulation of TNFα and NFκB signaling
T. Becker1, S. Voigt2,3, H. Hengel4, M. Trilling1, V. T. K. Le-Trilling1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Koch Institute, FG12 - Measles, Mumps, Rubella, and viruses affecting immunocompromised patients, Berlin, Germany
3Charité Medical School, Department of Pediatrics, Division of Oncology and Hematology, Berlin, Germany
4Albert Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
2Robert
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus establishing life-long latent infection. Changes in immune
homeostasis induce reactivation of lytic infection, mostly inapparent in healthy individuals, but often causing overt disease in
immuno-compromised hosts.
Analysis of phenotypic differences between the HCMV variants AD169varS (short UL region without ULb') and AD169varL
(long UL region containing most of the ULb' genes) revealed clear differences in TNFα-dependent NFκB signaling.
AD169varL-infected cells allowed TNFα-induced IΚBα degradation, whereas in AD169varS-infected cells it was inhibited. In
contrast, congruent IκBα amounts were found upon IL1β treatment, indicating independent IκBα regulation upon TNFα and
IL1β signaling, which is consistent with previously published data. Although IκBα became phosphorylated and degraded
upon TNFα treatment in AD169varL-infected cells, the negative feedback loop was not initiated to reexpress IκBα, a
prototypical NFκB target gene product.
To profit from the possibility of genetic manipulation, AD169varL was cloned into bacterial artificial chromosomes (BAC). This
technique yields clonal viral genomes and facilitates targeted mutagenesis. The analysis of high numbers of AD169varLderived BAC clones disclosed the existence of a fraction (5-10%) of viral genomes lacking the ULb' region. Sequence
determination showed that these genomes contained an IRL region with an IRL-UL intersection identical to the one in
AD169varS. This finding indicates that AD169varL is the ancestor of the widely used AD169varS laboratory strain. Using the
clones BAC2 and BAC20, which correspond to the AD169varL and AD169varS genomes, respectively, we recapitulated the
phenotypes observed for AD169varL and AD169varS. Complete sequencing of both BAC-cloned genomes allowed us to
annotate the coding capacity of both variants.
With these tools, we are currently examining the ULb'-encoded functions modulating TNFα and NFκB signaling.
Corresponding author:
Tanja Becker
[email protected]
Innate Immunity
P40
Regulation of interferon induction through sequestering IRF7 into inclusion bodies by nonstructual protein NSs in
SFTS Bunyavirus infection
Y. Hong1, Z. Xing2,1
1Nanjing
University Medical School , Nanjing, China
of Minnesota at Twin Cities, Saint Paul, United States
2University
Background: Induction of type I interferons (IFNs) during viral infection is crucial for innate immunity. IRF3 and IRF7, which
are highly homologous, play a critical role as the key transcription factors in the activation of the IFN induction. To survive
and replicate within a host, viruses have evolved a variety of strategies to evade innate immunity. Our previous studies have
shown that the nonstructural protein, NSs, of the severe fever with thrombocytopenia syndrome virus (SFTSV) can suppress
the IFN-β induction through its interaction with tank-binding kinase 1 (TBK1), indirectly sequestering the IKK complex,
including IKK ε and IRF3, into the inclusion bodies (IBs) formed by NSs in infected cells.
Question: While IRF3 played an antiviral response mainly at the early stage of infection, will IRF7 play any role in the
induction of innate immunity and how is IRF7 signaling regulated in SFTS bunyavirus infection?
Methods: Virus-host interaction was studied by using co-immunoprecipitation, confocal immunofluorescence microscopy,
and virological methods.
Results: IRF7 was significantly induced in response to SFTSV infection, promoted further induction of IFN-β and –a at the
late stage, and contributed to suppressing the viral replication. Our data indicated that NSs directly interacted and
sequestered IRF7 into IBs, different from IRF3 interacting with NSs indirectly. Phosphorylated IRF7 was trapped in the
cytoplasmic IBs, although interaction of NSs with IRF7 did not inhibit IRF7 phosphorylation, resulting in reduced IFN-a and -b
induction and enhanced viral replication.
Conclusion: Interaction of the nonstructural protein with both IRF3 and IRF7, and subsequent sequestration of these
transcription factors essential to IFN induction into the viral IBs, ensured the suppression and evasion of the host innate
immunity at both early or late stage of infection with this novel bunyavirus.
Corresponding author:
Zheng Xing
[email protected]
Innate Immunity
P41
Modulation of DAF and CD59 by influenza A viruses
Z. Vaz da Silva1, M. J. Amorim1
1Instituto
Gulbenkian de Ciencia, Cell Biology of Viral infection, Oeiras, Portugal
Influenza A viruses (IAV) are amongst the most relevant human pathogens. Present methods of surveillance, vaccination
and antiviral therapy are insufficient to abrogate yearly epidemics and recurrent pandemics, thus other strategies are
needed. The complement system is a host defence mechanism targeting infected cells and extracellular virions. When overstimulated it can lead to uncontrolled inflammation and damage, and so it is under tight regulation by Regulators of
Complement Activation (RCA). Being involved in cell and viral survival, RCA are perfect targets for viral modulation. Our goal
is to understand the interplay between IAV and two RCAs: decay accelerating factor (DAF) and CD59, exploring whether
these are hijacked by IAV to modulate viral persistence in the host.
To address this question, we have used both in vivo and in vitro models challenged with currently circulating pandemic IAV
strains.
Our data shows that upon IAV infection, mice lacking both DAF and CD59 have a better disease outcome then wild-type
(WT). Double KO mice have reduced weight loss and mortality, as well as decreased lung viral loads, damage and
inflammatory infiltrates. Experiments using single DAF or CD59 KO mice showed that the protection is due to absence of
DAF alone, and not CD59, contradicting what was previously published. We suggest that DAF is used by IAV to increase
viral persistence via two mechanisms: modulating host immune response, or directly protecting the virus against complement
attack by incorporation into viral envelope. The presence of DAF could hinder activation of host immune response, enabling
persistence of infection and damage. Incorporation of RCA into virions was observed for many viruses, but no data is
available for IAV. We show that both DAF and CD59 are incorporated into IAV, and that RCA-mediated protection operates
only against non-physiological levels of complement. Hence, DAF likely eases viral persistence by modulation of host
immune response, or protection of infected cells by a yet unidentified process. The survival of WT and single KO lung
epithelial cells in presence of activated complement is currently under investigation.
We believe that this work provides better understanding of the role of DAF and CD59 in IAV infection, opening new doors to
identify potential therapeutic pathways and targets.
Corresponding author:
Zoé Vaz da Silva
[email protected]
Innate Immunity
P42
HIV-1-mediated downmodulation of HLA-C impacts target-cell recognition and antiviral activity of NK cells
C. Körner1, C. R. Simoneau2, P. Schommers3,4, M. Granoff2, B. Corleis2, V. Naranbhai 2,5, D. S. Kwon 2,6, E. P. Scully2,6,7, S.
Jost2,8, M. Carrington2,5, M. Altfeld1
1Heinrich
Pette Institute, Virus Immunology, Hamburg, Germany
Institute of MGH, MIT and Harvard, Cambridge, United States
3University of Cologne, Department of Internal Medicine, Cologne, Germany
4German Center for Infection Research (DZIF), Cologne, Germany
5Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, Cancer and Inflammation Program, Frederick, United
States
6Massachusetts General Hospital, Division of Infectious Diseases, Boston, United States
7Johns Hopkins University School of Medicine, Division of Infectious Diseases, Baltimore, United States
8Beth Israel Deaconess Medical Center, Center for Virology and Vaccine Research, Boston, United States
2Ragon
Interaction of inhibitory receptors with their respective HLA class I ligands confers functional competence (licensing) to
natural killer (NK) cells. In contrast, NK cells lacking inhibitory receptors for self HLA class I are rendered hyporesponsive.
Licensed NK cells may sense HIV-1-mediated alterations of HLA class I expression through poor binding of self-inhibitory
receptors, thus potentially influencing the control of HIV-1 replication. Here we show that HIV-1-mediated downmodulation of
HLA-C was associated with reduced binding to its respective inhibitory receptors. Despite this binding difference, licensed
NK cells expressing self-inhibitory receptors for HLA-C displayed reduced antiviral activity in vitro as compared to their
unlicensed counterparts, potentially due to residual inhibitory KIR binding. Blocking of self-inhibitory receptors in turn
improved the antiviral activity of licensed NK cells. Nevertheless, NK cells were able to sense alterations of HLA-C
expression demonstrated by increased antiviral activity when exposed to a viral strain which robustly downmodulated HLA-C
in contrast to a strain that lacked this ability. Thus, our results suggest that the capability of HLA-C-licensed NK cells to
control HIV-1 replication is determined by the strength of KIR/HLA-C interactions and is thus dependent on both the hosts
genetic background and the extent of virus-mediated HLA downregulation.
Corresponding author:
Christian Körner
[email protected]
Innate Immunity
P43
Inhibition of type I interferon induction by Sandfly Fever Sicilian Virus virulence factor NSs
J. D. Wuerth1, M. Habjan2, A. Pichlmair2, G. Superti-Furga3, F. Weber1
1Justus
Liebig University Giessen, Institute of Virology, Giessen, Germany
Planck Institute of Biochemistry, Martinsried/Munich, Germany
3Center for Molecular Medicine, Vienna, Austria
2Max
Phleboviruses are a group of emerging viruses with a wide spectrum of virulence: For example, Rift Valley Fever virus
(RVFV) is highly pathogenic, whereas Sandfly fever Sicilian virus (SFSV) causes intermediate pathogenicity.
The major virulence factor of phleboviruses is the non-structural protein NSs, an inhibitor of the type I interferon (IFN)
system. The extensively studied NSs protein of highly pathogenic RVFV has been reported to shut off host cell transcription
by targeted sequestration and degradation of host factors. In contrast, the mode of action of the NSs protein of intermediately
pathogenic SFSV has remained elusive.
We therefore aimed to characterize the IFN-inhibitory capacity of and the mechanism employed by SFSV NSs.
Previously, our group has identified multiple candidate host interactors by tandem affinity purification and mass spectrometry
(Pichlmair et al. Nature 2012). Intriguingly, these included interferon regulatory factor 3 (IRF3). We confirmed the interaction
with IRF3, while other IRF family members were not targeted by SFSV NSs. Further, SFSV NSs alone was sufficient to
abrogate IRF3-dependent promoter activity induced by stimulation of the RIG-I and TLR3 signalling pathways, as well as
overexpression of constitutively active IRF3. Finally, we generated a SFSV NSs mutant that lost the ability of binding IRF3
and inhibiting IFN-β promoter activity.
In summary, we were able to show that the NSs protein of Sandfly fever Sicilian virus (SFSV) efficiently inhibits the induction
of type I interferon. Different to the highly pathogenic RVFV NSs, which induces a general host transcription shutoff,
however, SFSV NSs specifically targets IRF3.
Thus, although these phleboviruses are highly related, their NSs proteins display remarkably diverse strategies of
counteracting the type I interferon system. Hence, we hypothesize that the quality of NSs-host factor interactions correlates
with the virulence levels of phleboviruses.
Corresponding author:
Jennifer Deborah Wuerth
[email protected]
Innate Immunity
P44
Gene expression of innate immunity receptors, type I interferons and IL-10 in equine peripheral mononuclear blood
cells in vitro after infection with different strains of equine herpesvirus type 1 (EHV-1)
A. Golke1, R. Jacek1, J. Cymerys1, T. Dzieciatkowski2, A. Chmielewska1, A. Tucholska1, A. Cywinska3, K. Posyniak1, M. W.
Banbura1
1Warsaw
University of Life Sciences - SGGW, Department of Preclinical Sciences, Warsaw, Poland
University of Warsaw, Department of Medical Microbiology , Warsaw, Poland
3Warsaw University of Life Sciences - SGGW, Department of Pathology and Veterinary Diagnostics, Warsaw, Poland
2Medical
Question: Equine herpesvirus type 1 (EHV-1), the member of the Alphaherpesvirinae subfamily, is highly prevalent
pathogen in most equine populations worldwide. After replication in the respiratory epithelium EHV-1 enters lymphocytes in
which it spreads systemically via a cell-associated viremia. Host innate immune responses are essential to stimulate
appropriate adaptive immunity and prevent severe clinical signs.
Therefore, the aim of this study was to evaluate possible changes of TLR2, TLR3, IFNα, IFNβ and IL-10 mRNA expression in
primary cell culture of equine peripheral mononuclear blood cells (PMBCs) infected with neuropathogenic and nonneuropathogenic strains of EHV-1.
Methods: Total RNA was obtained from 106 uninfected or infected with EHV-1 equine PMBCs 24 hours post-infection (p.i.).
Real-time PCR was performed in LightCycler 2.0 instrument (Roche Diagnostics) using LightCycler FastStart DNA Master
SYBR Green I (Roche Diagnostics). The amount of specific mRNA was normalized to GAPDH mRNA levels.
Results: In the present study we have observed that in equine PMBCs both investigated EHV-1 strains up-regulated IFNβ
mRNA expression. However, neuropathogenic EHV-1 strain down-regulated the expression of TLR2 and IFNα mRNAs while
after infection with non-neuropathogenic strain, levels of these mRNAs were comparable to uninfected control. TLR3 and IL10 mRNA levels were up-regulated after infection with non-neuropathogenic strain and comparable to uninfected control after
infection with neuropathogenic strain.
Conlusions: Obtained results shown a significant differences in the ability to stimulate innate immune responses between
neuropathogenic and non-neuropathogenic EHV-1 strains. It may indicate that those may be the factors that contribute to
neurological manifestation.
Corresponding author:
Anna Golke
[email protected]
Innate Immunity
P45
Functional characterization of the porcine ISG15 system regarding expression and antiviral potential
M. Zickler1, N. Althof1, A. Beling1
1Charité
University Medicine Berlin, Institute of Biochemistry, Berlin, Germany
Introduction: ISG15 is a type I interferon-inducible ubiquitin-like modifier that acts in two ways: intra-, extracellular as a free
protein or conjugated to target proteins. Conjugation, called ISGylation, utilizes three proteins, similar to the ubiquitinating
enzymes E1, E2, and E3. ISG15 has been shown to play a major role in the innate immune response to pathogens e.g.
Influenza A, B virus, Coxsackievirus B3 (CVB3), M. tuberculosis.
Objective: The antiviral activity of ISG15 was shown for the murine system and is under debate for humans. Here, we aim to
characterize the porcine ISG15 system regarding its baseline and inducible expression as well as its antiviral activity against
a range of pathogens to assess species-specific differences and therapeutic potential.
Material & Methods: Upon stimulation with porcine IFN β (pIFN β), different porcine cell lines were characterized by
detecting ISG15 system components and ISGylation. Additionally, infection studies with different porcine viruses were carried
out to test susceptibility and sensitivity under different conditions.
Results: While porcine ISG15 was induced in all three tested porcine cell lines upon stimulation with pIFN β, ISGylation was
observed only in those, where also the E1 enzyme Ube1L could be detected. The E2 enzyme was present in all cell lines.
Infection with IFN β-sensitive porcine sapelovirus (PSV) and porcine enterovirus 9 (PEV-9) led to induction of ISG15: an
observation which could not be achieved after infection of HeLa cells with CVB3.
Conclusion: The porcine ISG15 system is induced by stimulation with IFN β and infection with porcine viruses. Assessment
of cellular mechanisms and antiviral potential may uncover possibilities to overcome therapeutic resistance.
Corresponding author:
Martin Zickler
[email protected]
Innate Immunity
P46
Interaction of the foot-and-mouth disease virus with the innate immune system
C. Luttermann1, K. Schult1, G. Meyers1
1Friedrich
Loeffler Institute, Institute of Immunology, Greifswald - Isle of Riems, Germany
Foot-and-Mouth Disease (FMD) is a highly contagious disease of cloven-hoofed animals (e.g. cattle, swine and sheep). The
viral infection causes lameness, fever and vesicular lesions. This results in a loss of productivity through reduced milk
production and weight loss of the animals. The etiologic agent of FMD, FMDV, belongs to the genus Aphthovirus in the
picornavirus family. The FMDV genome consists of a single stranded positive sense RNA and has a length of about 8500 nt.
It contains one open reading frame, resulting in the expression of one polyprotein that is post-translationally cleaved into 12
proteins by viral proteases. To survive in the host, FMDV has evolved strategies to antagonize the host type I interferon (IFN)
response. It inhibits cellular transcription and protein synthesis, including proteins of the immune system like IFN type I.
Furthermore, viral proteins are supposed to interfere directly with interferon signaling cascades. The underlying mechanisms
and the viral proteins acting as IFN-I antagonists are not well known. We have investigated the effect of viral proteins like the
Leader protease (LPro) , 3C protease and other nonstructural proteins on the IFN-I response. These analyses help to
elucidate the role of different viral proteins in interference of FMDV with the innate immune system through translational shut
down and specific inhibition of interferon signaling cascades.
Corresponding author:
Christine Luttermann
[email protected]
Innate Immunity
P47
Reduction of HIV-1 particle infectivity by the interferon-inducible guanylate-binding proteins 2 and 5 (GBP2 and 5) is
evolutionary conserved
E. Reith1, D. Hotter1, F. Kirchhoff1, D. Sauter1
1Ulm
University Medical Center, Institute of Molecular Virology, Ulm, Germany
Introduction: We recently identified guanylate-binding protein 5 (GBP5) as a cellular restriction factor of HIV-1 and related
primate lentiviruses (McLaren et al., Retrovirology, 2015; Krapp et al., Cell Host & Microbe, 2016). GBP5 interferes with the
processing and virion incorporation of the viral envelope (Env) glycoprotein and reduces the infectivity of viral particles.
Objectives: The main goal of this study was to determine whether other members of the GBP family also possess
antiretroviral activity and whether GBP-mediated restriction is evolutionary conserved. Furthermore, this study aims at
identifying the motifs and mechanisms underlying the antiretroviral activity of GBP5.
Material & Methods: Human, simian, murine, and feline GBP family members were transiently expressed in HEK293T cells
to investigate the antiretroviral activity of different GBP paralogs and orthologs. Infectious virus yield and Env incorporation
were monitored by infection of TZM-bl reporter cells and Western blot, respectively. To analyze the antiretroviral effect of
GBP5 in primary macrophages and CD4+ T cells, we performed knock-down experiments and generated an infectious HIV-1
clone, expressing GBP5 instead of Nef. Furthermore, GBP5 mutants and chimeras were characterized to map the
antiretroviral activity of this restriction factor.
Results: Analysis of different GBP family members revealed that, in addition to GBP5, GBP2 also displays antiretroviral
activity. Notably, GBP5-mediated impairment of virion infectivity is conserved from mice to men and not limited to HIV-1, as
SIV and MLV Env are also targeted. Furthermore, mutational analyses showed that C-terminal isoprenylation and the central
stalk domain are crucial for full antiretroviral activity. Finally, experiments in primary cells demonstrated that GBP5 restricts
viral replication in both macrophages and CD4+ T cells.
Conclusion: Our data demonstrate that two guanylate-binding proteins, i.e. GBP2 and 5, restrict replication of HIV-1 and
related retroviruses. Both proteins are expressed in primary target cells of HIV and strongly inducible by interferons. Together
with the observation that this inhibitory activity is evolutionary conserved, our findings suggest a relevant role of GBP2 and
GBP5 in antiretroviral interferon responses.
Corresponding author:
Elisabeth Reith
[email protected]
Innate Immunity
P48
Unbiased mass spectrometry based approach to identify protein complexes in interferon stimulated cells
L. Beatrice1, S. Alexey1, H. Nadine1, G. Virginie1, P. Andreas1
1Max
Planck Institute of Biochemistry, Martinsried/Munich, Germany
The innate immune system consists of a complex network of interacting proteins and has the ability to distinguish between
self and pathogen-derived molecular patterns. Such patterns are recognized by receptors that lead to the induction of
signaling cascades culminating in the expression of cytokines including type-I interferons (IFN-α/β). An autocrine and
paracrine feed-back loop leads to the expression of hundreds of interferon-stimulated proteins which cooperatively restrict
virus growth. For full functionality, formation of multi-protein complexes is required. To identify such multi-protein complexes
in response to type-I interferon stimulation in an unbiased manner, we combined size exclusion chromatopgraphy as
separation method with high resolution mass spectrometry. The relationship and interaction profile of individual proteins
could be reconstructed and visualized through a custom made bioinformatics analysis platform. This method allowed us to
identify interferon-induced changes in the interaction profile of proteins and enables visualization of an interferon-mediated
re-wiring process that links yet unappreciated proteins to the interferon response.
Corresponding author:
Laudenbach Beatrice
[email protected]
Innate Immunity
P49
The role of inhibitory ligands expressed on myeloid cells for the regulation of T cell responses
P. David1, M. Drabczyk-Pluta1, T. Werner1, U. Dittmer1, G. Zelinskyy1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Myeloid cells are a heterogeneous population of cells regulating the adaptive T cell response on different phases of T cell
differentiation. The Friend virus (FV) infection is a well characterized murine retrovirus model for studying the basic
mechanism of the acute and chronic immune response. In our earlier studies we have shown that PD-L1 expression is
increased on Gr1+ cells. The Gr1 molecule is expressed on most myeloid cells. The aim of this study is the kinetic analysis
of subpopulations of myeloid cells for the expression of inhibitory ligands during FV infection and analysis of the functional
immune regulatory properties of these cells. As first we will characterize the expansion of different subpopulations of myeloid
cells (dendritic cells, monocytes, macrophages, granulocytes) in FV infected organs. The expanded myeloid cells will be
characterized for the expression of inhibitory ligands (PD-L1, PD-L2, HVEM, CD48, and VISTA). Our preliminary results
demonstrate the increased expression of HVEM on macrophages, mDCs, and conventional DCs. The enhanced expression
of PD-L1 was observed on macrophages and mDC. In following experiments, the role of inhibitory ligands expressed on
myeloid cells will be functionally in vivo and in vitro analyzed. The knowledge received from this study will show the new
possible targets for the immunomodulatory therapy of acute and chronic viral infections.
Corresponding author:
Paul David
[email protected]
Innate Immunity
P50
HIV-1 infection leads to changes in the HLA class I-presented peptide repertoire of primary human CD4+ T-cells
M. Ziegler1, A. Nelde2, S. Stevanović2, M. Altfeld1
1Heinrich
Pette Institute, Virus Immunology, Hamburg, Germany
Hospital Tübingen, Immunology, Tübingen, Germany
2University
During viral infections altered presentation of peptides via HLA class I (HLA-I) molecules on the surface of infected cells is an
important mechanism to activate the immune system. It was shown that HLA-I molecules do not only present peptides to
CD8+ T-cells, but also interact with killer-cell immunoglobulin-like receptors (KIR) on the surface of natural killer (NK) cells.
NK cells are part of the innate arm of the immune system and as such play an important role in the early response to
infections. Through the interaction of KIRs and HLA-I molecules NK cells can distinguish between self and non-self or
altered-self. This engagement is not only dependent on the specific KIR and HLA molecule, but also on the peptide
presented by the HLA-I molecule. Multiple studies have provided evidence that NK cells and especially HLA-I/KIR
interactions play a crucial role in HIV-1 infection and that these interactions are furthermore influenced by the HLA-I bound
peptide. However, the precise molecular mechanisms that lead to differential HIV-1 disease outcome, and in particular the
role of HLA-I presented peptides in this process, remain unknown.
Identification of HLA-I presented peptides is traditionally performed using in vitro based screening approaches. In this project
we established a method to directly identify naturally HLA-I presented peptides of HIV-1 infected primary human CD4+ Tcells. To this end we isolated CD4+ T-cells from two healthy donors with specific HLA types. These cells were in vitro
infected with HIV-1. The HLA-I bound peptides of these HIV-1+ cells were subsequently isolated by immunoaffinity
chromatography and analysed by LC-MS/MS in comparison to uninfected cells. We identified an overlap in the presented
peptide repertoire of infected and uninfected cells of 50%. Infection with HIV-1 led to a presentation of unique peptides of up
to 27%. Nine out of ~9000 identified HLA-I presented peptides were of viral origin. Furthermore we observed a donordependent modulation of the presented peptides between HIV-1 infected and uninfected cells of up to 27% significantly (fold
change ≥4, p≤0.01) regulated HLA ligands.
In summary we show that only a minority of HLA-I presented peptides after HIV-1 infection is virus-derived and that the
majority of the presented peptides stem from host-proteins. If the presented peptides are able to interact with KIRs and
thereby modulate NK cell function needs to be investigated.
Corresponding author:
Maja Ziegler
[email protected]
Innate Immunity
P51
MicroRNA expression profile in NK cells in children with severe and/or recurrent infection with Herpes simplex
virus.
M. Lenart1, A. Gruca1, M. Rutkowska-Zapała1, A. Szaflarska1, M. Surmiak2, K. Kobylarz3, M. Majka4, M. Sanak2, M. Siedlar1
1Jagiellonian
University Medical College, Institute of Paediatrics, Faculty of Medicine, Department of Clinical Immunology, Cracow, Poland
University Medical College, Department of Internal Medicine, Cracow, Poland
3Jagiellonian University Medical College, Institute of Paediatrics, Faculty of Medicine, Department of Anesthesiology and Intensive Care, Cracow, Poland
4Jagiellonian University Medical College, Institute of Paediatrics, Faculty of Medicine, Department of Transplantation, Cracow, Poland
2Jagiellonian
Question: The pathomechanism of severe and/or recurrent infections with Herpes viruses is still largely unknown. Recent
findings indicate an important role of regulation of gene expression by microRNA (miRNA) in development and function of NK
cells. Nonetheless, the role of miRNA in regulation of NK cells function in anti-herpesviral response has never been
investigated. The aim of the study was to determine miRNA expression profile in NK cells in children suffering from severe
and/or recurrent infections with Herpes simplex virus (HSV).
Methods: Peripheral blood samples were obtained from 30 children with severe and/or reccurent HSV infection and 30 agematched control subjects. NK cells were isolated from peripheral blood mononuclear cells using MACS and then the
expression of 754 most common human miRNA was performed by TaqMan real-time PCR technique.
Results: In children with HSV infection, several miRNA were detected to be upregulated (with statistical significance), e.g.
miR-1247, miR-320, miR-146a, miR-128a, in comparison to healthy control subjects. Yet, only miR-146a was previously
reported to increase in chronic viral infections, in particular hepatitis B infection, while other miRNA have not yet been
described to take part in regulation of NK cell function.
Conclusion: Our findings may indicate, that upregulated expression of miRNA in HSV patients may be associated with
decreased antiviral immunity. Nonetheless, at this stage, it is not clear whether the observed alteration of the expression of
microRNAs is a cause of observed disturbance of antiviral response or a result of prolonged viral infection.
Corresponding author:
Marzena Lenart
[email protected]
Innate Immunity
P52
SAMHD1-dependent retroviral restriction and sensing in mice
A. Herrmann1, R. Behrendt2, S. Wittmann1, A. Roers2, T. Gramberg1
1Friedrich
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
of Technology Dresden, Institute of Immunology, Dresden, Germany
2University
SAMHD1 is a dNTPase that restricts retroviral infection in noncycling cells by reducing intracellular dNTPs below the level
necessary for reverse transcription. Mutations in samhd1 are associated with the autoimmune disease Aicardi-Goutières
syndrome (AGS), a hallmark of which are elevated type I interferon (IFN) levels in the absence of exogenous infection. In
AGS, loss of SAMHD1 is thought to cause accumulation of nucleic acids, which are detected by intracellular immune
sensors. Interestingly, also HIV infection in the absence of SAMHD1 has been shown to cause an enhanced innate immune
response in DCs. However, it is still unclear how and what kind of nucleic acids are sensed in the absence of SAMHD1 and
whether retroviral infection is detected by the same unknown sensors.
To analyze immune responses in greater detail we took advantage of our previously described mouse model. Infection of
BMDCs from wildtype (wt), SAMHD1, IFNAR, and SAMHD1/IFNAR double-KO mice with a VSVG-pseudotyped HIV-GFP
reporter virus revealed an enhanced infectivity in double-KO compared to SAMHD1 or IFNAR single KO cells. Similar
findings were obtained from in vivo infection experiments. Infectivity of HIV-GFP in splenocytes of double-KO mice was
enhanced ~20-fold, while the single KO mice only showed a moderate 3-5-fold enhancement of infectivity compared to wt.
These data point towards an IFN-dependent antiviral block in the absence of SAMHD1 both in vitro and in vivo.
Next, we compared the transcriptome of wt, SAMHD1, IFNAR, and double-KO BMDCs in the presence and absence of HIVGFP infection. Without viral challenge we found enhanced numbers and transcription levels of ISGs in SAMHD1 KO
compared to wt cells, resembling the spontaneous IFN signature. Infection resulted in elevated numbers of upregulated ISGs
together with higher relative transcription levels for SAMHD1 KO compared to wt cells. Moreover, analysis of double-KO cells
revealed that this immune response is IFN-dependent.
Currently, we are characterizing the ISGs upregulated in response to infection in SAMHD1 KO cells. To analyze the
involvement of the cGAS sensing pathway, we will analyze the immune response to infection in cGAS and STING KO mice.
Together, our results wills shed light on the nature of the antiviral and autoimmune response in the absence of SAMHD1.
Corresponding author:
Alexandra Herrmann
[email protected]
Innate Immunity
P53
Modified Vaccinia virus Ankara induced CCL2 is mediated by type I IFN receptor and recruits natural killer cells and
T cells to the lung
M. H. Lehmann1, L. E. Torres-Domínguez1, P. J. R. Price1, C. Brandmüller1, C. J. Kirschning2, G. Sutter1
1Ludwig
Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
of Duisburg-Essen, Institute of Medical Microbiology, Essen, Germany
2University
Innate immune responses influence the outcome of vaccination. Modified Vaccinia virus Ankara (MVA), which is used as a
versatile vector for antigen delivery, has been proven effective in preclinical and clinical studies. To understand why
vaccination with non-replicating MVA induces immunity without need for an adjuvant, we study MVA-induced cytokine
expression in macrophages and leukocyte recruitment to the site of inoculation.
We and others have shown that MVA, but not other vaccinia virus strains, induces robust innate immune responses including
recruitment of leukocytes to the lung within hours (Lehmann et al. 2009 J. Virol., Lehmann et al. 2015 Virol. J.). During the
early stages of MVA infection neutrophils represent the largest cell population of the infiltrating leukocytes, which are
recruited by a mechanism involving chemokine (C-C motif) receptor 1 (CCR1) (Price et al. 2014 J. Virol.) and complement
component 5 (Price et al. 2015 J. Immunol.).
Recently, we revealed that the type I interferon receptor but not TLR2 mediates MVA-induced CCL2 expression and the
recruitment of natural killer (NK) cells and T cells to lung (Lehmann et al. 2016 J. Leukoc. Biol.). We elucidated in murine
bone marrow-derived macrophages (BMDM) that the initiation of interferon-alpha and interferon-beta mRNA transcription
precedes the upregulation of CCL2 mRNA by about two and four hours, respectively. The importance of type I interferons for
MVA-induced CCL2 expression was demonstrated in macrophages from type I interferon receptor deficient mice (Ifnar1-/-).
Using these mice, we confirmed a critical role of type I interferons for CCL2 production in the lung. Consequently, the
recruitment of NK cells and T cells to the lung after intranasal infection with MVA was severely affected in Ifnar1-/- mice
compared to wild type mice.
In summary, MVA-induced CCL2 expression and respiratory recruitment of NK cells and T cells in the murine system is a
consequence of type I interferon induction by MVA.
Corresponding author:
Michael H. Lehmann
[email protected]
Innate Immunity
P54
IFN-λs-induced human Mx2 protein inhibits Hantaan virus infection through the JAK-STAT pathway
N. Li1, Q. Z. Chen1, N. Zhu1,2, H. Wang1, L. Xie1, F. Luo1, H. Xiong1, Y. Feng1, M. Yue3, Y. Zhang4, W. Hou1
1Chinese
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
of Basic Medical Sciences, Hubei University of Science and Technology, Department of Microbiology, Xianning, China
3The First Affiliated Hospital of Nanjing Medical University, Department of Infectious Diseases, Nanjing, China
4Institute of Military Medical Sciences,Nanjing Command, Nanjing, China
2School
Hantaan virus (HTNV), a member of the Bunyaviridae family, can cause a hemorrhagic fever with renal syndrome that has
high fatality rates in humans. However, there are no specific antivirals available for HTNV infection. Although interferon
lambdas (IFN-λs) have been shown to induce an antiviral state against HTNV, the molecular mechanisms remain to be
determined. We found that IFN-λs exerted its anti-HTNV function by activating JAK-STAT pathway-mediated antiviral
immunity in A549 cells. Simultaneously, IFN-λs downregulated SOCS proteins, which are the known negative feedback
regulators of the JAK-STAT signaling pathway. Furthermore, we used both ectopic expression and gene-silencing
techniques to show the roles of myxovirus resistance 2 (Mx2, also known as MxB) protein as a potential inhibitor of HTNV
infection. These findings indicate IFN-λs play a role in cellular defenses against HTNV infection at an early stage of the
infection and that Mx2 may represent a potential therapeutic target.
Corresponding author:
Wei Hou
[email protected]
Innate Immunity
P55
Suppression of chikungunya virus and differential innate responses of human peripheral blood mononuclear cells
during co-infection with dengue virus
J. A. Aguilar Briseño1, M. Ruiz Silva1, H. van der Ende- Metselaar1, J. Smit1, I. A. Rodenhuis-Zybert1
1University
Medical Center Groningen, Medical Microbiology, Groningen, Netherlands
Dengue and chikungunya are viral diseases transmitted to humans by infected mosquitoes. It is estimated that dengue virus
(DENV) infects 390 million people per year making it the most prevalent arboviral infection. During the last decade
chikungunya virus (CHIKV) has caused large outbreaks and has expanded its circulation territory causing millions of cases in
Asia, Africa and America. The viruses share a common mosquito vector and cause potentially severe and/or debilitating
chronic disease. The growing overlap in the geographical distribution of these mosquito-borne infections has led to an
upsurge in reported cases of DENV/CHIKV co-infections. Unfortunately as yet, we have little understanding of the
consequences of co-infection. The overall aim of this study was to explore the effects of the DENV/CHIKV co-infection on
viruses replication and on the innate immune responses of infected peripheral blood mononuclear cells (PBMCs). We
demonstrate that the concomitant infection resulted in the significant reduction of CHIKV with a moderate enhancement of
DENV replication. Notably, the inhibitory effect of DENV on CHIKV infection occurred regardless of DENV replication.
Analysis of the immune mediators produced during mono vs co-infections by multiplex immunoassay showed changes in
type I IFN, IL-6, IL-8, TNF-α, MCP-1 and IP-10 production under these conditions. This study is the first step in determining
the interplay of DENV/CHIKV during concomitant infection.
Corresponding author:
José Alberto Aguilar Briseño
[email protected]
Innate Immunity
P56
Control of human cytomegalovirus infection by murine and human monocyte-derived dendritic cells
B. Kasmapour1, J. Holzki1, T. Kubsch1, U. Rand1, C. Sinzger2, L. Cicin-Sain1
1Helmholtz
2Ulm
Center for Infection Research, Braunschweig, Germany
University Medical Center, Institute of Virology, Ulm, Germany
Cytomegalovirus (CMV) is a ubiquitous beta-herpesvirus whose reactivation from latency is a major cause of morbidity and
mortality in immunocompromised hosts. We have previously shown that myeloid dendritic cells (mDC) secreted soluble
antiviral factors upon contact with CMV (MCMV) infected endothelial cells, but not upon direct infection of mDC in
monocultures. Supernatants blocked CMV replication at the level of immediate-early gene expression, but upon DC removal,
CMV infection rapidly re-initiated, consistent with latency onset.
Here we report that human monocyte derived dendritic cells (moDCs) are also capable of controlling human CMV (HCMV)
infection. The moDCs reversibly block HCMV replication and spread in co-culture with human fibroblasts in an effector to
target ratio dependent manner. The cell free supernatant of this co-culture maintained the antiviral effect for days, arguing
that this is driven by soluble factors secreted by the moDCs into the co-culture supernatant. Blocking interferon receptor
function in the infected target cells showed no rescue of viral growth, suggesting that IFN type I is not the main mechanism of
the antiviral action. Furthermore, virus control or spread was not rescued by nitric oxide (NO) donors or inhibitors, arguing
that NOs are also not a significant factor.
Remarkably, the co-culture supernatant generated in the murine system was capable of controlling the spread of HCMV
infection in human cells. This cross-species effect strongly argued that the active antiviral compound is an innate immune
mechanism that was conserved through evolution and acts on functionally similar targets in mice and men. We are currently
working on identifying the antiviral compound(s) and characterizing the dynamics and mechanism of their action during
MCMV and HCMV infection.
Corresponding author:
Bahram Kasmapour
[email protected]
Innate Immunity
P57
Identification and molecular characterization of a novel inhibitory feedback loop of RIG-I signaling mediated by
DAPK1
J. Willemsen1, O. Wicht2, J. Wolanski1, N. Baur2, S. Bastian1, D. A. Haas3, P. Matula4, B. Knapp5, L. Meyniel-Schicklin6, C.
Wang7, R. Bartenschlager8, V. Lohmann8, K. Rohr5,9, H. Erfle5, L. Kaderali5,10, J. Marcotrigiano7, A. Pichlmair3, M. Binder1
1DKFZ
Heidelberg, Group "Dynamics of early viral infection and the innate antiviral response" (F170), Heidelberg, Germany
Faculty Heidelberg, Dept. Infectious Diseases, Molecular Virology (AG Binder), Heidelberg, Germany
3Max Planck Institute of Biochemistry, Martinsried/Munich, Germany
4Masaryk University, Brno, Czech Republic
5University of Heidelberg, Heidelberg, Germany
6ENYO Pharma, Lyon, France
7Rutgers University, Piscataway, NJ, United States
8University of Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
9DKFZ Heidelberg, Heidelberg, Germany
10Ernst Moritz Arndt University Greifswald, Institute of Bioinformatics, Greifswald, Germany
2Medical
The pattern recognition receptor RIG-I is a pivotal sensor of viral infections. Its activation by 5′-triphosphorylated or doublestranded RNA leads to subsequent signaling via MAVS, TBK1 and IKK epsilon resulting in IRF3 nuclear translocation.
Activated IRF3 induces transcription of type I and type III interferons and several interferon stimulated genes. Despite
intensive investigations on the RIG-I signaling pathway, its regulatory network still remains largely elusive.
To gain more insight into the complex regulation of this pathway a kinome-wide siRNA screen was performed. The primary
screen revealed over 100 siRNAs that significantly altered the translocation of IRF3 to the nucleus upon RIG-I stimulation.
The top 50 candidates were further analyzed in three independent validation screens based on IRF3-sensitive promoter
reporter assays or Rift-valley-fever virus replication. Taking all three validation screens into account, 21 novel regulators of
the RIG-I signaling pathway could be identified. Relevance of the identified hits in regulating the host-cell antiviral defense
was demonstrated by analyzing their impact on Influenza A virus replication.
In the course of this screen, DAPK1 was identified as an inhibitor of RIG-I mediated IRF3 activation. DAPK1 was further
shown to be activated during RIG-I stimulation, therefore constituting a bona fide negative feedback loop. Accordingly,
knock-down of DAPK1 resulted in increased and prolonged production of interferon beta and IFIT1. Extensive mapping
experiments revealed a minimal construct, including the kinase domain, to be sufficient for inhibiting IRF3 reporter activation
in over-expression experiments. Furthermore, interaction studies revealed binding of DAPK1 to RIG-I, suggesting that a
DAPK1 mediated phosphorylation of RIG-I inhibits its activity. In fact, by means of an in vitro kinase assay we could
demonstrate that RIG-I is a substrate of DAPK1. Subsequent phospho-specific mass spectrometry analysis identified sites in
RIG-I that are phosphorylated by DAPK1. Using phosphomimetic mutants of the identified sites we could show that
phosphorylation at a single amino acid completely abrogated RIG-I signaling and thus strongly increased influenza virus
infections.
In conclusion, by means of high throughput screening we identified a novel feedback regulation of the antiviral RIG-I
signaling pathway in which DAPK1 gets activated and subsequently inhibits signaling by phosphorylation of the sensor RIG-I.
Corresponding author:
Marco Binder
[email protected]
Figure 1
Innate Immunity
P58
kinetic characterization of the RIG-I signal transduction pathway
J. Frankish1, D. Schweinoch2, L. Kaderali2, M. Binder1
1DKFZ,
2Ernst
Virus Associated Carcinogenesis, Heidelberg, Germany
Moritz Arndt University Greifswald, Institute of Bioinformatics, Greifswald, Germany
A group of pattern recognition receptors (PRRs), known as the RIG-I-like-Receptors (RLRs), are responsible for recognizing
and responding to RNA derived from pathogens, such as viruses. One member of this family of receptors is Retinoic Acid
Inducible Gene I (RIG-I), which responds to 5-triphosphorylated double stranded RNA (5 3P dsRNA). RIG-I then initiates a
signaling cascade that results in the activation of specific transcription factors (including IRF3 and NFKB) to activate an
innate antiviral response. While many characteristics of the specific signaling network have been described in detail, the
dynamics and the regulation of specific signaling events is still poorly understood. By examining each major factor within the
signaling network in terms of activation and regulation dynamics, a better understanding can be made of how viruses interact
with the host. Mathematical modelling of both, viral replication dynamics and dynamics of the antiviral signaling, will allow for
studying the intricate functional relationship between viral processes and host cellular responses. This will furthermore
deepen our understanding of the molecular foundation for the establishment of chronicity in certain viral infections, such as
Hepatitis C virus, in contrast to efficient viral clearance in other cases, for example Influenza A virus.
Corresponding author:
Jamie Frankish
[email protected]
Innate Immunity
P59
DAPK family members are negative regulators of RIG-I signalling
J. Wolanski1, J. Willemsen1, M. Binder1
1DKFZ,
Virus Associated Carcinogenesis, Heidelberg, Germany
We recently identified death-associated protein kinase (DAPK) 1 as a potent negative feedback inhibitor of the retinoic acid
inducible gene I (RIG-I) signalling pathway. This signalling cascade, initiated upon RNA virus recognition, results in type I
interferon production and transcription of interferon stimulated genes (ISGs). Further analyses of closely related DAPK family
members DAPK2 and DAPK3 showed that these kinases also negatively regulate RIG-I-mediated antiviral signalling.
Although we observed direct interaction with RIG-I only for DAPK2, both kinases potently inhibited IRF3 activation upon overexpression, independent of endogenous DAPK1. SiRNA-mediated down-regulation of DAPK3 not only enhanced IRF3
activation upon stimulation, but this increase in signalling activity was also significantly more pronounced when combined
with DAPK1 knockdown. Mutational analyses revealed that the effect was kinase-dependent for both DAPK2 and DAPK3
since truncation mutants comprising only the kinase domains, as well as constitutively kinase-active mutants, acted in the
same fashion as the full-length proteins, whereas kinase-deficient mutants did not confer inhibition of IRF3 activation.
Indeed, RIG-I was phosphorylated by DAPK2 and DAPK3 in vitro. Notably, while all DAPKs induced apoptosis when overexpressed in target cells, there was no effect on IRF3 activation upon pharmacological inhibition of apoptosis, arguing that
the effect observed on RIG-I signalling was not due to induction of cell death. Additionally, none of the examined cell types
showed pronounced induction of autophagy upon DAPK over-expression, although this pathway has been described to be
activated by DAPKs.
Taken together, these results demonstrate a role for DAPK2 and DAPK3, in addition to that previously found for DAPK1, as
strong negative regulators of RIG-I signalling. This regulation happens independent of known DAPK functions such as
apoptosis and autophagy induction. Although they seem to be acting independently of each other, there is most probably
interaction between DAPKs, allowing flexible and exact regulation of RIG-I signalling.
Corresponding author:
Julia Wolanski
[email protected]
Innate Immunity
P60
Dissecting molecular mechanisms of inhibition of HCV RNA replication by interferon
A. Pandey1, C. Dächert1, D. Schweinoch2, J. Frankish1, C. Zitzmann2, M. Schulze3, L. Kaderali2, M. Binder1
1DKFZ,
Virus Associated Carcinogenesis, Heidelberg, Germany
Moritz Arndt University Greifswald, Institute of Bioinformatics, Greifswald, Germany
3Technical University of Dresden, Dresden, Germany
2Ernst
Chronic Hepatitis C virus (HCV) infection carries a significant clinical burden with an estimated 150 million infected people
world-wide and more than 1 million deaths every year. It is a major causative agent of hepatitis and infection can progress to
chronic liver inflammation, liver cirrhosis and cancer. Previously established interferon (IFN)-based therapy is associated with
adverse effects and often fails to induce a sustained response. New generation of treatment options offer cure rates of above
90%, but those are still very expensive and have limited availability.
HCV triggers the host immune responses during its course of infection. However, the virus manages to evade the host
defense mechanisms and is able to establish a low-level replication leading to decades-long persistence. Our studies
therefore aim at deciphering this intricate and dynamic interplay between antiviral host factors and HCV replication. A
particularly interesting question is, how HCV successfully persists in a context of an intact type I IFN system despite its
generally high sensitivity towards IFN. Addressing open questions in such a complex system by experimentation is often
limited by technical feasibility. Thus, we utilize a previously developed detailed mathematical model describing the
intracellular life cycle of HCV RNA to complement our experimental results. We are using this model for dissecting the
molecular mechanisms by which the IFN response inhibits HCV RNA replication. For this purpose, we quantify viral
components in a time-resolved manner (luciferase/viral protein by Western blot; RNA by qPCR and Northern blot) in the
presence or absence of IFN. We further compare the observed effects to the effects of direct-acting antiviral drugs with
known and well-defined points of attack (e.g. protease inhibition, polymerase inhibition). Thereby, we complement and
confirm model predictions about the mode of action of HCV inhibition by IFN. Ultimately, we aim at expanding our model to
include the whole viral life cycle and the host cells antiviral response, which will allow us to better grasp the underlying
mechanisms allowing for viral persistence in the face of an operational antiviral response.
Corresponding author:
Aparna Pandey
[email protected]
Innate Immunity
P61
In vitro evolution of persistent rabies virus – a role for pattern receptors?
M. Eizinger1, A. A. Hennrich1, K. M. Sparrer1,2, A. Ghanem1, K. K. Conzelmann1
1Ludwig
Maximilian University Munich and Max von Pettenkofer Institute and Gene Center, Munich, Germany
of Chicago, Department of Microbiology, Chicago, United States
2University
Little is known about the molecular mechanisms by which RNA viruses can establish prolonged and persistent infections and
why intrinsic immune responses fail to recognize and clear such infections.
The attenuated neurotropic rabies virus (RABV) SAD L16 does not exhibit a strong CPE in cell culture or in vivo, but longer
infection is harmful to the cell. We have generated non-toxic, "persistent" RABV variants by continuous passage of infected
293T cells and selection of surviving cells. By deep sequencing of the virus pool we identified several abundant point
mutations, which were introduced individually or in combination into recombinant viruses. A single site mutant (A) showed a
persistent, nontoxic phenotype both in 293T cells and in mESC-derived neurons, similar to a double mutation (A+B).
Remarkably, the single point mutant (B) virus revealed high cytotoxicity, suggesting that the A mutation is dominant with
respect to preventing or counteracting cell killing.
Notably, the persistent viruses induced very little IFN-β in infected cells, suggestive of a failure of recognition by pattern
receptors. However, as revealed by infection of RIG-I- and MAVS-KO cells, RLR signaling is not required for cell death
induced by the cytotoxic mutant viruses. By comparing the transcriptomes of infected cells, a typical stress-signature was
observed. Current experiments, including CRISPR/Cas9 genome-wide KO screens, are aimed at identification of the killing
pathways involved and how persistent viruses can avoid cytotoxicity.
Corresponding author:
Karl-Klaus Conzelmann
[email protected]
Innate Immunity
P62
IFN inhibition by rabies and other lyssaviruses – identification of critical residues in the viral phosphoprotein P
M. Wachowius1, T. Halbach1, K. K. Conzelmann1
1Ludwig
Maximilian University Munich and Max von Pettenkofer Institute and Gene Center, Munich, Germany
The phosphoprotein P of the neurotropic rabies virus (RABV) is a potent antagonist of the host interferon response in human
and murine cells, preventing both PAMP-mediated IFN induction and IFN-mediated JAK/STAT signaling. In addition, P is an
essential factor for genomic RNA encapsidation and transcription of viral RNA. We have previously identified RABV P
deletion mutants defective for inhibition of IFN induction but retaining all other functions. RABVs carrying the deletion are
attenuated in mice after intracerebral injection, indicating a critical role of IFN escape in pathogenesis.
We here assessed the ability of the P proteins of diverse RABV and other lyssaviruses to interfere with IFN induction in
human cells. We have identified P proteins from all biotypes that completely failed in preventing IRF3 activation and IFN
induction upon expression from plasmids or in the context of recombinant RABV. Bioinformatics analyses of the proteins led
to the identification of potentially relevant individual residues. By pull down experiments, mass spectrometry, and IFN assays,
we could identify two single amino acid residues which are essential for binding of P to its target protein and IFN inhibition,
respectively. Recombinant viruses carrying the individual mutations induced high levels of IFN while exhibiting growth
kinetics of the parental virus in cell culture and therefore represent promising candidates for more immunogenic life vaccines.
Corresponding author:
Karl-Klaus Conzelmann
[email protected]
Innate Immunity
P63
Phenylalanines 75 and 78 of IFITM3 are dispensable for normal membrane targeting and IFITM3-IFITM3 interactions
M. Winkler1, F. Wrensch1, P. Bosch1, S. Gärtner1, M. Knoth1, M. Schindler2, S. Pöhlmann1
1German
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
2University
Introduction: The expression of interferon-induced transmembrane proteins (IFITMs) is induced by interferons and IFITM 13 from different species have been shown to block host cell entry of diverse enveloped viruses. IFITMs inhibit virus entry at
the step of membrane fusion but the molecular mechanism has not been fully established. Several hypotheses have been
proposed, among them IFITM-induced accumulation of cholesterol in endosomal compartments and enhancement of
membrane rigidity via interactions between IFITM molecules.
Objectives: We have previously shown for influenza A virus that sensitivity to cholesterol accumulation in endosomes does
not correlate with sensitivity to the antiviral activity of IFITMs. Therefore, we addressed the hypothesis that interactions
between IFITM molecules are required for antiviral activity.
Material & Methods: Interactions between membrane proteins are difficult to analyze since they are influenced by
membrane lipid composition. With biochemical methods being prone to artifacts, we sought to establish a method amenable
to study close localization or interaction between membrane proteins in intact cells.
Results: For the analysis of IFITM-IFITM interactions, we established a FACS-based FRET assay (FACS-FRET), for which
potentially interacting proteins are fused to fluorescent proteins, CFP or YFP. Upon excitation of CFP, energy can be
transferred in a radiation-free form to YFP, if both molecules are located in close proximity, and the resulting YFP signal
serves as a measure for co-localization. Employing appropriate controls for membrane associated proteins we obtained
robust signals in cells co-expressing IFITM1-3-CFP and IFITM1-3-YFP, while signals measured for IFITM5 were reduced.
This finding is compatible with extensive homo- and heterotypic interactions between IFITM1-3. However, mutation of
phenylalanines 75 and 78, which were previously described to be required for self-interactions of IFITM3, did not reduce the
FACS-based FRET signal and did not interfere with IFITM3-IFITM3 co-immunoprecipitation. However, mutation of
phenylalanines 75 and 78 markedly reduced with IFITM3 antiviral activity.
Conclusion: Our results suggest that phenylalanines 75 and 78 are required for IFITM3 antiviral activity but dispensable for
IFITM3-IFITM3 interactions.
Corresponding author:
Michael Winkler
[email protected]
Innate Immunity
P64
Rhesus macaque IFITM3 gene polymorphisms and SIV infection
M. Winkler1, S. Gärtner1, F. Wrensch1, M. Krawczak2, U. Sauermann3, S. Pöhlmann1
1German
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
Albrechts University, Institute of Medical Informatics and Statistics, Kiel, Germany
3 German Primate Center GmbH, Infection Models Unit, Göttingen, Germany
2Christian
Introduction: Interferon-induced transmembrane proteins (IFITMs) are important antiviral effectors of the innate immune
system, which inhibit cell entry of a broad range of enveloped viruses. IFITM3 polymorphisms can modulate influenza A virus
disease severity and AIDS progression in human patients, indicating that IFITM3 contributes to innate antiviral defenses in
the infected host. The experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) is an important
model for HIV infection of humans. However, it is at present unknown whether the IFITM3 gene of rhesus macaques has
sequence polymorphisms and whether polymorphisms impact SIV infection.
Objectives: The aim of our study was to determine whether the IFITM3 gene of rhesus macaques has sequence
polymorphisms and whether these polymorphisms impact viral load and disease progression in the context of SIV infection.
Material & Methods: We determined sequences of IFITM3 genes from rhesus macacques, which were experimentally
infected with SIV, and correlated polymorphisms with survival time and virus load.
Results: We found that host cell entry of MLV-particles pseudotyped with the SIVmac239 envelope (Env) protein was
inhibited by rhesus macaque (rh) IFITM3, albeit with reduced efficiency as compared to its human counterpart. Sequencing
of the IFITM3 gene from 94 SIV-infected animals revealed 15 polymorphisms, most of which were located in non-coding
regions. In addition, three synonymous changes were detected in the coding region. When analyzing the relationship
between these polymorphisms and AIDS-free survival time, three polymorphisms showed a nominally significant association
in the logrank test. The associations found for two of these polymorphisms were not significant when analyzed by the
Wilcoxon-test, while the association between polymorphism g.279 G>A and survival time was significant in both tests.
Finally, no significant association between rhIFITM3 polymorphisms and virus load at set point were discovered.
Conclusion: Our findings suggest that some polymorphisms in rhIFITM3 may impact AIDS-free survival time but not virus
load. However, a larger number of animals needs to be analyzed to substantiate the potential importance of these
polymorphisms.
Corresponding author:
Michael Winkler
[email protected]
Diagnostic Tools
P65
Rapid and sensitive detection of the coat protein gene of red seabream iridovirus (RSIV) and nervous necrosis virus
(NNV) by loop-mediated isothemal amplification (LAMP)
J. Hwang1,2, S. Y. Park1, T. K. Lee1
1Korea
Institute of Ocean Science & Technolgy, South Sea Environment Research, Geoje, South Korea
Introduction: Loop-mediated isothermal amplification (LAMP) of DNA is currently one of the commonly used molecular
diagnostic tools due to the speed and ease with which it can amplify target DNA under isothermal conditions. In the present
study, a LAMP test was designed for the coat protein gene of Red seabream iridovirus (RSIV) and Nervous necrosis virus
(NNV), which has been responsible for major epidemics in the aquaculture industry in many Asian countries resulting in huge
economic losses at fish farm. Using a set of synthesized primers matching the sequences of a specific region of the RSIV
and NNV genes from the NCBI database, not originating from RSIV and NNV-infected fish.
Objective: The aim of this study, we developed a novel and highly specific LAMP assay for the identification of RSIV and
NNV infection.
Material & Methods: Using a set of synthesized primers matching the sequences of a specific region of the RSIV and NNV
genes from the NCBI database, not originating from RSIV and NNV-infected fish, the efficiency and specificity of LAMP were
measured dependent on the concentration of DNA polymerase and the reaction temperature and time. Also, In order to
compare the sensitivity of the LAMP and PCR assays, the synthesized RSIV and NNV template was serially diluted. In
addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between other virus
families.
Results: The optimized LAMP reaction carried out at 54~64 °C for 60 min, and above 4 U Bst DNA polymerase for RSIV
and NNV. The sensitivity of LAMP for the detection of RSIV and NNV were thus about 10 times greater than the sensitivity of
PCR. Finally, we demonstrated that the LAMP assay primers were specific for the detection RSIV and NNV infection in wild
fish.
Conclusion: In this study, using synthesized nucleotides for an RISV and NNV genes, we established optimal LAMP
conditions for the detection of RSIV and NNV infection in wild fish. Furthermore, we observed that the synthesized
sequences could be efficiently used to determine the presence of RSIV and NNV. Collectively, these findings indicate that
the design of LAMP primers based on genetic information from a public database may provide a simple method for detecting
viral infections in marine organisms.
Corresponding author:
Jinik Hwang
[email protected]
Diagnostic Tools
P66
Efficient detection of pathogen virus in sand dabs, Paralichthys olivaceus using loop-mediated isothermal
amplification (LAMP)
J. Hwang1, S. Y. Park1, T. K. Lee1
1Korea
Institute of Ocean Science & Technolgy, South Sea Environment Research, Geoje, South Korea
Introduction: Viral hemorrhagic septicemia virus (VHSV) and marine birnavirus (MABV) are the causative pathogens for
some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses
in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the
prevalence of pathogen infection. The loop-mediated isothermal amplification (LAMP) of DNA is currently one of the most
commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal
conditions.
Objective: The aim of this study is to establish a novel and highly specific loop-mediated isothermal amplification (LAMP)
assay for the identification of VHSV and MABV infection.
Material & Methods: Using a set of synthesized primers matching the sequences of a specific region of the VHSV and
MABV genes from the NCBI database, not originating from VHSV and MABV-infected fish, the efficiency and specificity of
LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In
addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between VHSV and
MABV and other virus families, Red seabream iridovirus (RSIV) and Nervous necrosis virus (NNV).
Results: The optimized LAMP reaction carried out at 54~64 °C for 60 min, and above 4 U Bst DNA polymerase for VHSV
and MABV. The sensitivity of LAMP for the detection of VHSV and MABV were thus about 10 times greater than the
sensitivity of PCR. Finally, we demonstrated that the LAMP assay primers were specific for the detection VHSV and MABV
infection in wild fish, Paralichthys olivaceus.
Conclusion: These finding suggest that the development of LAMP primers based on genetic information from a public
database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic
organisms.
Corresponding author:
Jinik Hwang
[email protected]
Diagnostic Tools
P67
HIV drug resistance in 2 and 1% proportions with NGS in virologic relevant quantities
R. Ehret1, M. Schütze1, M. Obermeier1
1MIB,
Berlin, Germany
Background: NGS technologies have made their way into routine-diagnostics.. The relevance of mutations detected in low
frequencies is still a subject of debate. Clinical data is rare. The report of mutations of at least 10% of the viral population is
chosen by many labs due to its equivalency to Sanger-sequencing minority detection. We here report the frequency of
additional mutations in population-proportions of greater than 2% and 1% in routine laboratory testing and correlate them to
viral load.
Methods: 645 HIV-1 resistance tests (reverse transcriptase/protease) performed between 10/2014 and 04/2016 with an in
house PCR followed by NGS (Illumina MiSeq, sequences reported with >100 reads only) were analyzed. Sequences were
interpreted by HIV-GRADE (http://www.hiv-grade.de) for resistance mutations using 10%, 2% and 1% minority cut-offs.
Besides the subtype and the overall increase in mutations, a specific focus laid on differences in reported resistance
associated mutations and resistance levels (e.g. additional drug class or further drugs same class). The proportion of
subpopulations harbouring additional mutations with greater than 1000 and 2000 c./mL(= mutational load) were calculated.
Results: 483 (74.9%) samples were identified as subtype B. No drug resistance associated mutations were reported by HIVGRADE for 44% with a 10% cut-off, 29.5% and 19.7% with 2% and 1% respectively. This also correlates with an increase of
resistance level in the interpretation, especially for NNRTIs. With a cut-off of 10% in 148 samples (105 non-B subtype) only
PI relevant mutations were detected. We found mutations only relevant for NRTIs in 21 samples and for NNRTI in 100
samples. Additional mutations could be detected in 94 of the samples using a 2% minority cut-off. This corresponds to an
additional mutational load of >1000 c/mL in 49 and >2000c/mL in 25 samples.
Conclusion: A relative high proportion (56%) of investigated sequences showed resistance mutations at a minority cut-off of
10%.. This high percentage of resistance increases substantially lowering the cut-off range to 2 or 1% not only by number of
mutation but also regarding resistance-levels. Despite the low percentage these viral quasispecies can be detected in a
relevant absolute quantity. There is a clear need for clinical evaluation of the relevance of mutations in the low percentage
range for resistance interpretation due to its broader use in clinical routine.
Corresponding author:
Robert Ehret
[email protected]
Diagnostic Tools
P68
Development of neutralization assay using an eGFP chikungunya virus based on a newly isolated strain
S. Q. Liu1, C. L. Deng1, L. L. Xu1, B. Zhang1
1Chinese
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
Abstract: Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging
pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an
infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage)
from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the
levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could
be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virusbased neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.
Figure 1: Isolation and characterization of CHIKV from clinical human cases. (a) the flow chart of virus isolation on C6/36
cells from human serum; (b) the CHIKV strain that was passaged for seven rounds on C6/36 cells showed apparent CPE on
BHK-21 cells; (c) plaque morphology of the CHIKV strain on BHK-21 cells on the four days post-inoculation; and (d)
phylogenetic analyses of CHIKV genome sequences using the neighbor-joining method.
Figure 2: Confirmation of the availability of eGFP-CHIKV in neutralization assay with the serum samples from CHIKVinfected patients (a) detection of eGFP expression by fluorescent microscope with different dilutions of human sera; (b)
quantification of neutralization assay with increasing serum dilution; (c) neutralizing activity of the human sera against WT
CHIKV based on PRNT assay; (d) detection of CHIKV genome copy number in BHK-21 cells treated with virus or virusantiserum mixture by real-time RT-PCR.
Corresponding author:
Si-Qing Liu
[email protected]
Figure 1
Figure 2
Diagnostic Tools
P69
CpG oligonucleotides increase HBV specific cytokine responses in whole blood and enhance cytokine release
assay sensitivity
W. Dammermann1,2, J. Dornbrack1,2, K. Bröker1,2, S. Lüth1
1Brandenburg
2University
Medical School, Center for Internal Medicine II, Brandenburg an der Havel, Germany
Medical Center Hamburg-Eppendorf, Department of Anatomy and Experimental Morphology, Hamburg, Germany
Question: Hepatitis B is one of the most common chronic infectious diseases worldwide. Many patients develop chronic
hepatitis B leading to liver cirrhosis or hepatocellular carcinoma. To develop a therapeutic vaccine for chronic hepatitis B
patients it is necessary to assess not only humoral, but also cellular immune responses to hepatitis B virus (HBV) antigens.
In this context our whole blood based cytokine release assay demonstrates a powerful, easy and rapid screening tool to
analyze HBV specific cellular immunity.
We investigated the potential of Toll like receptor (TLR) 9 agonists, i.e. CpG oligonucleotides, as costimulators in our HBV
specific cytokine release assay in order to increase diagnostic sensitivity and specificity.
Methods: Whole blood from 80 healthy individuals (n=51 hepatitis B vaccinated, n=29 unvaccinated) was stimulated with
hepatitis B surface antigen (HBsAg) or hepatitis B core antigen (HBcAg) in presence or absence of CpG oligonucleotides.
Subsequently, IL-2 and IFNγ secretion in plasma was assessed using ELISA.
Results: Our data demonstrate that CpG oligonucleotides specifically enhance HBsAg-mediated IL-2 (276 ± 79 pg/ml vs.
320 ± 82 pg/ml) and IFNγ (77 ± 35 pg/ml vs. 401 ± 121 pg/ml) responses in whole blood. When IFNγ release was
considered as readout depicting the hepatitis B vaccination status, the assay reached a diagnostic sensitivity of 61% without,
but of 76% with additional CpG oligonucleotide stimulation at a diagnostic specificity of 90%. IL2 release after HBsAg
stimulation depicted hepatitis B vaccination status unaltered with a diagnostic sensitivity and specificity of 85 % and 90 %.
Conclusion: Taken together, our results show that innate signals mediated for instance via TLRs contribute to HBV-specific
cellular immune responses. The improved diagnostic sensitivity of the cytokine release assay for IFNγ after addition of CpG
oligonucleotides demonstrates that these effects can be exploited in order to make whole blood based cytokine release
assays even more powerful as screening tools in HBV immunology and HBV vaccination studies.
Corresponding author:
Werner Dammermann
[email protected]
Diagnostic Tools
P70
Detection of viruses in environmental samples using NGS
A. Bogoyavlenskiy1, M. Alexyuk1, P. Alexyuk1, A. Turmagambetova1, V. Berezin1
1Institute
of Microbiology and Virology, Virology, Almaty, Kazakhstan
Diagnostics of viral infections is on the verge of creating of new theories, hypotheses and discoveries with the advent of
NGS. This is due to several reasons, the most important of which are a multiple increase of data about the availability of
viruses in the environment, including soil, water, feces, air, etc., as well as the ability to analyze of viruses without their
cultivation.
In our research, we studied the biodiversity of viruses in the water reservoirs of Almaty region. Sequencing was carried out
by a double-barrel shotgun method. In this case the useful information could be obtained by paired-end sequencing of DNA
fragment. These two sequences are oriented in opposite directions and along of the length of the fragment can be separated
from each other, and also can be used for genome assembling using different software. In our research was used the HiSeq
sequencing system and Edena software. Total contigs were 447,000 with a length of 200 to 80,000 bp. The Metavir2
program selected the 184,431 contigs and the 249,780 of which was identified as viral gene sequences, and 157,000 of
which are previously unknown viral sequences.
Bacteriophages, algae viruses and viruses of the protozoa were the 97% of total viruses of this water sample. Other 3%
included the viruses capable of causing of the disease of animals, higher plants and humans. Among them: 2 families of
retro-transcribing viruses (Retroviridae, Caulimoviridae), 2 families of single-stranded RNA viruses (ssRNA viruses), family of
single-stranded DNA virus (ssDNA viruses - Inoviridae), family of double-stranded RNA virus (dsRNA viruses Endornaviridae) and 20 families of double-stranded DNA viruses (dsDNA viruses, among them Herpesviridae, etc) were
detected.
Thus, the NGS is opening a new era in the development of monitoring of viral infections that allows take a different look at
the ecology of viruses.
Corresponding author:
Andrey Bogoyavlenskiy
[email protected]
Diagnostic Tools
P71
Development and validation of an immunohistochemistry procedure for the detection of a neurotropic bovine
astrovirus
C. Boujon1, S. Selimovic-Hamza1, I. Bouzalas1, T. Seuberlich1
1University
of Bern, Division of Experimental Clinical Research, Bern, Switzerland
Members of the Astroviridae family are best known to cause diarrhea in different mammalian species. Lately, some
strains have been associated with encephalitis in humans, minks and cattle. In this study, we developed an
immunohistochemistry (IHC) procedure for the detection of a neurotropic bovine astrovirus (BoAstV-CH13/NeuroS1),
which is associated with non-suppurative encephalitis in cattle. We expressed five recombinant antigens
corresponding to different putative viral proteins of BoAstV-CH13/NeuroS1. Antigens were then used for the
production of hyperimmune sera in rabbits. Out of the five hyperimmune sera, the one directed against the conserved
N-terminus of the viral capsid protein, termed ORF2-con, clearly surpassed the others in the detection of viral
antigens in IHC in terms of strong signal intensity and low background staining. The accuracy of the ORF2-con IHC
protocol was then evaluated using different sets of brain tissue samples: 30 samples from 9 animals with confirmed
BoAstV-CH13/NeuroS1 infection, 30 samples from 8 animals with non-suppurative encephalitis of another etiology
and 30 samples from apparently healthy slaughtered animals. The IHC was positive only with tissue samples from
animals with a known positive BoAstV-CH13/NeuroS1 status, but not with those from negative ones, indicating a good
diagnostic sensitivity and specificity of the assay. The ORF2-con IHC procedure is therefore an adequate tool for the
detection of BoAstV-CH13/NeuroS1 infections in cattle.
Corresponding author:
Céline Boujon
[email protected]
Figure 1
Figure 2
Diagnostic Tools
P72
Comparison of two immunoassays for simultaneous detection of HCV antigen and antibodies among HCV/HIV Coinfected patients in dried serum spots
A. Eshetu1, B. Bartmeyer2, V. Bremer2, D. Schmidt2, C. T. Bock3, N. Bannert1, A. Hauser1
1Robert
Koch Institute, Division of HIV and Other Retroviruses, Berlin, Germany
Koch Institute, Division of HIV/AIDS, STI and Blood-borne Infections, Berlin, Germany
3Robert Koch Institute, Division for Viral Gastroenteritis and Hepatitis Pathogens and Enteroviruses, Berlin, Germany
2Robert
Introduction: Hepatitis C virus (HCV) antigen and antibody combination assays have been launched as a cost-effective
alternative to nucleic acid testing (NAT) for early/rapid diagnosis by reducing the window period (~ 60 days).
Objective: To evaluate the clinical performance of two HCV antigen/ antibody combination assays during window period
using HCV seroconversion panels followed by NAT for ELISA reactive samples.
Material & Methods: The performance of the Monolisa HCV antigen-antibody Ultra (Monolisa, Bio-Rad) and the Murex
Ag/Ab test (Murex, Abbott) were compared by using two sets of serially diluted commercial HCV seroconversion panels
[Genotypes (Gt) 1a & 2b]. Moreover, both assays were used to screen out HCV co-infection in filter dried serum spots (DSS)
of newly diagnosed HIV-cases (n=1683). All samples positive for both tests were further evaluated in the quantitative RTPCR (qRT-PCR) developed to amplify the 5' non-coding region of HCV genome.
Results: For lower dilutions of commercial HCV seroconversion panels, the Murex detected HCV infection 9 (Gt 1a) and 2
(Gt 2b) days earlier than the Monolisa. However, Murex showed fluctuating and lower OD values below the cut-off for higher
dilutions until days 28 (Gt 1a) and 135 (Gt 2b) compared with the Monolisa which showed a progressive increase in the OD
value for all dilutions in each panel. Accordingly, 219/1683 (12.9%) and 193/1683 (11.4%) DSS samples were found positive
in the Murex and Monolisa, respectively. Further testing of all ELISA reactive samples by qRT-PCR revealed 167 NAT
positive results. Of these, 74 (44.3%) were also positive for both, the Murex and the Monolisa. Moreover, 10/167 (5.9%) of
samples positive in the HCV RNA assay were only positive in the Monolisa while 5/167 (2.9%) were only positive in the
Murex ELISA.
Conclusion: The Monolisa provides more reliable results for the detection of HCV infections in all dilutions compared to the
Murex indicating its potential use for HCV screening in DSS. However, with its reactivity at earlier days of HCV infection, the
Murex may serve as an additional diagnostic tool in HIV/HCV co-infected patients to narrow the window period. Moreover,
the application of NAT on ELISA positive samples can be used to confirm these early infections and to distinguish between
active/chronic and resolved HCV infections.
Corresponding author:
Amare Eshetu
[email protected]
Diagnostic Tools
P73
Elaboration of diagnostic tool for detection of antibodies to Newcastle disease virus
O. Blotska1
1The
State Scientific Control Institute of Biotechnology and Strains of Microorganisms, Biotechnology and Control of Quality of Viral Preparations, Kyiv,
Ukraine
Objective: Newcastle disease (ND) is one of the most important infectious viral diseases of poultry. The world-wide
economic loss from it is 2-3 billion USD per year.It is necessary to control the intensity of post-vaccination immunity in poultry
and the timing of revaccinations. OIE recommends enzyme linked immunosorbent assays(ELISA)and haemagglutination
inhibition (HI)test for these purposes. Advantages that are favorably distinguishes HI test are:quick, reliable, cheap, accurate
and valuable.
Methods: During the development of a new diagnostic tool, we used the reference strain "La-Sota". We have produced
haemagglutinating antigen using SPF fowl eggs. For NDV inactivation, we used aminoethyleneimine. Positive serum was
prepared by immunizing 60-day-old chickens with active virus once and by inactivated virus twice. Negative serum was
obtained from healthy birds. Samples of 1% suspension of chicken erythrocytes in phosphate buffered saline (pH 7.0-7.1)
were used.
Results: For estimation of sensitivity and specificity of HI test kit on development stage were used antigen and sera from
commercial diagnostic kits. We also used certified negative control serum and samples of International Standard Sera, which
were obtained from Reference Laboratories against the variety pathogens.
Comparison between the national diagnostic test kit for HI and commercial ELISA kit (IDEXX) in the evaluation of humoral
immune response to ND in vaccinated chickens was investigated by examining of serum samples (n=152). Statistical
analysis of data showed that the correlation coefficient for the results of both tests was 0.92. The relative sensitivity of HI test
kit was 93.5% and the relative specificity - 91.5%.
In order to ensure a high degree of specificity for the antigen, special attention was given to the selection of a stabilizer for
freeze-drying (patent pending).
We have developed the regulatory documents, completed the procedure of validation and registration in Ukraine of the
commercial HI test kit for the detection of antibodies to NDV.
Conclusion: Has been elaborated highly sensitive and a highly specific diagnostic tool for the detection of antibodies to NDV
based on HI assay that helps to maintain the disease-free status of the Ukrainian poultry industry with regard to ND.
Corresponding author:
Oksana Blotska
[email protected]
Diagnostic Tools
P74
A cell-based ELISA testing for HIV-1 neutralizing antibodies
D. Ivanusic1, J. Denner1
1Robert
Koch Institute, Berlin, Germany
Introduction: HIV-positive patients develop antibodies against the HIV envelope protein, only a few of them, termed
neutralizing antibodies can block viral entry into HIV target cells, among them a few broadly neutralizing antibodies which
binds to the membrane proximal ectodomain region (MPER) of the transmembrane envelope protein gp41 of HIV-1 such as
2F5 and 4E10. At present most attempts to induce such antibodies failed.
Objectives: In order to detect such antibodies a cell-based ELISA was developed which is based on the expression of the
MPER epitopes on the surface of HeLa cells. The developed cell-based ELISA was developed expressing either very small
proteins or larger proteins up to 28 kDa.
Material & methods: To express the MPER epitopes on the cell surface, sequences of the tetraspanins CD63 or CD82 were
used as an anchor for translocation to the plasma membrane. Expression vectors with sequences corresponding to the
epitopes of the broadly neutralizing antibodies 2F5 and 4E10 together with teraspanin sequences were transfected
transiently into HeLa cells seeded in a 96-well plate using MetafectenePro. After 24 h post-transfection binding of HIV-1
neutralizing antibodies anti-2F5 or anti-4E10 was tested and in addition protein expression of the HIV-1 epitopes were
analysed using confocal laser scanning microscopy and Western blot analysis.
Results: The short proteins corresponding to the epitopes of the broadly neutralizing antibodies 2F5 and 4E10 were
successfully transported using tetraspanin anchors to the cell surface and expressed in the correct conformation allowing
binding of both antibodies. 2F5 and 4E10 binding was observed at high specificity and affinity. Binding of anti-2F5 antibodies
is more effective than of anti-4E10 antibodies in this newly developed cell based ELISA. Interestingly, the cell surface protein
Claudin-1 was not able to share the same protein surface presentation feature as representatives of the tetraspanin
superfamily.
Conclusion: The newly developed cell based ELISA allows for screening for broadly neutralizing antibodies binding to the
MPER such as 2F5 and 4E10. Due to the native conformation of the epitopes, the cell-based assay offers a major advantage
compared to the use of synthetic peptides. With this approach costs for testing were reduced, because there is no need for
expensive coating with peptides or expressed and purified recombinant protein. The assay can be used for the presentation
of other conformational epitopes.
Corresponding author:
Daniel Ivanusic
[email protected]
Diagnostic Tools
P75
Real-time PCR-based, sequencing-independent pathotyping of potentially zoonotic avian influenza A viruses of the
subtypes H5 and H7
A. Graaf1, M. Naguib1, M. Beer1, T. C. Harder1
1Friedrich
Loeffler Institute, Institute of Diagnostic Virology, Greifswald - Isle of Riems, Germany
Introduction: avian influenza viruses (AIV) threaten poultry production worldwide. Vast majority of AIV subtypes are classified
as low-pathogenic (LPAIV), while only viruses of the subtypes H5 and H7 are known to have the ability to mutate to highly
pathogenic phenotypes (HPAIV). The latter are closely monitored as they cause devastating losses in poultry and, due to
zoonotic propensities of some strains, also pose a threat to public health. As Europe witnesses another wave of incursion of
H5 HPAIV since November 2016 and also outbreaks of H7 LPAIV and HPAIV in poultry in Europe continue to occur, it is of
utmost importance to differentiate between LPAI and HPAI virus strains during H5/H7 surveillance and outbreaks.
Method: a set of four quantitative reverse transcription real-time PCR assays (RT-qPCR) was developed that allows
sequencing-independent pathotype distinction of these viruses. To this end, different RT-qPCRs for each sub- and pathotype
were developed to amplify a fragment that spans the HA cleavage site (CS) region of the haemagglutinin gene (HA). A total
of 130 and 81 poultry samples positive for H5 or H7, respectively, were used to determine diagnostic sensitivity and
specificity relative to an influenza A virus generic matrix (M) gene RT-qPCR.
Results: all RT-qPCR pathotyping data fitted the results achieved by sequencing of the HA cleavage site. There was no
significant difference between the median Cq (cycle of quantification) values of each specific assay and the generic M RTqPCR, indicating that the newly developed RT-qPCRs have similar diagnostic sensitivity. Consequently, these new RTqPCRs allow a sensitive and highly specific detection and distinction of LPAIV and HPAIV of the subtypes H5 and H7
circulating in European countries.
Conclusion: the developed molecular diagnostic assays is a useful tool for rapid simultaneous detection and pathotyping of
AIV of subtypes H5 and H7 without sequencing to be used in the surveillance of domestic and wild bird populations.
Corresponding author:
Annika Graaf
[email protected]
Diagnostic Tools
P76
Development of multiplex real-time PCR method for cell-culture based testing of antivirals with potential activity
against human adenovirus C
M. Przybylski1, R. Zahorska1, T. Dzieciatkowski1, A. Gorski2,3
1Medical
University of Warsaw, Department of Medical Microbiology, Warsaw, Poland
University of Warsaw, Department of Clinical Immunology, Warsaw, Poland
3Institute of Immunology and Experimental Therapy of Polish Academy of Sciences, Bacteriophage Laboratory, Wroclaw, Poland
2Medical
Question: We developed multiplex real-time PCR (qPCR) for detection of early and late genes of human adenoviruses
(HAdV) belonging to C species, for application in antiviral activity testing in in-vitro studies. Method was developed for use
with regular cell-culture based methods, and our goal was to omit observer-dependent bias observed often in end-point
reading methods relying on CPE formation, especially for viruses which dont form easy-to-read plaques in cell culture.
Methods: For the experiments, we used HAdV-5, propagated in A549 cell line. For the experiments, we used cell culture
infected with the virus in titers ranging from 10 to 1000 TCID50/ml, and qPCR was performed after 24 and 48 hours after
infection, using DNA isolated both from supernatant and frozen/thawed cells. We designed three sets of primers and TaqMan
hydrolysis probes, detecting early adenoviral gene, late adenoviral gene and human glucose-6-phosphate dehydrogenase.
The latter gene can be used both as an internal control in regular qPCR and as reference gene in gene expression studies.
We evaluated basic parameters of developed qPCR methods and performed multiplexing optimalization. For quantitative
experiments, serial ten-fold dilutions of calibrated plasmids containing amplicon sequences were used. We also performed
series of experiments regarding utility of developed methods for time-dependent gene-expression studies, using RNA
isolated from frozen/thawed cells every 8 hours till 48 hours after infection.
Results: Validation of developed methods revealed 100% product identity, and fairly fulfilled premises required for qPCR
technique. Methods proved their efficacy also in multiplex variant. Surprisingly, quantitative detection of early and late genes
of HAdV in cell line infected with the virus revealed similar levels of viral DNA copies, regardless of HAdV infective dose, but
significant differences were observed in supernatants taken after 24 and 48 hours.
Conclusion: Application of the methods for the detection of differences in viral load in inhibition experiments revealed good
correlation between viral titer measured with end-point CPE-based methods and number of viral copies detected with qPCR.
Detection of G6PD gene in infected cell culture supernatant seems to be good indicator of number of cells lysed by the virus,
thus may be used as an additional measure of cell culture condition.
This work was partially supported with grant 3D1702G of Polish National Science Center.
Corresponding author:
Maciej Przybylski
[email protected]
Diagnostic Tools
P77
Can plasma free DNA levels be a diagnostic tool to rule out severe postpartal sepsis
G. O. Ajayi1
1University
Of Lagos, Prenatal Diagnosis and Therapy Centre, Dept of Obstetrics & Gynaecology, Lagos, Nigeria
Setting: A Prenatal Diagnostic and Therapy Centre of a Tertiary Hospital in Lagos/Nigeria
Material & Methods: A total of n=13 unbooked women admitted through accident and emergency unit with the history of
severe sepsis post delivery per C/S formed ground 1 and in group 2 were n=11 women with elective C/S without
complications. On admission venous blood were taken for PF-DNA concentration, serum C-reactive protein (CRP),
procalcitonin (PCT), viral and serum lipid concentration analysis. For statistical analysis Student t test was used.
Result: Admission PF-DNA concentrations were significantly higher in septic when compare to non septic
women[15.904(37-191,288)GE/mL versus 469.6(0-8863)GE/ mL, p<0.0001]. PF-DNA concentration showed a significant
correlation with CRP,PCT and HDL concentration.
Conclusion: Plasma DNA seems to be potentially valuable tool to confirm sepsis in a gynecological obstetrical emergency
care.
Corresponding author:
G.O. Ajayi
[email protected]
Diagnostic Tools
P78
Mechanism research for rion disease
Q. Shi1
1Chinese
Center for Disease Control and Prevention, National Institute of Viral Disease Control and Prevention, Beijing, China
Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimers
disease and Parkinsons disease. However, the brain proteomics of human prion disease remains less understood. In the
study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial
insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with
multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global
protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with
quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and downregulated proteins (727proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and
G114V geneticCJD. Viral myocarditis, Parkinsons disease, Alzheimersdisease, lysosome, oxidative phosphorylation, protein
export,and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases.
Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate
that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics
changes at their terminal stages, which show the similarities not only among human prion diseases but also with other
neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will
be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.
Corresponding author:
Qi Shi
[email protected]
Diagnostic Tools
P79
VAmpSeq: Ultrahigh multiplex PCRs for virus detection
A. Brinkmann1, A. Radonic1, A. Nitsche1
1Robert
Koch Institute, Centre for Biological Threats and Special Pathogens (ZBS) 1 - Highly Pathogenic Viruses, Berlin, Germany
Next-generation sequencing offers great chances for the diagnosis of viral infections in humans and animals, not requiring "a
priori" knowledge of the pathogen. However, depending on the expected depth of the analysis and the specimen numbers,
the sensitivity can be limited, given that the viral load of patient samples can be low and in a high background of human
genetic material. Recent attempts to increase the sensitivity of virus detection by NGS have led to the development of
targeted sequence enrichment techniques, including sequence capture and pre-amplification.
We assessed the feasibility of multiplexed sequence amplification prior to NGS to facilitate the simultaneous identification of
various viral pathogens in patient samples. We developed a combination of ultrahigh-multiplex PCRs and NGS (VAmpSeq),
which, exemplarily, is able to target more than 450 viral genomic regions of 45 viruses causing haemorrhagic fevers in a
single approach.
We were able to identify viral genomes present in as little as one genomic copy in a complex sample matrix. Furthermore, we
performed VAmpSeq with real-time nanopore sequencing, generating sufficient viral read numbers for species identification
within 10 minutes of sequencing and a total sample-to-result time of <5 hours.
VAmpSeq offers a new approach for broad virus genome identification in patient specimens, offering the benefits of
multiplexed reactions and concurrently decreasing the processing time significantly.
Corresponding author:
Annika Brinkmann
[email protected]
Viral Replication
P80
The Y-domain of hepatitis E virus (HEV) ORF1 – role in RNA replication and virion infectivity
M. Parvez1
1King
Saud University College of Pharmacy, Riyadh, Saudi Arabia
Objective: In HEV, while the 5mRNA capping activity of non-structural ORF1 MTase-domain is well characterized and
implicated in RNA replication, the function of Y-domain remains completely unexplored. The present study therefore,
investigates a potential role of Y-domain sequences in HEV life cycle.
Methods: In silco analysis and site-directed mutation of HEV (genotype 1) Y-domain was carried out in the viral genomic
replicon (pSK-GFP) and studied in the baculovirus- S10-3 cell model. In vitro transcribed mRNA constructs were transfected
to S10-3 cells and flow cytometry was employed to score the viral RNA replicating GFP positve cells. Virion infectivity was
assayed on naïve HepG2 cells, followed by flow cytometry..
Results: Analysis of human HEV Y-domain (a.a. 216-442) and closely-related virus sequences identified potential
palmitoylation-site (C336C337) and a-helix segment (L410Y411S412W413L414F415E416) counterparts. Molecular analysis of C336A,
C337A and W413A mutants of the three universally-conserved residues showed non-viability. Further, of the ten consecutive
saturation mutants covering the entire Y-domain (nts. 650-1339), three constructs (nts. 788-994) severely affected virus
replication. This revealed the indispensability of the internal but not the up or downstream sequences at transcriptional level.
Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation,
indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed
formation of stable hairpins by nts. 778-994 where saturation mutation drastically inhibited virion infectivity.
Conclusion: This is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, probably through
gene regulation and/or membrane-binding in intracellular replication-complexes.
Corresponding author:
Mohammad Parvez
[email protected]
Viral Replication
P81
Epstein-Barr Viruses (EBVs) deficient in EBV-encoded RNAs (EBERs) display reduce lytic replication in EBVtransformed lymphoblastoid cell lines
Z. Li1, M. H. Tsai1, R. Poirey1, H. J. Delecluse1
1German
Cancer Research Center (DKFZ), Pathogenesis of virus-associated tumors F100, Heidelberg, Germany
Introduction: The Epstein-Barr virus (EBV) infects more than 90% of the world population. All EBV strains can transform
primary B-lymphocytes, but some strains, such as M81, also induce spontaneous virus replication in transformed B cells [1].
EBV expresses two noncoding RNAs called EBER1 (EBV-encoded RNA 1) and EBER2 (EBV-encoded RNA 2), that are 167
and 173 nucleotides (nts) long, respectively. They are expressed during all forms of EBV latency and also during lytic
replication. EBER1 accumulates to 106 and EBER2 to 2.5 x 105 copies per infected cell. The high copy number and the
evolutionary conservation of EBERs point to a fundamental role of EBERs in the EBV life cycle.
Objectives: to study the role of EBV-encoded small RNAs (EBERs) during these processes.
Methods: We constructed an EBV mutant that lacks both the encoded small RNAs (EBERs) (M81∆E1/2) by using the EBVBAC system in 293 cells [2].
Results: The M81∆E1/2 mutant displays lower levels of lytic replication in transformed B cells. We also generated single
EBER mutants in the M81 strain, and tested their role in lytic replication. We found that EBER2 plays a predominant role, but
EBER1 is also involved in the control of lytic replication. In addition, we found that B-cells infected by the virus that lacks
these small RNAs (EBERs) grew more slowly in the first few weeks post infection, relative to wild type controls. We finally
found that the B cell transforming capacity of the M81∆E1/2 mutant is reduced by 50%, relative to wild type viruses.
Conclusion: the small RNAs (EBERs) play important roles in crucial viral functions.
References:
[1] Tsai MH, et al. (2013) Spontaneous lytic replication and epitheliotropism define an Epstein-Barr virus strain found in
carcinomas. Cell Rep 5: 458–470.
[2] Feederle R, et al. (2010) Epstein-Barr virus genetics: talking about the BAC generation. Herpesviridae 1(1): 6.
Corresponding author:
Zhe Li
[email protected]
Viral Replication
P82
Cooperative enhancement of translation by two adjacent microRNA-122/Argonaute 2 complexes binding to the
5´untranslated region of Hepatitis C Virus RNA
A. Nieder-Röhrmann1, N. Dünnes1, G. Gerresheim1, L. Shalamova1, A. Herchenröther1, M. Niepmann1
1Justus
Liebig University Giessen, Institute of Biomedicals, Giessen, Germany
The liver-specific microRNA-122 (miR-122) binds to two conserved binding sites in the 5´-untranslated region (5´UTR) of
Hepatitis C Virus (HCV) RNA. This binding was reported to enhance HCV RNA replication, translation and stability. We have
analyzed binding of miR-122/Argonaute 2 (Ago2) complexes to these sites using anti-Ago2-co-immunoprecipitation of
radioactively labeled HCV RNAs along with ectopic miR-122 in HeLa cells. Our results show that the miR-122 target sites
can be addressed separately. When both target sites were addressed simultaneously, we observed a synergistic binding of
both miR/Ago2 complexes. Consistently, simultaneous binding of both miR-122/Ago2 complexes results in cooperative
translation stimulation. In the binding assays as well as in the translation assays, binding site 1 has a stronger effect than
binding site 2. We also analyzed the overall RNA stability as well as the 5´end integrity of these HCV RNAs in the presence
of miR-122. Surprisingly, using short HCV reporter RNAs we did not find effects of miR-122 binding on overall RNA stability
or 5´end integrity over up to 36 hours. In contrast, using full-length HCV genomes that are incapable of replication, we found
a positive influence of miR-122 on RNA stability, indicating that features of the full-length HCV genome that do not reside in
the 5´- and 3´UTRs may render HCV RNA genome stability miR-122-dependent.
Corresponding author:
Michael Niepmann
[email protected]
Viral Replication
P83
RNA structure dynamics and sequence context influence microRNA-122 binding in the NS5B coding region and the
3´untranslated region of Hepatitis C Virus RNA
G. Gerresheim1, N. Dünnes1, A. Nieder-Röhrmann1, L. Shalamova1, M. Fricke2, I. Hofacker3, C. Höner zu Siederdissen4, M.
Marz2, M. Niepmann1
1Justus
Liebig University Giessen, Institute of Biomedicals, Giessen, Germany
Schiller University Jena, Faculty of Mathematics and Computer Science, Jena, Germany
3University of Vienna, Institute of Theoretical Chemistry, Vienna, Austria
4University of Leipzig, Department of Computer Science, Leipzig, Germany
2Friedrich
microRNAs (miRNAs) regulate gene expression mostly at a post-transcriptional level. Binding of miRNAs to target RNAs is
mediated by Argonaute (Ago) proteins. We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three
conserved target sites of Hepatitis C Virus (HCV) RNA, two in the non-structural protein 5B (NS5B) protein coding region and
one in the 3´untranslated region (3´UTR), using co-immunoprecipitations of the HCV RNA along with Ago2 and miR-122. We
show that miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of
flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the
particular sequence feature that contributes most to the correlation between target site accessibility and binding strength
varies between different target sites. In site 5B.2 this main feature is the nucleation sequence (the target nucleotides
opposite to miRNA nucleotides 2 – 5), in site 5B.3 it is mainly an additional A residue directly downstream of the seed target
sequence that can bind a pocket in Ago2 protein, and in the 3´UTR site it is the complete seed target sequence. This
suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also depends on flanking
sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on
coding sequence and overlapping cis-signals and model the accessibility of cis-signals.
Corresponding author:
Michael Niepmann
[email protected]
Viral Replication
P84
Dissecting the requirements for Hepatitis C virus RNA synthesis using a split replication system
L. Shalamova1, N. Dünnes1, A. Nieder-Röhrmann1, G. Gerresheim1, D. Metzger1, S. Mueller2, Y. Yutong3, E. Wimmer3, O.
Rossbach4, M. Niepmann1
1Justus
Liebig University Giessen, Institute of Biomedicals, Giessen, Germany
Inc., Stony Brook, United States
3Stony Brook University, Molecular Genetics and Microbiology, Stony Brook, United States
4Justus Liebig University Giessen, Institute of Biochemistry, Giessen, Germany
2Codagenix
Replication of the Hepatitis C Virus (HCV) single-stranded RNA genome requires cis-signals at both genome ends for
initiating the synthesis of antigenome minus strand and progeny plus strand RNAs. However, it is difficult to decide if a ciselement exerts its function when physically present in the plus strand or in the minus strand, and to assign the effect of a
mutation in a region with multiple overlapping cis-elements to a specific step of RNA replication. We developed a split
replication system to analyse the role of each single HCV genome end in minus or plus strand RNA synthesis, thereby
uncoupling the functions of the involved sequences from overlapping functions in other steps of the viral life cycle. This
system provides new and unprecedented insights into HCV replication, including interference of translation at the genome
5´end with RNA minus strand synthesis at the genome 3´end, long-range interactions and microRNA action. We show that
HCV RNA minus strand synthesis at the 3´end of the plus strand is possible in the absence of any HCV 5´-end sequences.
Similarly, RNA synthesis initiation from the HCV antigenome 3´end is possible in the absence of antigenome 5´end
sequences. Partial HCV plus strand 5´-end sequences strongly enhance minus strand synthesis, whereas insertion of the
complete 5´untranslated region (5´UTR) including a short open reading frame interferes with enhanced minus strand
synthesis. The liver-specific microRNA-122 stimulates minus strand initiation independent of its functions in the 5´-UTR. Cissequences in the coding region stimulate minus strand initiation but not plus strand initiation. The concept of this split
replication system may be applicable to investigate replication of any RNA virus.
Corresponding author:
Michael Niepmann
[email protected]
Viral Replication
P85
Specific NS1 residues enhance viral replication and pathogenicity of avian influenza A virus in mammalian systems
P. Kanrai1, A. Mostafa1, R. Madhugiri1, M. Lechner2, E. Wilk3, K. Schughart3, L. Ylösmäki4, K. Saksela4, J. Ziebuhr1, S.
Pleschka1
1Justus
Liebig University Giessen, Institut for Medical Virology, Giessen, Germany
University Marburg, Department of Pharmaceutical Chemistry, Marburg, Germany
3Helmholtz Centre for Infection Research, Department of Infection Genetics, Braunschweig, Germany
4University of Helsinki, Department of Virology, Helsinki, Finland
2Philipps
Influenza A virus (IAV)-encoded NS1 proteins have diverse functions in viral replication and pathogenesis. Previously, we
reported that sequence variations in the NS segment affect the growth characteristics of avian IAVs in mammalian systems.
Efficient replication of the strictly avian influenza virus A/FPV/Rostock/34 (FPV, H7N1) in mammalian cells was observed for
FPV-derived reassortants containing the NS segment from the highly pathogenic avian influenza virus (HPAIV)
A/Goose/Guangdong/1/1996 (GD, H5N1), but not for reassortants containing the NS segment of the low pathogenic avian
influenza virus (LPAIV) A/Mallard/NL/12/2000 (MA, H7N3). The NS1 proteins of GD and MA differed only in 8 (out of 230)
positions. Here we investigated the role(s) of specific NS1 residues in causing the previously observed growth differences.
We generated FPV-NSMA reassortants carrying specific NS1 mutations via reverse genetics and analysed virus replication in
different cell systems and in vivo. The data show that efficient replication of FPV-NSMA-derived mutants was linked to the
presence of specific triple or single substitution(s) in the NS1-MA protein (P3S+R41K+D74N and D74N, respectively). The
latter substitution(s) led to (i) increased virus titers, (ii) larger plaque sizes, and (iii) increased levels and faster kinetics of viral
mRNA and protein accumulation in mammalian cells. Interestingly, the NS1 substitutions did not affect viral growth
characteristics in avian cells. To assess the presumed critical role of the NS1 D74N substitution for viral replication in vivo,
we characterized an FPV mutant containing the single D74N substitution in the FPV NS1 protein context. Infection
experiments revealed that the FPV mutant encoding this single substitution is able to replicate and cause disease in mice.
Our results demonstrate the key role of NS1 in the adaptation of avian IAV to mammalian hosts.
Corresponding author:
Stephan Pleschka
[email protected]
Viral Replication
P86
PIAS2 and PIAS1, novel restriction factors of filoviruses?
M. Klüver1, S. Becker1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae and cause severe, often fatal, illness in
humans and non-human primates. The recent epidemic of Ebola virus in West Africa with over 28.000 cases revealed the
need for a deeper understanding of filovirus infections in order to develop novel vaccines and antivirals. The latter might be
achieved e.g. by identifying new cellular targets that play crucial roles during filovirus infections. Here we present the results
of a study to identify cellular interaction partners of MARV VP30, which is an important filovirus transcription factor.
A high-throughput yeast-two hybrid assay was performed with VP30 as the bait protein and the identified interactors were
confirmed by co-immunoprecipitation. Using reverse genetics-based filoviral minigenome assays the function of the identified
MARV VP30 interactors for filoviral transcription and replication was tested.
We were able to show that the host cell proteins Protein inhibitor of activated stat (PIAS) 2 and PIAS1 interact with MARV
and EBOV VP30. These proteins are E3-SUMO-ligases that play a role in different cellular pathways including JAK-STAT
and transcription regulation. A conserved region in the N-Terminus of VP30 appeared to be essential in mediating the
interaction with PIAS2. Furthermore, we were able to show that PIAS2 and PIAS1 negatively regulate the transcription and
replication in filovirus minigenome assays. For PIAS2 a dose-dependent effect was shown. When PIAS2 function was
abandoned using CRISPR/Cas9 technology, filoviral transcription was elevated.
The results of our study revealed that MARV and EBOV VP30 interact with the host cell proteins PIAS2 and PIAS1, which
were able to negatively regulate filoviral transcription. These results indicated PIAS2 and PIAS1 to assume a role as cellular
restrictions factors during filovirus infections.
Corresponding author:
Michael Klüver
[email protected]
Viral Replication
P87
Influence of bovine hepacivirusIRES domains on translation efficiency
A. L. Baron1, C. Baechlein1, A. Gleich2, P. Becher1
1University
2University
of Veterinary Medicine Hanover, Institute of Virology, Hanover, Germany
of Leipzig, Institute of Veterinary Physiological Chemistry, Leipzig, Germany
Recently, a novel hepatitis C virus (HCV) related virus was identified in bovine serum samples and was termed Bovine
hepacivirus (BovHepV). This single-stranded RNA virus contains an internal ribosomal entry site(IRES) sequence in the 5`
non translated region (5`NTR) which enables a cap-independent translation. Compared to HCV, the IRES structure of
BovHepV is composed of three domains (II, III and IV) including one miR-122 binding site, whereas the 5`NTR of HCV
contains two miR-122 binding sites. MiR-122 is highly expressed in the human liver and promoteshepacivirusRNA
accumulation and replication. Our aim was to gain detailed insights into cap-independent translation initiation of BovHepV.
We analyzed the influence of the IRES domains and partial core sequences on the translation efficiency by using a dual
luciferase system. Thereby IRES specific firefly luciferase activity was normalized against renilla luciferase activity serving as
a translation control. HeLa, BFH12 (bovine foetal hepatocyte-derived cell line) and Huh-7 cells were transfected with
plasmids harboring parts of BovHepV IRES or IRES-core constructs and luciferase activities were measured 48 hours post
transfection.It was shown, that all three domains are needed for initiation of BovHepVtranslation.Inclusion of 36 nucleotides
of the core sequence upstream of the firefly luciferase leads to increased translation whereas larger parts of core sequences
had an inhibitory effectwhich was independent of the cell line. Deletion of the miR-122 binding site had no effect on
translation in HeLa and BFH12 cells but lead to decreased translation in Huh-7 cells, where miR-122 is highly expressed.
Corresponding author:
Anna Lena Baron
[email protected]
Viral Replication
P88
Three-dimensional cultivation of primary human cells affects virus-host interaction on transcriptome level (RNASeq)
R. Koban1, M. Neumann1, A. Daugs2, O. Bloch2, H. Ellerbrok1
1Robert
2Auto
Koch Institute, Centre for Biological Threats and Special Pathogens (ZBS) 1 - Highly Pathogenic Viruses, Berlin, Germany
Tissue Berlin GmbH, Berlin, Germany
Virus infection studies are mainly carried out in vitro in monolayer (2D) cell cultures. Cell-cell interaction and cell morphology
in such cultures are known to differ considerably from those in a three-dimensional (3D) network. Therefore, biological
relevance of data from virus infection studies generated in 2D might be limited and hardly transferable to the in vivo situation.
In contrast, 3D models might reflect more realistically the in vivo situation during virus infection.
In this study, a new 3D cell culture system was established for infection studies. Epithelial primary human cells (NHEK) were
grown on decellularized equine pericardial extracellular matrix (ECM, AutoTissue GmbH) and were subsequently infected
with Cowpoxvirus (CPXV). Cell growth, various parameters of cellular activity and virus infection were monitored on protein
and mRNA level. Cell morphology, expression and localization of cellular proteins and virus spread were visualized via
immunohistochemistry (IHC). Cell viability was screened with the CellTiter-Glo® assay (Promega). Comparative
transcriptome analysis (RNA-Seq) of infected 3D cultures and corresponding 2D cultures was performed on Illumina
HiSeq1500 platform. GO-Term analysis of RNA-Seq results was carried out with the BiNGO tool (Cytoscape).
The established cultivation protocol reproducibly generated polarized 3D cell cultures with a strongly increased number of
cells per surface area compared to 2D while ATP, RNA and protein levels per cell were reduced distinctly in 3D. Virus
replication and cell viability during infection were comparable in both models. Comparative RNA-Seq of uninfected 3D and
2D cultures revealed significant differential expression for 17,5% of all transcripts detected. Infection in 2D culture resulted in
distinctly higher numbers of regulated cellular genes than infection in 3D culture. RNA-Seq results were verified via qPCR,
Western Blot and IHC for selected targets.
The established 3D model represents an easy to handle reproducible cell culture system for CPXV infection studies in
primary human cells. Analysis of gene expression may allow identification of new relevant cellular targets for the
development of host-directed antiviral therapies. This system can be used with different cell types and is suitable for study of
various viruses.
Corresponding author:
Robert Koban
[email protected]
Viral Replication
P89
A seven-segment influenza A virus packages eight riboucleoprotein cmplexes
T. Noda1, S. Murakami2, H. Imai2, S. Nakatsu2, Y. Muramoto1, K. Shindo1, H. Sagara2, Y. Kawaoka2
1Kyoto
University, Institute of Frontier Life and Medicacl Sciences, Kyoto, Japan
of Tokyo, Institute of Medical Science, Tokyo, Japan
2University
The influenza A virus genome is composed of eight single-stranded negative-sense RNAs. Eight distinct viral RNA segments
(vRNAs) are known to be selectively packaged into progeny virions, with eight vRNAs in ribonucleoprotein complexes
(RNPs) arranged in a specific 1+7 pattern, i.e., one central RNP surrounded by seven RNPs. Here, we artificially generated a
seven-segment virus that lacks the hemagglutinin (HA) vRNA and examined its genome packaging. Electron microscopy
(EM) of the seven-segment virions showed that, even in the presence of only seven vRNAs, the virions efficiently packaged
eight RNPs arranged in the same 1+7 pattern as wild-type virions do. Intriguingly, next generation sequencing (NGS) of
RNAs isolated from the seven-segment virions revealed that the virions specifically incorporate host-derived 18S and 28S
ribosomal RNAs (rRNAs), likely present as the eighth RNP, instead of the omitted HA vRNAs. These findings highlight the
importance of the assembly of eight RNPs into a specific 1+7 configuration for genome packaging in progeny virions.
Corresponding author:
Takeshi Noda
[email protected]
Viral Replication
P90
Examining herpes simplex virus Type 1-induced host shutoff at the transcriptional level
A. Whisnant1, L. Dölken1
1Julius
Maximilians University Würzburg, Institute of Virology and Immunbiology, Würzburg, Germany
Infection with Herpes Simplex Virus type 1 (HSV-1) is characterized by a profound shutoff of host gene expression. This is
largely mediated by two viral proteins: virus host shutoff (VHS), a virion-associated ribonuclease that degrades activelytranslated mRNA immediately upon infection; and infected cell protein 27 (ICP27), an RNA-binding protein which facilitates
expression of intronless viral genes but inhibits processing of cellular mRNA. Furthermore, our lab has recently demonstrated
during lytic infection that newly synthesized RNAs suffer from defective transcriptional termination for the vast majority of
cellular – but not viral – genes.
In order to understand the modulation of VHS activity and the differential effects of ICP27 on host versus viral transcripts, we
have established an improved version of the Photoactivatable Ribonucleoside-Enhanced Crosslinking and
Immunoprecipitation (PAR-CLIP) technique to globally identify RNA binding sites for both proteins. We have also profiled
global RNA Polymerase II activity throughout timepoints of infection using native elongating transcript sequencing technology
for mammalian chromatin (mNET-Seq). Here we describe our current findings and future directions.
Corresponding author:
Adam Whisnant
[email protected]
Viral Replication
P91
First characterization of the pestiviral replication factories
I. Romero Brey1, O. Isken2, R. Bartenschlager1, N. Tautz2
1University
2University
of Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
of Lübeck, Institute of Virology and Cell Biology, Lübeck, Germany
Pestiviruses (genus Pestivirus) belong to the family Flaviviridae, which also includes the genera Hepacivirus, Flavivirus and
Pegivirus.
Among these viruses two main architectures of remodeled membranes can be found in the cytoplasm of infected cells that
may reflect two alternative strategies to induce the membranous replication factories. On one hand, the replication factories
of members of the genus Flavivirus (including Dengue virus and Tick borne encephalitis virus) correspond to invaginations of
ER membranes forming vesicle packets and remaining connected to the cytosol via 10 nm-pores. In stark contrast, Hepatitis
C virus (HCV), prototype of the genus Hepacivirus, provokes mainly the formation of double membrane vesicles (DMVs) that
seem to be formed as ER protrusions and that remain connected to ER membranes via neck-like structures.
In this study we applied different high-resolution imaging techniques, including correlative light electron microscopy (CLEM),
to elucidate the architecture and composition of the pestivirus replication factories. Our data show that the intracellular
membrane rearrangements induced by pestiviruses have a DMV morphology, similar to the one found in HCV-infected cells.
Expression of the pestiviral replicase proteins (NS3-5B), in the absence of viral RNA replication, was sufficient for the
formation of DMVs. Along these lines, expression of the NS3-5A cassette (lacking the 5B polymerase) also led to the
formation of DMVs. However, the membrane alterations induced by the expression of the replicase components NS3-4B or
NS3-4A did not resemble the DMVs observed in pestivirus-replicating cells.
Within the family Flaviviridae, with respect to genome structure, organization and mechanism of RNA translation, pestiviruses
have much higher similarity to hepaciviruses than to flaviviruses. The results obtained in this study argue that pestiviruses
utilize as well an HCV-like membrane remodeling strategy to build up their replication factories.
Corresponding author:
Inés Romero Brey
[email protected]
Viral Replication
P92
The host factor AUF1 supports Flavivirus RNA replication by destabilizing stem-loop structures in the 5 and 3
untranslated regions
S. Friedrich1, S. Höpfner1, T. Schmidt1, G. Szczepankiewicz2, S. Bergs2, U. G. Liebert2, B. Kümmerer3, R. Golbik1, S. E.
Behrens1
1Martin
Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Halle/Saale, Germany
of Leipzig, Institute of Virology, Leipzig, Germany
3University of Bonn, Institute of Virology, Bonn, Germany
2University
In earlier studies we demonstrated that the RNA-binding protein AUF1 acts as a host factor that supports the replication of
the important human pathogen West Nile virus (WNV) [1]. AUF1 was demonstrated having an RNA chaperone activity as
well as an RNA annealing activity which supports the structural rearrangement and cyclization of the WNV genomic RNA that
is required for the initiation of (-)-strand RNA synthesis [2]. Here, we show that the intracellular level of AUF1 also crucially
affects the propagation of Dengue virus (DENV) and Zika virus (ZIKV) indicating a general role of AUF1 in Flavivirus
replication. Investigating DENV by RNA structure probing, we were able to visualize the AUF1-induced restructuring of the
viral RNA. It turned out that specific nucleotides within the DENV 3-terminal stem-loop (3SL) are more flexible in the
presence of AUF1. AUF1 further modulates the secondary structure of the 5-terminal stem-loop B (SLB) of the DENV
genome. Thus, by destabilizing specific stem-loop structures within the 5- and 3-end of the flaviviral RNA AUF1 triggers the
structural rearrangement that is essentially required for genome cyclization and RNA replication.
References:
[1] Friedrich et al. 2014. J Virol. 88:11586-99
[2] Friedrich et al. 2016. RNA. 22(10):1574-91
Corresponding author:
Susann Friedrich
[email protected]
Viral Replication
P93
Human respiratory 3D-tissue models to study the pathogenesis of respiratory syncytial virus
T. Kreuzahler1, M. Steinke2, B. Weißbrich1, J. Liese3, H. Walles2, C. Krempl1
1Julius
Maximilians University Würzburg, Institute of Virology and Immunobiology, Würzburg, Germany
Maximilians University Würzburg, Department for Tissue Engineering and Regenerative Medicine, Würzburg, Germany
3Julius Maximilians University Würzburg, Department of Pediadrics, Würzburg, Germany
2Julius
Introduction: Humans are the only fully susceptible host for the important pathogen respiratory syncytial virus (RSV).
Conventional cultured cell lines often do not reveal attenuated phenotypes of RSV mutants, and none of the RSV animal
models reflect the full spectrum of human RSV infection. Consequently, primary 3D-tissue models of the human
tracheobronchial mucosa are closest to natural host tissue for RSV. However, they are time consuming and expensive to
build up, and differentiation of respiratory epithelial cells is highly dependent on the patient material obtained. Using 3Dculture techniques with a biological scaffold the more convenient permanent respiratory cell lines may acquire a phenotype
reflecting features of respiratory tissue.
Objectives: We aimed to establish and evaluate 3D-tissue models of the human respiratory mucosa for characterization of
RSV mutants.
Methods: 3D-tissue models consisted of the human respiratory cell lines A549, Calu-3 or HCC-827 apically seeded onto a
biological scaffold containing primary human fibroblasts. Following culture under air lift conditions for 3 weeks, 3D-tissue
models were infected with RSV A2 or the attenuated rgRSV expressing GFP. Apical and basolateral virus release was
analyzed over time. In parallel, virus replication in conventional 2D-cultures of the same cell lines was determined. In
addition, 3D-cultures were subjected to histological examination.
Results: The susceptibility of Calu-3- and HCC-827-based 3D-tissue models for RSV A2 infection was comparable to the
corresponding conventional 2D-cultures, whereas virus titers released from the A549 3D-tissue models were significantly
lower. All 3D-tissue models, but none of the conventional 2D-tissue models revealed the attenuated replication phenotype of
rgRSV, with a minimum of 10-fold differences in titers. In 3D-cultures Calu-3 cells formed a consistent epithelial monolayer
with virus release detectable only from the apical surface, whereas in HCC 3D-tissue models a significant amount of RSV
was detected in the basolateral medium. A549 cells, however, displayed a clearly tumorous, even invasive growth.
Conclusion: Calu-3-based 3D-models composed of the human respiratory mucosa appear to be a convenient and suitable
test system for the replication efficacy of RSV mutants.
Corresponding author:
Christine Krempl
[email protected]
Viral Replication
P94
Phenotypic lentivirus screens to identify functional single domain antibodies to interfere with virus replication
F. I. Schmidt1,2, L. Hanke2, B. Morin3, R. Brewer2, V. Brusic3, S. P. J. Whelan3, H. L. Ploegh2
1University
of Bonn, Institute of Innate Immunity, Bonn, Germany
Whitehead Institute, Cambridge, United States
3Harvard Medical School, Department of Microbiology and Immunobiology, Boston, United States
2MIT,
Question: Manipulation of proteins is key in assessing their in vivo function. While genetic ablation is straightforward,
reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as
camelid-derived VHHs (variable domains of heavy chain-only antibodies), can be expressed in the cytosol to serve as
inhibitors or activators of intracellular protein function. Yet, functional testing of identified VHHs is laborious. We set out to
develop a functional screening approach to identify VHHs with desired functions in a rational manner.
Methods: We immunized two alpacas with inactivated influenza A virus (IAV) and vesicular stomatitis virus (VSV), isolated
peripheral blood lymphocytes, extracted RNA, and amplified VHH coding sequences by PCR using VHH-specific primers.
The repertoire of VHHs was then cloned into a lentiviral vector. Using the obtained plasmid library, we then produced selfinactivating lentivirus and transduced human lung ephitelial A549 cells, so that each cell inducibly expressed a different VHH
in the cytosol. Cells were subsequently challenged with a lethal dose of IAV or VSV, allowing only survival of cells expressing
VHHs that interfered with virus infection. VHH sequences from resistant cell clones were recovered, and further analyzed to
reveal their mechanism of action.
Results: We identified 19 antiviral VHHs that protect A549 cells from lethal infection with IAV or VSV, respectively. All 15
anti-IAV VHHs targeted the viral nucleoprotein NP and prevented nuclear import of viral ribonucleoproteins; three of them in
addition impaired replication and transcription of a model genome segment. The 4 anti-VSV VHHs recognize the viral
nucleocapsid N and inhibited mRNA transcription in cells. Yet, only two of the VHHs directly impaired mRNA transcription in
vitro, suggesting that the other VHHs interfere indirectly with mRNA transcription, e.g. by inhibiting genome replication and
thus limiting the amount of available templates.
Conclusion: Both viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins, revealing a novel
vulnerability of negative-sense RNA viruses. In principle, the screening approach described here should be applicable to
identify inhibitors of any pathogen or biological pathway.
Corresponding author:
Florian Ingo Schmidt
[email protected]
Figure 1
Viral Replication
P95
Sequence requirements for avian ANP32A to overcome influenza A virus polymerase host restriction
P. Domingues1, B. Hale1
1University
of Zurich, Institute of Virology, Zurich, Switzerland
In a previous proteomic screen, we found that the host-range determining factor ANP32A was SUMOylated during Influenza
A Virus (IAV) infection. Here, we assessed the contribution of the host SUMO system on ANP32A activities and their
mechanistic consequences on avian viral polymerase adaptation to human hosts. A recent study showed that expression of
an avian ANP32A (avANP32A) protein, but not the human homologue (huANP32A), was sufficient to rescue the ability of
avian-derived IAV polymerase (carrying the PB2 627E avian signature) to replicate in mammalian cells (Long et al., 2016;
Nature). It was also shown in this study that an additional 33 amino acid domain present in the avANP32A was responsible
for this activity. Within this domain we observed 6 unique amino acids containing a motif of hydrophobic residues (VLSLV),
which are commonly found in SUMO-Interacting Motifs (SIM). After performing mutagenesis analyses of this motif we found
that the VL-LV residues are crucial for avANP32A activity during avian IAV polymerase replication. To further examine
whether SIM-like sequences play a role in avANP32A activity, we generated chimeric constructs by inserting wellcharacterized heterologous SIM peptides into huANP32A. Interestingly, these constructs were partially active in supporting
avian polymerase activity, boosting activity by ~10 fold, compared to the levels observed in cells expressing the huANP32A
protein. These results indicate that hydrophobic SIM-like sequences contribute, at least to some extent, to avANP32A
activities in enhancing avian IAV polymerase function. However, biochemical analyses revealed no strong interaction
between avANP32A and SUMO, suggesting a SUMO-independent role for the hydrophobic SIM-like sequence in
determining avANP32A function. Nonetheless, further analysis showed that the avANP32A hydrophobic motif is required for
its interaction with the IAV polymerase. We also observed that the IAV polymerase interacts more strongly with avANP32A
when compared with huANP32A, and that this enhanced interaction is independent of the IAV polymerase signature (avian
PB2 627E or human PB2 627K). Our current work is focused on understanding the significance of this enhanced binding for
avANP32A activities on IAV host adaptation.
Corresponding author:
Patricia Domingues
[email protected]
Viral Replication
P96
Virus-specific antibodies allow viral replication in the marginal zone, thereby promoting CD8 + T-cell priming and
viral control
V. Duhan1, V. Khairnar1, K. S. Lang1
1University
Duisburg-Essen, University Hospital Essen, Institute of Immunology, Essen, Germany
Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a
pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we
found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used
the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced viral replication occurs in the presence of
virus-specific antibodies or virus-specific CD8+ T cells. We found that after systemic recall infection with LCMV-WE the
presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast,
specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain
LCMV-Docile, viral replication in spleen was essential for the priming of CD8+ T cells and for viral control. In contrast to
specific antibodies, memory CD8+ T cells inhibited viral replication in marginal zone but failed to protect mice from persistent
viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of
LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control
of persistent virus.
Corresponding author:
Vikas Duhan
[email protected]
Viral Replication
P97
Establishment and functional characterization of a fully infectious Hepatitis Delta Virus genotype 3 clone
F. Lempp1,2, Z. Zhang1, S. Urban1,2
1University
2German
Hospital Heidelberg, Heidelberg, Germany
Centre for Infection Research (DZIF), Heidelberg, Germany
Chronic Hepatitis Delta represents the most severe form of viral hepatitis that is associated with an increased risk of patients
to develop liver cirrhosis and hepatocellular carcinoma. Hepatitis Delta is caused by chronic co-infection with Hepatitis Delta
Virus (HDV) and Hepatitis B Virus (HBV). HDV, being a satellite virus of HBV, requires the HBV envelope proteins for viral
packaging and release. Eight different genotypes for HDV have been described with distinct geographical distributions: while
genotype 1 (gt1) is the most prevalent genotype and can be found worldwide, genotype 3 (gt3) is endemic exclusively in the
Amazonian Region in South America, and genotypes 5-8 are predominant in Central Africa. The discovery of the cellular
receptor sodium-taurocholate cotransporting polypeptide (NTCP) and the subsequent development of novel cell culture
infection models have provided important tools to study HDV infection and pathogenesis in vitro. However, almost all studies
published for HDV have been performed using a gt1 isolate, while only little is known about other genotypes mostly due to
the lack of the respective cell culture clones. Using HDV gt3 as a prototype, we developed an in silico approach to establish
HDV clones: a more-than-genome length antigenomic HDV sequence was designed based on a clinical isolate, the
nucleotide sequence was chemically synthesized and cloned in a eukaryotic expression vector. HDV particles were produced
by co-transfection of Huh7 cells with the antigenomic constructs and a plasmid encoding for the HBV envelope proteins and
virus was purified by heparin affinity chromatography. The purified HDV gt3 has shown to be fully infectious on both NTCPreconstituted human hepatoma cells and primary human hepatocytes (PHH). Interestingly, subcellular localization of
Hepatitis Delta antigen (HDAg) in infected cells differed between gt1 and gt3 with gt3 HDAg being localized exclusively in the
cell nuclei. Virus-dependent induction of the innate immune system was similar between both genotypes as determined by
RNA-levels of interferon stimulated genes. In summary, we have developed a method to construct HDV clones of all different
genotypes and/or clinical isolates. These different genotypes can be used to further study viral pathogenesis, to examine
novel treatments and evaluate possible drug resistances.
Corresponding author:
Florian Lempp
[email protected]
Viral Replication
P98
HIV-1 Vpr increases HCV replication through VprBP in cell culture
Y. Yan1, F. Huang1, T. Yuan1, B. Sun1, R. Yang1
1Chinese
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
Coinfection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs at a high frequency, in which HIV
shows a promotion of HCV-derived liver diseases. However, the mechanism of how this occurs is not well understood. Our
previous work has demonstrated that the HIV-1 accessory protein Vpr enhances HCV RNA replication in cell culture.
Because Vpr performs most of its functions through host protein VprBP (DCAF1), the role of VprBP in the regulation of HCV
by Vpr was investigated in this study. We found that the Vpr mutant Q65R, which is deficient in VprBP binding, could not
enhance HCV replication. Furthermore, Vpr-mediated enhancement of HCV replication was severely diminished in VprBP
knockdown cells. In addition, an inhibitor of CRLs, MLN4924, impaired the function of Vpr during HCV replication. Together,
these results suggest that Vpr promotes HCV replication in a VprBP-dependent manner, and that the activity of CRLs is
essential to this process. In conclusion, our findings demonstrate that HIV-1 Vpr makes the cellular environment more
suitable for HCV replication, which might relate with the host ubiquitination system.
Corresponding author:
Rongge Yang
[email protected]
Viral Replication
P99
Dynamic Phosphorylation of VP30 is essential for Ebola virus life cycle
C. Lier1, S. Becker1, N. Biedenkopf1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The recent
Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities.
Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. An
important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral
nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with
NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30
phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase
cofactor VP35 and/or the viral RNA, presumably leading to increased viral replication, which is supported by NP, L and VP35
alone. Together, these results underlined the significance of VP30 dephosphorylation for its function in viral transcription.
However, though the non-phosphorylatable VP30 perfectly supported viral transcription, it was impossible to generate a
recombinant EBOV encoding VP30 with no N-proximal phosphorylation sites, implicating a critical role for VP30
phosphorylation at certain time points of infection.
By employing different phosphorylation mutants of VP30, we show that the dynamic phosphorylation of VP30 is critical for
the co-transport of VP30 with nucleocapsids to the sites of viral RNA synthesis where VP30 is required to initate primary viral
transcription. We further demonstrate that one single serine residue at the VP30 phosphorylation sites, amino acid position
29, was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics
comparable to wildtype virus. In contrast, rescue of a recombinant virus with a single serine at position 30 at the VP30
phosphorylation site was unsuccessful. Our results indicate a critical role for the dynamic phosphorylation of VP30 during the
first steps of the viral life cycle.
Corresponding author:
Nadine Biedenkopf
[email protected]
Viral Replication
P100
Identification of NS3 Surface residues that are critical for HCV replicase assembly or virion morphogenesis
H. Schwanke1, N. Tautz1, O. Isken1, C. Erfurth1
1University
of Lübeck, Institute of Virology and Cell Biology, Lübeck, Germany
The assembly of hepatitis C virus (HCV) replicase complexes (RCs) is a complicated process in which many viral and
cellular factors are involved. Attempts to understand the assembly of the HCV RC have been challenging owing to limitations
in our understanding of the protein-protein interactions leading to the formation of functional RCs.
Replication complex maturation is intensively controlled at multiple levels to prevent the non-functional assembly of these
multiprotein structures. At the center of this process is NS3, a multifunctional protein with roles in polyprotein processing,
RNA replication and virion morphogenesis. Recently we demonstrated that a hydrophobic NS3 protease surface patch is
critical for this temporal regulation of RC assembly. This NS3 surface promotes NS2 protease stimulation to allow NS2-NS3
cleavage most likely via interactions with NS2 surface residues. Only after NS2-3 cleavage has occurred, this NS3
surface/area can become engaged in protein-protein interactions which lead to a functional replicase assembly.
In this study, we extended our earlier revers-genetic characterization of this multifunctional NS3 surface. These efforts led to
the identification of additional surface residues which are critical for functional RC assembly and RNA replication.
Interestingly, some of these determinants reside in the NS3 helicase domain showing the importance of this region in RC
maturation. Together with our recently identified surface patch these residues form a contiguous surface area on NS3 that
most likely functions as interaction platform for the first step of replicase assembly.
NS3 is also involved in virion morphogenesis and its helicase domain has been mainly attributed to have a role in this
process. Interestingly, we identified one residue within this surface region on the helicase domain, which inhibited viral
production while it had no apparent effect on RNA replication and thus RC maturation. This illustrates the multipurpose use
of protein surfaces to assemble functionally different multiprotein complexes. Together, our present work is providing the
basis to continue to dissect the interactions between the HCV nonstructural proteins during HCV life cycle and to gain more
insights about the molecular architecture of HCV RCs.
Corresponding author:
Norbert Tautz
[email protected]
Viral Replication
P101
Role of M1 tyrosine 132 phosphorylation in the influenza A virus life cycle
A. Mecate-Zambrano1, Y. Börgeling1, D. Anhlan1, T. Shi2, S. Ludwig1
1University
of Münster, Institute of Virology, Münster, Germany
Innovation Center, Immunology, Inflammation and Infectious Diseases (I3) DTA, Roche Pharma Research and Early Development, Basel,
Switzerland
2Roche
Question: Influenza viruses still represent a major health threat worldwide, however, the antiviral arsenal to fight these
pathogens is currently quite limited. The highly conserved influenza virus matrix (M1) protein is a master regulator of the
virus life cycle controlling virus replication on several stages such as uncoating, RNP transport and budding. The different
functions of this protein are most likely regulated by posttranslational modifications. Therefore, identification of essential
regulatory phosphorylation sites as well as respective kinases is key towards development of novel broadly active antiviral
approaches. Different studies identified the tyrosine on position 132 (Y132) of M1 to be phosphorylated. Wang and
colleagues reported in 2013 by overexpression approaches that this phosphorylation is indispensable for virus replication,
mediating M1 import into the nucleus. The aim of this study was the characterization of replicating viruses carrying a
phospho-mimetic mutation or non-phosphorylatable aminoacids in M1 Y132.
Methods: For functional analysis, phosphosite-mutant viruses were generated by reverse genetics technology. Prospective
analyses included assessment of replication fitness and deeper characterization of phenotypes.
Results: M1 Y132A mutant virus was successfully rescued, while M1 Y132D resulted in no production of progeny virions.
Analysis of M1 Y132A showed a significantly decreased viral replication compared to wildtype virus. Surprisingly, there was
no impairment of M1 Y132A import into the nucleus observable, although a small fraction was found at the plasma
membrane already at early stages of infection, resulting in coarse aggregates of M1 at later stages. This was accompanied
by a vast production of defective particles.
Conclusion: This study highlights the importance of replicating viruses to fully characterize the function of posttranslational
modifications of viral proteins. Furthermore, phosphorylation of M1 Y132 mediated by cellular kinases is needed in late
stages of viral replication such as budding or segment packaging. Blocking cellular activities indispensable for virus
propagation will ensure interference with replication of a broad range of influenza viruses with a high barrier towards
generation of resistant variants.
Corresponding author:
Angeles Mecate-Zambrano
[email protected]
Viral Replication
P102
The role of the PB2 627-domain in influenza virus polymerase function
B. Nilsson1, A. te Velthuis1, E. Fodor1
1University
of Oxford, Sir William Dunn School of Pathology, Oxford, United Kingdom
Question: The RNA-dependent RNA polymerase of influenza viruses is a complex structure consisting of the subunits PA,
PB1 and PB2. Viral genomic vRNA is transcribed in a cap-dependent manner and replicated de novo via a complementary
(cRNA) replicative intermediate. Avian influenza virus replication is usually severely restricted in mammalian hosts. In order
to enable efficient viral replication, the avian influenza virus needs to acquire adaptive mutations in its polymerase. An E to K
substitution at position 627 of the PB2 subunit is able to overcome this host restrictive effect and enhance avian polymerase
activity to the level of mammalian-adapted influenza polymerases. PB2-627 lies on the surface of a structurally distinct
domain in the C-terminal region of PB2 called the 627-domain, close to the proposed RNA-exit channel of the influenza virus
polymerase. Several other adaptive mutations have been described that cluster around the 627-domain and have been
shown to compensate for the for the host-restrictive effect in a similar manner as E627K. However, the mechanism by which
these mutations enable host adaptation remains unclear. Moreover, whereas functions can be attributed to most domains of
PB2, the role of the 627-domain in the viral replication cycle is uncertain.
Methods: We used recombinant influenza virus polymerase with C-terminally truncated PB2 mutants to investigate the role
of the C-terminal two thirds, including of the 627-domain, in polymerase function in vitro and cell based transcription and
replication assays.
Results: We found that the PB2 627-domain is neither required for the assembly of the heterotrimeric polymerase complex,
nor for in vitro RNA promoter binding. It is also not required for capped RNA-primed transcription initiation on a vRNA
template and for primer-independent de novo and ApG-primed replication initiation on vRNA and cRNA templates.
Nevertheless, in a cellular context we found that the 627-domain is essential for viral RNA replication, transcription and cRNA
stabilisation in an NP-independent manner.
Conclusion: These results suggest that the PB2 627-domain does not contribute to the core functions of the polymerase but
performs an auxiliary function potentially facilitating the assembly of the newly produced RNA with polymerase.
Corresponding author:
Benjamin Nilsson
[email protected]
Viral Replication
P103
The pestiviral non-structural protein 4A is critically involved in RNA replication and virion morphogenesis
D. Dubrau1, A. E. Kiss1, J. Janz1, N. Tautz1
1University
of Lübeck, Institute of Virology and Cell Biology, Lübeck, Germany
A specific feature of the Flaviviridae is the critical function of non-structural (NS) proteins for virus particle formation. Within
the pestiviral life cycle the multifunctional protein NS3 together with its cofactor NS4A is essential for polyprotein processing
and RNA replication. For virion morphogenesis pestiviruses generally rely on uncleaved NS2-3/4A. For the closely related
hepatitis C virus (HCV) the NS3 protease cofactor NS4A has been shown to be critical not only for polyprotein processing
and RNA replication but also for virion morphogenesis. In the C-terminal part of NS4A residues which were essential
specifically for virion morphogenesis have been described. For pestiviruses such a detailed knowledge is not available so far.
In our present study, we conducted a reverse genetic analysis by introducing alanine mutations into bovine viral diarrhea
virus (BVDV) NS4A with the aim to elucidate the functional importance of its C-terminal part in RNA replication and virion
formation. The NS4A mutants were characterized with regard to their function in polyprotein processing, RNA replication and
virion morphogenesis. Some of the mutations in the NS4A kink region were affecting RNA replication without obvious effects
on polyprotein processing. Interestingly, most of the mutations in the pestiviral acidic domain do neither inhibit RNA
replication nor virion morphogenesis which is in contrast to findings in the HCV system. Furthermore, the analysis revealed
that an amino acid motif at the C-terminal end of the trans-membrane domain of NS4A is critical for virion morphogenesis but
not for RNA replication. Surprisingly, this phenotype could be functionally rescued by three compensatory mutations residing
in NS2, NS3 and NS5A indicating an interaction of these proteins with NS4A in the process of virion formation. These novel
observations provide a basis for the future characterization of protein complexes involved either in RNA replication or virion
morphogenesis.
Corresponding author:
Norbert Tautz
[email protected]
Viral Replication
P104
Functional and biochemical characterization of norovirus nonstructural protein 3
Z. Menne1, P. Schult1, S. Doerflinger1, A. Koromyslova1, G. S. Hansman1, V. Lohmann1
1University
of Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
Introduction: Human norovirus (HuNoV) is the leading cause of acute gastroenteritis (AGE) worldwide. Since the
emergence of the genogroup II (GII) strains in the late 1990s, GII strains have been responsible for over 95% of all cases of
sporadic AGE in children under 18 years of age caused by HuNoV. However, investigation of HuNoV nonstructural (NS)
protein function has been hindered by the lack of a robust and widely available cell culture model.
Objectives: We sought to characterize the enzymatic activity of recombinant NS3 (rNS3) protein from the HuNoV GII.4 New
Orleans outbreak strain produced and purified from a prokaryotic expression system. Additionally, we aimed to identify amino
acid residues in the NS3 protein critical for norovirus replication using a murine norovirus (MNV) reverse genetic system.
Material & Methods: rNS3 was produced in a prokaryotic expression system and purified via immobilized metal affinity
chromatography (IMAC). rNS3 was assayed for its ability to hydrolyze ATP by colorimetric detection of released inorganic
phosphate (Pi) in assay mixtures. A library of MNV NS3 mutants in an infectious DNA clone of MNV was constructed via
overlap PCR and subsequently transfected into a permissive cell line. Recovery of MNV was measured by virus titration in an
endpoint dilution assay.
Results: rNS3 showed ATPase activity in a protein concentration dependent manner while an NS3 mutant previously shown
unable to bind ATP, K171Q, did not show detectable ATPase activity. The ATPase activity of rNS3 was sensitive to varying
assay buffer conditions including temperature, pH, and NaCl concentration. Reverse genetic analysis of conserved residues
in the amino acid sequence of NS3 showed sensitivity to mutagenesis to alanine. Of 11 point mutants assayed, MNV NS3
only tolerated a mutation from isoleucine to alanine at position 17.
Conclusion: Our results indicate that the NS3 protein from the GII.4 New Orleans HuNoV strain functions as an ATPase in
vitro. Additionally, our results suggest that ATPase function is indispensible for norovirus replication in an MNV reverse
genetic model.
Corresponding author:
Zach Menne
[email protected]
Adaptive Immunity
P105
Improved effector functions of CD8+ T cells by IFNα subtypes are mediated by antigen-specific DCs
J. Dickow1, U. Dittmer1, K. Sutter1
1Ludwig
Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
Introduction: The expression of type I interferons (IFN) is the immediate host response during most viral infections. These
cytokines induce an antiviral state in cells by the expression of antiviral proteins. Additionally, type I IFNs can also modulate
immune cell functions as well as elicit antiproliferative responses. The type I IFN family is composed of multiple IFNα
subtypes (up to 14 in mice), and although they all bind the same receptor, they differ in their biological activity.
Objectives: Here, we are interested in the immunoregulatory functions of IFNα subtypes on antigen-specific CD8+ T cell
responses to further use them as an immunotherapeutic agent during chronic retroviral infection.
Methods: To investigate the influence of different IFNα subtypes on CD8+ T cell proliferation, activation and effector
functions we performed antigen-specific T cell proliferation assays in vitro. The impact of IFNα subtypes on the cytotoxicity of
CD8+ T cells was analyzed by an in vitro kill assay. IFNAR-deficient CD8+ T cells and dendritic cells (DCs) were used to
determine how IFNα subtypes regulate antigen-specific CD8+ T cell responses. Phenotypic analysis of CD8+ T cells and
DCs were performed by flow cytometry.
Results: We detected a direct antiproliferative capacity of all tested IFNα subtypes on CD8+ T cells. In particular, IFNα4 and
IFNα9 strongly induced the expression of cytokines like IL-2, TNFα and IFNγ in vitro. IFNα subtype-treated DCs were highly
activated and matured and showed increased production of TNFα, IL-6 and IL-10. The induction of cytokines in CD8+ T cells
by IFNα subtypes required IFNAR expression on DCs. Furthermore, IFNα subtype stimulation (especially IFNα4 and IFNα6)
induced highly activated CD8+ effector T cells which exhibit an improved cytotoxic phenotype (Granzyme B, Eomes)
whereby killing of target cells by CD8+ T cells was significantly enhanced.
Conclusion: These results demonstrate the distinct immunostimulatory properties of each IFNα subtype which can be used
to develop future immunotherapies against retroviruses.
Corresponding author:
Julia Dickow
[email protected]
Adaptive Immunity
P106
Mesenchymal stem cells induce immune response and protect against lethal Herpes Simplex Virus Type 1 infection
in an animal model
E. Momotyuk1,2, R. Klimova2, A. Kushch2
1Moscow
state academy of veterinary medicine and biotechnology named K.I. Skryabin, The Russian Federation Ministry of Agriculture, Moscow, Russian
Federation
2N. F. Gamaleya Federal Research Center of Epidemiology and Microbiology, The Russian Federation Ministry of Health , Moscow, Russian Federation
Introduction: Mesenchymal stem cells (MSC) have been used in regenerative medicine and as vectors in gene therapy.
These cells demonstrate anti-inflammatory and immunomodulatory activities after allogenic transplantation.
Objectives: Examine the effect of MSC injection on immune response and protection against herpes simplex virus (HSV)
infection in a mouse model.
Material & Methods: MSC culture was isolated from bone marrow of DBA mice. Group I mice (n=5) were given 2 injections
of 2х105 MSC into tail vein at a 3-week interval. Group 2 mice (n=5, control) received 2 injections of saline solution. Both
groups were infected with HSV1 (10 LD50 intraperitoneally). The mice were observed for 14 days. Sera were pooled in each
group. Anti-HSV1 antibodies were studied by IFA and microneutralization test in vitro. Т-cell response was assessed in blast
transformation test in vitro.
Results: All group I mice survived HSV1 injection, while in control group all mice died (Р<0.05). Titer of anti-HSV antibodies
in group 1 was 1:800, in control group: 1:50 (Р=0.03). Titer of virus-neutralizing antibodies in group 1 mice was 1:160, and
1:15 in group 2 mice (Р=0.007). Statistically significant intergroup difference was demonstrated for blast number in
splenocyte population: 32±3 (group 1) vs. 9.3±2.3 (group 2), P=0.005.
Conclusion: The results of this pilot study indicate that MSC induce B and T cell immune response to HSV and provide
100% protection of mice against 10 LD50 HSV infection. Immunostimulatory and protective activities of MSC in viral
infections require further investigation.
Corresponding author:
Ekaterina Momotyuk
[email protected]
Adaptive Immunity
P107
Naïve T cells are activated by autologous HCMV-infected endothelial cells through NKG2D and can control HCMV
transmission in vitro
N. Subramanian1, Z. Wu1, F. Reister2, K. Laib Sampaio1, G. Frascaroli1, L. Cicin-Sain3, T. Mertens1
1Ulm
University Medical Center, Institute of Virology, Ulm, Germany
University Medical Center, Department of of Obstetrics and Gynecology, Ulm, Germany
3Helmholtz Centre for Infection Research, Department of Vaccinology and Applied Microbiology, Braunschweig, Germany
2Ulm
Human cytomegalovirus (HCMV) is a species specific β-herpesvirus. Endothelial cells (ECs) are important target cells of
HCMV and are involved in the trafficking of the T cells. The aim of this work is to study the interaction of naïve T cells from
umbilical cord with HCMV infected and uninfected autologous ECs. Naïve T cells became activated when exposed to HCMV
infected human umbilical vein endothelial cells (HUVECs) within 24 hours of exposure and the activation percentage
increased over time. In contrast to the current dogma that T cells need classical antigen presenting cells (APCs) for
activation, the activation of naïve T cells occurred even in the absence of classical APCs, without addition of any cytokines.
The activation was HCMV specific; occurring only upon exposure to HCMV-infected HUVECs and not uninfected HUVECs.
Using a functional cell culture assay, we showed the naïve T cells, which were activated upon exposure to HCMV-infected
HUVEC, specifically inhibited the HCMV transmission. Finally we could show that only infected HUVECs activate naïve T
cells, whereas infected autologous fibroblasts isolated from the same cord did not. This activation of naïve T cells was
inhibited when the NKG2D receptor was blocked on the T cells. Based on our observations, we propose a novel mechanism
for activation of umbilical cord naïve T cells by HCMV-infected autologous HUVECs that is independent of the presence and
function of classical APCs.
Corresponding author:
Narmadha Subramanian
[email protected]
Adaptive Immunity
P108
HBV-mediated Nrf2 activation in host cells and its implication for CTL-mediated immune response
M. Biehl1, K. Wisskirchen2,3, U. Protzer2,3, E. Hildt1,4
1Paul
Ehrlich Institute, Virology, Langen, Germany
University Munich, Institute of Virology, Munich, Germany
3Helmholtz Centre Munich, Munich, Germany
4German Centre for Infection Research (DZIF), Braunschweig, Germany
2Technical
The human hepatitis B Virus (HBV) activates the host cellular nuclear factor, erythroid 2-like 2 (Nrf2), which regulates the
expression of antioxidant proteins and enzymes involved in the detoxification of electrophiles and radicals. HBV-mediated
Nrf2 activation leads to an increased activity of the constitutive proteasome, ultimately leading to a decreased activity of the
immune proteasome [1] which is important for generation of peptides presented on MHC class I molecules for the recognition
by cytotoxic T cells.
Question: We hypothesize that, by activation of Nrf2, HBV protects the host cells from the adaptive immune system by downmodulation of antigen-processing and thereby -presentation and by protecting against cell-mediated cytotoxicity.
Methods: The effect of HBV-regulated Nrf2 activity on T cell-mediated cytotoxicity was assessed by the modulation of Nrf2
activity in HBV replicating cells, using the small molecular inhibitor brusatol before and during the co-culture with transgenic
HBV-specific CD8+ T cells. The CD8+ T cell response was assessed by quantifying the release of lactate dehydrogenase
from dying target cells as well as IFNγ release by specifically activated CTLs.
Results & Conclusion: The presence of brusatol leads to a significantly decreased sensitivity of target cells towards T cellmediated cytotoxicity, whereas the IFNγ secretion by T cells was increased compared to the controls. Since brusatol has
more recently been proposed to be an inhibitor of global protein synthesis rather than a specific Nrf2 inhibitor [2], we were
seeking for a more specific way of modulating Nrf2 activity. Therefore, lentiviral constructs were generated, expressing a
dominant negative mutant of Nrf2 (tdnNrf2) which has been described to inhibit Nrf2 target genes [3]. HBV replicating
hepatoma cells were transduced with lentiviral vectors expressing tdnNrf2. In these cells, we could detect a significantly
reduced mRNA expression of the Nrf2 target gene nqo1 compared to GFP-transduced and untransduced control cells.
These HBV replicating and tdnNrf2 expressing cells will be co-cultured with HBV-specific CD8+ T cells in order to evaluate
the role of Nrf2 activity for CTL-mediated immune responses.
References:
[1] Schädler et al., 2009 PMID 20956535
[2] Vartanian et al., 2016 PMID 26711467
[3] Auf dem Keller et al., 2006 PMID 16648473
Corresponding author:
Marlene Biehl
[email protected]
Adaptive Immunity
P109
Zika virus cross-neutralization by polyclonal sera after dengue virus infection
S. Malafa1, J. Aberle1, S. Aberle1, F. X. Heinz1, K. Stiasny1
1Medical
University of Vienna, Department of Virology, Vienna, Austria
The explosive spread of Zika virus to Pacific Islands as well as South- and Middle America underscores the potential threat
of newly emerging arthropod-borne viruses. At present it is unclear which factors contributed to this emergence, potentially
including mutational adaptations that changed the character of this virus. In addition, however, there is increasing evidence
that pre-existing cross-reactive flavivirus (especially dengue) antibodies can enhance Zika virus replication and thus might
play a role in pathogenesis. Recent structural and monoclonal antibody studies have identified epitopes involved in infection
enhancement as well as cross-neutralization between Zika and dengue viruses and provided new insights into their antigenic
structures.
Information on cross-neutralization of Zika virus by polyclonal antibodies induced by other flavivirus infections and/or
vaccinations is currently lacking. We have therefore conducted a systematic analysis of Zika virus neutralization by serum
samples from dengue virus-infected people to obtain new and quantitative data on the extent and individual variation of
cross-neutralization between the two viruses.
We analyzed a panel of 31 serum samples from travelers returning to Austria from South America and Asia that were
diagnosed with dengue. For measuring virus neutralizing antibodies, we have established high-throughput
microneutralization tests (NTs) with Zika and dengue viruses.
Four of the 31 dengue serum samples (13%) showed considerable Zika virus-neutralizing activity (50% neutralization titers >
100), but the titers were still significantly lower than the homologous dengue NT values. These data indicate that Zikadengue cross-neutralizing antibodies represent a rare subset in only a few dengue virus-infected individuals. Currently, we
are investigating the reciprocal situation, i.e. whether and to what extent polyclonal sera obtained from Zika virus-infected
patients can neutralize dengue virus.
Corresponding author:
Stefan Malafa
[email protected]
Adaptive Immunity
P110
Characterization of endogenous MHC Class II presentation pathways from modified Vaccinia Virus Ankara infected
dendritic cells
S. Tao1, F. Thiele2, Y. Zhang2, A. Muschaweckh2, T. Zollmann3, U. Protzer2, R. Abele3, I. Drexler1
1Heinrich
Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
University Munich and Helmholtz Centre Munich, Institute of Virology, Munich, Germany
3Johann Wolfgang Goethe University Frankfurt, Institute of Biochemistry, Frankfurt a.M., Germany
2Technical
CD4 T lymphocytes play a central role in the immune system and mediate their function after recognition of their respective
antigens presented on major histocompatibility complex II (MHCII) molecules on antigen-presenting cells (APCs).
Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4 T cells. Certain epitopes, however, can
be processed directly from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we
characterized the MHCII antigen presentation pathways that are possibly involved in the immune response upon vaccination
with modified vaccinia virus Ankara (MVA), a promising live viral vaccine vector. We established CD4 T-cell lines specific for
MVA-derived epitopes as tools for in vitro analysis of MHCII antigen processing and presentation in MVA-infected APCs. We
provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to
efficiently activate CD4 T cells. By using knockout mice and chemical inhibitory compounds, we further elucidated the
molecular basis, showing that among the various subcellular pathways investigated, proteasomes and autophagy are key
players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays
an important role, neither TAP nor LAMP-2 was found to be involved in the peptide transport. Defining the molecular
mechanism of MHCII presentation during MVA infection provides a basis for improving MVA-based vaccination strategies by
aiming for enhanced CD4 T-cell activation by directing antigens into the responsible pathways.
Corresponding author:
Sha Tao
[email protected]
Adaptive Immunity
P111
Quantification of the TBEV neutralising antibody response to antibody subsets in human and mouse sera after
depletion
V. Bradt1, S. Malafa1, G. Tsouchnikas1, J. Zlatkovic1, K. Stiasny1, F. X. Heinz1
1Medical
University of Vienna, Department of Virology, Vienna, Austria
Introduction: Tick-borne encephalitis virus (TBEV) is an important human-pathogenic flavivirus endemic in large parts of
Europe, Central and Eastern Asia. Long-lasting protection after natural infection or vaccination with TBEV is mediated by
neutralising antibodies, recognising the viral envelope protein E. Recently, recombinant virus particles carrying a luciferase
reporter gene provide a new possibility for sensitively measuring antibody-mediated neutralisation in polyclonal sera.
Objectives: In order to characterise the neutralising antibody responses against TBEV in human and mouse serum samples
and to quantify the contribution of defined antibody subsets to overall neutralisation, we wanted to establish a novel
neutralisation test (NT) using single-round infectious TBE reporter virus particles (RVP).
Methods: We designed and optimised a neutralisation test with TBE RVPs in which infection of cells was measured as a
function of luciferase activity. For further quantification of antibody subpopulations with different fine specificities and their
contribution to overall neutralisation, different test sera were depleted with recombinant antigens representing viral surface
protein domains and domain combinations.
Results and Conclusion: The novel reporter virus neutralisation test proved to be a robust, sensitive and fast assay,
allowing us to analyse human and murine neutralising antibody responses. By using the reporter NT before and after
depletion with different recombinant protein domains of E, we could define the contribution of distinct antibody subsets to the
overall neutralisation and thereby distinguish differences in the immunodominance patterns of individual test sera.
Corresponding author:
Victoria Bradt
[email protected]
Adaptive Immunity
P112
Identification of blood donors with exceptional neutralizing responses against the human cytomegalovirus
F. Jessica1, S. Dagmar1, S. Christian1, W. Martina2, L. Ramin2, A. Krawczyk3, A. Mira3
1Ulm
University Medical Center, Institute of Virology, Ulm, Germany
Red Cross Blood Transfusion Service Baden-Württemberg – Hessen and Ulm University Medical Center, Institute of Clinical Transfusion Medicine
and Immunogenetics, Ulm, Germany
3University Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
2German
Attempts to exploit hyperimmunoglobulins for prevention and treatment of human cytomegalovirus (HCMV) infections were
disappointing so far. One possible explanation is that the quality of transfused antibodies was too low to induce sufficient
neutralization titers in treated individuals. To improve the efficacy of anti-HCMV immunoglobulins, we aimed to identify
exceptional "elite" blood donors with high and broad neutralization capacities. Using a Gaussia-luciferase reporter virus,
plasma samples from 9000 HCMV-seropositive blood donors were screened in fibroblast cultures regarding their capacities
to neutralize HCMV infection. An initial analysis of 1000 samples had shown that high neutralization on fibroblasts is always
accompanied by high neutralization capacity in endothelial cells. A subset of the top neutralizers were retested by an ELISAbased assay concerning the broadness of the inhibitory effect against various HCMV strains and compared with standard
hyperimmunoglobulins. All highly and broadly neutralizing plasmas were analyzed concerning their mode of action, i.e. their
effect on virus attachment and penetration using fluorescent-tagged viruses. 240 out of 9000 plasma samples were identified
as highly neutralizing in the screening procedure. In 80 samples that were further analyzed, the half-maximal neutralization
titers ranged between 115 and 3467 (mean 1061, SD 726). 80% were broadly effective against all tested HCMV strains.
While most of the plasma samples preferentially inhibited the penetration of attached particles, three exceptional donors
could almost completely block adsorption. A combination of the respective antibodies resulted in a synergistic inhibition of
HCMV. As expected, such elite neutralizing plasmas were superior to commercially available hyperimmunoglobulins.
Immunoglobulins composed of "elite" plasma preparations may provide a basis for improved antibody-based treatment of
HCMV infections. To test whether such preparations can induce sufficiently high neutralizing titers in treated patients we are
currently preparing a dose-finding study.
Corresponding author:
Falk Jessica
[email protected]
Adaptive Immunity
P113
Regulation of antiviral CD8 T cell response by CD100 and its soluble form in HBV infection
L. Wang1, S. Yang1, W. Bayer2, M. Lu2, U. Dittmer2, J. Timm3, D. Yang1, J. Liu1
1Union
Hospital, Tongji Medical College Huazhong University of Science and Technology, Department of infectious Disease, Wuhan, China
of Duisburg-Essen, Institute of Virology, Essen, Germany
3Heinrich Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
2University
CD100 was the first semaphorin described to have immune functions and serves important roles in T cell priming. Proteolytic
cleavage of CD100 from the cell surface gives rise to a soluble fragment of CD100 (sCD100), which also has
immunoregulatory properties. In this study, we characterized the expression and the possible role of CD100/sCD100 in
regulating antiviral response during HBV infection in patients and HBV-replicating mouse model. We could show that surface
CD100 expression on T cells of chronic Hepatitis B (CHB) patients was significantly increased compared to that of healthy
controls (HC). Meanwhile, CHB patients showed significantly lower concentrations of serum sCD100 than HC.
Correspondingly, decreased surface CD100 expression on T cells in PBMCs and elevated serum sCD100 levels were
observed in mice which eliminated HBV from their livers but not in HBV-tolerant mice. In vivo sCD100 treatment led to
accelerated viral clearance in HBV-tolerant mice. Interestingly, activation of liver sinusoidal endothelial cells by sCD100
treatment was observed. In vitro sCD100 treatment also resulted in enhanced HBV-specific CD8 T cell response in PBMCs
of some CHB patients. In contrast, blockade of the interaction of CD100 and its ligand CD72 by CD72 blocking antibody
treatment resulted in decreased HBV-specific CD8 T cell response and HBV persistence in mice. Next, we investigated the
possible mechanism involved in membrane CD100 shedding during HBV infection. Our results demonstrated that matrix
metalloproteinase (MMP) 2 and MMP9 are responsible for the cleavage of CD100 from the surface of T cells. In CHB
patients, significantly lower serum MMP2 concentration was observed than that of HC. Compared to HBV-tolerant mice, mice
which eliminated the virus showed elevated MMP2 expression in the liver during the acute phase of infection. In conclusion,
our results indicated CD100/sCD100-CD72 interaction is important for the induction of antiviral T cell response and viral
clearance during HBV infection. We hypothesize that insufficient liver MMP2 production during HBV infection results in
impaired membrane CD100 cleavage, which leads to lack of activation stimulation for intrahepatic antiviral T response and
thus the persistence of HBV infection.
Corresponding author:
Jia Liu
[email protected]
Adaptive Immunity
P114
Presentation of a conserved adenoviral epitope on HLA-C*0702 allows evasion of NK but not T-cell responses
A. Keib1, P. Günther1, B. Faist2, A. Halenius3, D. Busch2, M. Neuenhahn2, G. Jahn1, K. Dennehy1,4
1University
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
University Munich, Institute of Medical Microbiology, Immunology and Hygiene, Munich, Germany
3Albert Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
4German Center for Infection Research (DZIF), Tübingen, Germany
2Technical
Infection with adenovirus is a major cause of infectious mortality in children following haematopoietic stem cell
transplantation. While adoptive transfer of epitope specific T-cells is a particularly effective therapeutic approach, there are
few suitable adenoviral peptide epitopes described to date. Here we describe the adenoviral peptide epitope FRKDVNMVL
from hexon protein, and its variant FRKDVNMIL, that is restricted by HLA-C*0702. Since HLA-C*0702 can be recognized by
both T-cells and NK cells, we characterized responses by both cell types. T-cells specific for FRKDVNMVL were detected in
PBMC expanded from eight of ten healthy HLA-typed donors by peptide-HLA multimer staining, and could also be detected
by cultured IFNg ELISpot assays. Surprisingly, HLA-C*0702 was not down-regulated during infection, in contrast to the
marked down-regulation of HLA-A*0201, suggesting that adenovirus cannot evade T-cell responses to HLA-C*0702restricted peptide epitopes. By contrast, NK responses were inhibited following adenoviral peptide presentation. Notably,
presentation of the FRKDVNMVL peptide enhanced binding of HLA-C*0702 to the inhibitory receptor KIR2DL3, and
decreased NK cytotoxic responses suggesting that adenoviruses may use this peptide to evade NK responses. Given the
immunodominance of FRKDVNMVL-specific T-cell responses, apparent lack of HLA-C*0702 down-regulation during
infection, and the high frequency of this allotype, this peptide epitope may be particularly useful for adoptive T-cell transfer
therapy of adenovirus infection.
Corresponding author:
Kevin Dennehy
[email protected]
Adaptive Immunity
P115
Genetic background shapes the fine-specificity and individual variation of antibody responses to a tick-borne
encephalitis virus vaccine
G. Tsouchnikas1, V. Bradt1, J. Zlatkovic1, K. Stiasny1, F. X. Heinz1
1Medical
University of Vienna, Department of Virology, Vienna, Austria
Introduction: Serum antibodies comprise an essential arm of antiviral immunity by mediating virus neutralization and other
antibody-dependent effector mechanisms. Typically, swarms of antibodies are induced upon infection or vaccination that
target different epitopes on viral proteins, but usually only few of them dominate the antibody response. Immunodominance
may be influenced by the genetic background of the host and by stochastic processes of clonal B cell selection during affinity
maturation. Flaviviruses such as dengue, Zika or tick-borne encephalitis (TBE) viruses provide important model systems to
study the implications of immunodominance: 1. Antibodies to the envelope protein E mediate long-term protection, but can
also even enhance infection. 2. Knowledge of the atomic structure of E allows the use of recombinant sub-structures for
dissecting the biological roles of distinct antibody specificities in polyclonal sera.
Objective: The goal of our study was to analyze the fine-specificities and variation of antibody responses to tick-borne
encephalitis (TBE) virus in three groups of mice with different genetic backgrounds.
Material & Methods: Groups of thirty C57BL/6, CD1, and Balbc mice were immunized subcutaneously with inactivated TBE
virus and serum antibodies from individual mice were analyzed using virus neutralization and immunoassays with whole TBE
virus, recombinant E proteins as well as subdomains thereof (domain III, domains I+II).
Results: The genetic background of mice had a striking influence on the extent and fine-specificities of antibody responses.
CD1 mice mounted the strongest response and most of the neutralizing antibodies were directed to domain III. In contrast,
antibodies to domains I+II were dominant in C57BL/6 mice, whereas Balbc mice produced a significantly higher proportion of
cross-reactive, non-neutralizing antibodies. In addition to these overall differences we found an extensive variation of
antibody fine specificities among individuals in the same group of mice.
Conclusion: Our results highlight the influence of genetic background on the quality of antibody responses and point to the
individual variability of immunodominance in polyclonal sera as an important factor to be taken into account in the analysis of
post-infection or –vaccination sera.
Corresponding author:
Georgios Tsouchnikas
[email protected]
Adaptive Immunity
P116
Genome-wide analysis of T cell responses in patients with ongoing and resolved hepatitis E
J. Al-Ayoubi1, P. Behrend1, B. Bremer1, A. Gisa1, F. Rinker1, M. P. Manns1, M. Cornberg1, H. Wedemeyer1, A. Kraft1
1
Hanover Medical School, Department of Gastroenterology, Hepatology und Endocrinology, Hanover, Germany
Hepatitis E is an inflammatory liver disease caused by infection with the hepatitis E virus (HEV). With at least 3 million
symptomatic cases per year accounting for 70,000 HEV-related deaths is HEV a major cause of acute viral hepatitis. Usually
an HEV infection is self-limiting but can lead to chronicity in immunocompromised individuals. T cell responses against HEV
have been investigated against the structural proteins expressed by open reading frames (ORF) 2 and 3. However, little is
known about T cell responses against non-structural HEV proteins encoded by ORF1. The aim of this study was to
determine HEV ORF1-specific T cell responses in comparison to ORF2/3 in patients infected with HEV.
HEV genotype 3-specific CD8+ and CD4+ T cell responses were studied in patients with acute and chronic hepatitis E as
well as in HEV IgG seropositive and seronegative healthy individuals. Therefore, peripheral blood mononuclear cells
(PBMCs) were stimulated with HEV-specific overlapping peptide pools spanning all whole HEV genome. HEV-specific T cell
responses were determined by proliferation and intracellular cytokine assay. HEV-antigen was measured using an anti-HEV
antigen-specific ELISA.
HEV ORF1-specific T cell responses were detected in patients with acute, resolved and chronic hepatitis E without distinct
dominant regions. The magnitude and frequency of HEV ORF1-specific T cell responses were similar compared to
responses against HEV ORF2/3. Longitudinal studies of HEV-specific T cell responses displayed similar behaviour of HEVspecific T cell responses against structural and non-structural proteins. HEV-antigen levels were inversely correlated with
HEV-specific T cell responses.
Conclusion: HEV-specific T cell responses were detectable against the whole HEV genome. In terms that HEV-specific T cell
responses are associated with control of HEV infection these findings have implications for vaccine design against HEV. Our
data suggest that vaccine induced T cell responses against structural as well as non-structural HEV-proteins might be of
importance.
Corresponding author:
Anke Kraft
[email protected]
Adaptive Immunity
P117
Antibody-mediated virus control in tolerant congenitally infected virus carriers
P. Reuther1, K. Martin1, Y. I. Ertuna1, D. Pinschewer1
1Experimental
Virology, Department of Biomedicine, Basel, Switzerland
Neonatal infection of mice with lymphocytic choriomeningitis virus (LCMV) results in central antiviral CD8+ T cell tolerance
and thereby results in a life-long co-existence of virus and host, resembling the immune tolerant phase in perinatally hepatitis
B virus (HBV)-infected humans. Potential antiviral effects of antibody responses to congenital infection remain, however,
undefined.
We infected newborn mice, either wild type (wt) or B cell receptor repertoire-restricted (BCRres), with a fluorescently labeled
LCMV (LCMV-tomato). After 4-6 weeks of high-level viremia in both groups, viral titers in wt but not BCRres mice dropped
dramatically, and RNA levels in the liver were 100-fold reduced. Partial virus control in wt but not BCRres was accompanied
by LCMV glycoprotein-specific IgG production. Large foci of infected hepatocytes in the liver of BCRres mice contrasted with
randomly scattered individual virus-infected cells the liver of wt animals, suggesting antibody-mediated inhibition of cell-tocell spread. Analogously to BCRres mice also MHC class II-deficient and activation-induced deaminase (AID)-deficient mice
failed to control congenital LCMV infection. This indicated that conventional T helper cells escaped central tolerance, and
together with isotype class switch and/or somatic hypermutation were required for virus control. Analogous results on partial
antibody-dependent control of (non-fluorescent) wt LCMV in congentially infected mice corroborated and generalized these
findings.
Altogether, these observations indicated that neonatally infected mice mount protective T cell-dependent antibody
responses, which partially control the infection by limiting viral dissemination in tissues. Accordingly, congenital viral
infections are subject to humoral immune pressure, which escapes tolerance mechanisms.
Corresponding author:
Peter Reuther
[email protected]
Adaptive Immunity
P118
Analysis of the adaptive immune response to Nipah virus infection in mice and pigs
E. Stroh1, L. Sauerhering2, A. Maisner2, U. Blohm3, M. Groschup1, S. Diederich1
1Friedrich
Loeffler Institute, Institute of Novel and Emerging Infectious Diseases, Greifswald - Isle of Riems, Germany
University Marburg, Institute of Virology, Marburg, Germany
3Friedrich Loeffler Institute, Institute of Immunology, Greifswald - Isle of Riems, Germany
2Philipps
Introduction: Nipah virus (NiV), a BSL-4 classified zoonotic paramyxovirus, causes severe respiratory disease in pigs and
fatal encephalitis in humans. It has been shown that NiV evades the innate immune response by an interferon antagonistic
activity. Furthermore, the humoral CD4+ helper T-cell dependent immunity is a critical factor in surviving NiV infection and
future protection. However, how the cellular adaptive immune system of different hosts responds to NiV infection remains
poorly understood.
Objective: Aim of this study was to investigate if Nipah virus-like particles (NiVLPs) are a tool to investigate both humoral
and cellular immune response in different hosts.
Methods & Results: For this purpose, C57BL/6 and BALB/c mice as well as German Landrace pigs were immunized 3
times in a 3-week-interval with NiVLPs composed of the surface glycoproteins G and F and the matrix protein M. For
serology, mouse serum was collected after euthanasia, whereas porcine serum was collected weekly. NiVLPs induced the
production of antibodies against all 3 proteins with neutralizing activity in C57BL/6 and BALB/c mice and swine. To examine
the cellular immune response, murine splenocytes were isolated at various days post prime (dpp) for in vitro restimulation.
NiVLPs were able to induce antigen-specific proliferation of CD8+ T cells at 42 and 63 dpp in C57BL/6 mice. Further,
interferon-γ expression in CD8+ T cells after antigenic restimulation at the indicated times was measured. In contrast, none
to only marginal proliferation or IFN-y expression was observed in BALB/c mice. In the pig study, porcine peripheral blood
mononuclear cells (PBMC) were isolated weekly and analyzed for IFN-y response by ELISpot. After antigen-specific
restimulation in vitro, porcine PBMC respond with IFN-y expression and secretion. Whereas there is a measurable antigenspecific B-cell proliferation of PBMC and porcine splenocytes at 63 dpp, a specific proliferation was also observed by the
γδT-cell subpopulation in the blood at 21, 42, and 63 dpp.
Conclusion: To conclude, NiVLPs are a great tool to investigate the adaptive immune response, since those induce humoral
and cellular immune response in the model system mouse and the porcine host. In the future, the NiVLP system has to be
compared with life virus infection studies under BSL-4 conditions.
Corresponding author:
Eileen Stroh
[email protected]
Adaptive Immunity
P119
IL-1β as mucosal vaccine adjuvant – Specific induction of tissue-resident memory T cells and enhanced protection
against heterologous IAV
D. Lapuente1, A. Maaske1, V. Stab1, W. Bayer2, C. Ehrhardt3, M. Tenbusch1
1Ruhr-University
Bochum, Department for Molecular and Medical Virology, Bochum, Germany
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
3Westfaelische Wilhelms University Münster, Institute of Molecular Virology, Center of Molecular Biology of Inflammation, Münster, Germany
2University
Introduction: Influenza A viruses (IAV) are the most common cause of severe viral respiratory tract infections. Most licensed
IAV vaccines are designed to induce strain-specific antibodies to the highly variable surface protein hemagglutinin (HA).
These vaccines provide protection against the strains included in the vaccine but do not protect against unexpectedly
occurring viruses. Recombinant adenoviruses (rAd) are a promising vaccine platform, as they can induce cross-reactive T
cell responses against conserved virus proteins, e.g. the nucleoprotein (NP). Especially tissue-resident memory T cells
(TRM) in the lung are of particular importance for an efficient immunity against divergent IAV strains.
Objectives: rAd-IL-1β and rAd-IL-18 were evaluated as adjuvants in intranasal immunizations with adenoviral vectors. The
mucosal expression of the cytokines should increase mucosal T cell responses and thereby improve the vaccine efficacy
against divergent IAV strains.
Methods: Adenoviral vectors encoding the antigens HA and NP in combination with the respective adjuvant (IL-1β, IL-18 or
empty vector) were given intranasally to BALB/c mice. The immunogenicity and efficacy of the vaccinations were evaluated.
Results: rAd-IL-18 showed practically no adjuvant activity in our experiments. In contrast, rAd-IL-1β enhanced HA-specific
humoral responses, which was mostly evident as a 32-fold increased neutralization titer against the homologous IAV. T cell
responses in the lung, as characterized by tetramer staining and functional intracellular cytokine staining, were elevated up
to 15-fold and showed a specific increase of TRM. Mechanistic studies revealed that rAd-IL-1β initiates several important
checkpoints in the formation of lung TRM including (i) lung tissue inflammation, (ii) priming of committed TRM precursors and
(iii) local differentiation into TRM. In consequence, the inclusion of rAd-IL-1β in the vaccine led to significantly enhanced
protection against H1N1, H3N2 and H7N7 strains.
Conclusion: rAd-IL-1β revealed a hitherto unprecedented ability to stimulate local T cell responses and thereby induced a
broad immunity against several unrelated IAV strains. Considering the importance of TRM-mediated protection at mucosal
barriers, this study has profound implications for the vaccine development.
Corresponding author:
Dennis Lapuente
[email protected]
Adaptive Immunity
P120
In vivo imaging of the interplay between regulatory and effector T cells during retroviral infection
L. Otto1,2, A. Hasenberg2, G. Zelinskyy1, W. Bayer1, U. Dittmer1, M. Gunzer2
1University
2University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Duisburg-Essen, University Hospital Essen, Institute of Experimental Immunology and Imaging, Essen, Germany
Cytotoxic CD8 T cells play a crucial role in the control of acute retroviral infections. During chronic infections like HIV, these
cells become dysfunctional. Regulatory T cells (Tregs) that expand strongly in the late acute phase of infection promote this
process through immunosuppressive effects. In order to study this issue including expansion, migration and interactions of
Tregs with virus-specific CD8+ T cells we applied the well-established murine Friend Virus (FV) model. Therefore, we used
DEREG mice that express a green fluorescent protein (GFP) under the control of the Treg-specific Foxp3 promoter.
Additionally, red fluorescent (tdTomato) FV-specific CD8+ T cells were adoptively transferred in this model to illustrate their
interaction with Tregs in vivo. Both cell types were visualised in the bone marrow, a typical centre of FV infection, via
intravital 2-photon microscopy. First results indicated a frequent interaction of Tregs and virus-specific CD8+ T cells that
seems to form synapse-like structures at their contact sites. New modified FV-variants allowed the visualisation of infected
cells as well. This tool completed the present system and enabled the observation of Tregs, FV-specific CD8+ T cells and FVinfected cells simultaneously in vivo to understand their interactions and network. The results of our studies will shed a new
light on the cellular mechanisms that underlie the development of chronic retroviral infections. New therapeutic strategies
based on Treg-targeted immunotherapies might ultimately result from our work in the future, potentially opening alternatives
for clinical intervention in chronic infectious diseases.
Corresponding author:
Lucas Otto
[email protected]
Adaptive Immunity
P121
Cross-reactivity profiles of CD8+ T cells targeting an immunodominant epitope in the core protein of HBV
J. Brinkmann1, T. Schwarz1, H. Kefalakes2, A. Walker1, G. Gerken2, J. Timm1
1Heinrich
Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
Hospital Essen, Department of Gastroenterology and Hepatology, Essen, Germany
2University
Introduction: CD8+ T cells are important for immune control of hepatitis B virus (HBV) infections. Particularly, CD8+ T cells
directed against HBV core are associated with recovery and are therefore interesting for T cell based immune therapy.
Objectives: Presence of viral sequence polymorphisms may interfere with CD8+ T cell responses and thereby impair T cellbased immune therapies. Here, the degree of HBV sequence variation of the immunodominant HLA-A*02-restricted epitope
core18-27 (FLPSDFFPSV) and the cross-reactivity of CD8+ T cells was studied in detail.
Patients & Methods: The epitope region was sequenced from 228 patients with HBV-infection and PBMCs of 30 HLA-A*02positive individuals with chronic HBV infection were analyzed for their response to prototype and a total of 26 variant
sequences of the core18-27 epitope.
Results: Consistent with the concept of immune escape from HLA-A*02-restricted CD8+ T cells, substitutions in position 7
and 10 of the epitope are significantly enriched in HLA-A*02-positive patients. Interestingly, position 10 is, in addition,
significantly more variable in HBV genotype A than in genotype D. A total of 14 of 30 patients (46.7%) showed prototypespecific CD8+ T cell responses. Despite evidence for immune escape at the sequence level, there was substantial crossreactivity of prototype-specific CD8+ T cells with the variant peptides. In turn, for nearly all variant peptides variant-specific
CD8+ T cells cross-reacting with the prototype were detectable. Notably, in patients where the exact epitope sequence was
available, CD8+ T cells were also cross-reactive with the epitope sequence in the autologous virus arguing against functional
immune escape.
Conclusion: CD8+ T cells directed against the epitope core18-27 appeared to be broadly cross-reactive against naturally
occurring variants. Given the strong evidence for immune selection at the viral sequence level, this degree of cross-reactivity
was unexpected. In ongoing experiments the impact of viral sequence variants on endogenous processing of the epitope is
studied.
Corresponding author:
Janine Brinkmann
[email protected]
Adaptive Immunity
P122
Immune evasion strategies of human cytomegalovirus – functional characterization of the novel HCMV-encoded Fcγ
receptor gp95/RL12
K. Hoffmann1, E. Mercé-Maldonado2, H. Reinhard2, E. Corrales-Aguilar2,3, V. T. K. Le-Trilling2,4, P. Lacher2, H. Hengel1
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
3Virology CIET, San José, Costa Rica
4University Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
2Heinrich
Question: Human Cytomegalovirus (HCMV) evolved immune evasion strategies by expressing virus-encoded FcγRs
(vFcγRs) interfering with the activation of host FcγRs.
We identified and characterized a novel HCMV-encoded vFcγR, the type 1 transmembrane glycoprotein gp95 encoded by
the RL12 gene and report here on its distinctive features.
Methods: Functional testing of gp95 was performed using either BACmid-based HCMV mutants or recombinant VacciniaViruses expressing gp95 using a recently developed reporter cell-based FcgR-activation assay (Corrales-Aguilar et al, 2013).
Results: HCMV was reported to encode several IgG-Fc-binding proteins, specifically vFcγRs gp34 (RL11), gp68 (UL119UL118) (Atalay et al, 2002), gp95 (RL12) (Cortese et al, 2012) and gpRL13 (Cortese et al, 2012). For gp34 and gp68 we
have demonstrated a powerful inhibitory potential regarding all activating human FcγRs (Corrales-Aguilar et al, 2014).
Compared to gp34 and gp68, gp95 exhibits a clearly different pattern regarding IgG-subclass binding. Moreover differences
were also observed with respect to its efficiency to antagonize IgG-mediated immune responses by human FcγRs. While
gp34 and gp68 bind readily to all human IgG-subclasses, gp95 binding is restricted to IgG1 and IgG3. In comparison with
gp68 we identified Rituximab subclass-dependent inhibitory effects by gp95 in a hierarchical manner (FcγRIII > FcγRII and
only minor effects on FcγRI), while gp68 blocked all human FcγRs with a comparable efficiency.
In clear contrast to RL11 and UL119-UL118, the RL12 gene sequence is highly variable between HCMV strains and clinical
isolates. Remarkably, substantial differences in its inhibition potential towards human FcγRIII responses were noted. FcγR
activation assays using recombinant Vaccinia-expressed RL12 alleles revealed gp95 from HCMV TB40/E strain to be more
efficient compared to gp95 derived from the HCMV strains AD169 and Toledo.
Conclusion: Here we provide evidence for a novel HCMV-encoded vFcγR, gp95, antagonizing human FcγRs but with
substantial differences in its inhibition potential towards human FcγRIII depending on the RL12 allele tested. gp95 exhibits an
unique binding to Fcγ and acts in an additive and synergistic manner with the previously characterized vFcγRs gp34 and
gp68.
Corresponding author:
Katja Hoffmann
[email protected]
Adaptive Immunity
P124
B cell-dependent protection during acute pneumovirus infection in immunodeficient mice
L. Prager1, C. Krempl1
1Julius
Maximilians University Würzburg, Institute of Virology and Immunobiology, Würzburg, Germany
Introduction: In addition to its well-known role as important pediatric pathogen the pneumovirus human respiratory syncytial
virus (RSV) is a significant cause of morbidity and mortality in immunocompromised individuals, e.g. patients after
hematopoietic stem cell transplantation. Whereas i. a. lymphopenia is considered a risk factor for progression to the lower
respiratory tract, the detailed pathogenesis of RSV infection in these patients is not understood.
Objectives: In the present study we start out to explore the pathomechanisms of pneumovirus infection in the
immunocompromised host by using the pneumonia virus of mice (PVM) infection model, an established surrogate model for
severe RSV infection, in mouse strains of defined immunodeficiencies.
Methods: T cell- (TCRβδ-/-), T/B cell- (Rag1-/-) and T/B/NK cell-deficient (Rag1.Cγ-/-) mice were infected with PVM and
analyzed with respect to clinical symptoms, viral replication, cellular pulmonary and PVM-specific humoral immune
responses.
Results: Infection of TCRβδ-/- mice was asymptomatic throughout the experiment (duration 14 days) although mice failed to
clear the virus. However, after peaking on day 7 p.i. a reduction of virus replication was observed. During the infection B cells
were recruited to the lung similarly to control mice and a limited PVM-neutralizing antibody response was determined in
serum and lung. In contrast, Rag1-/- and Rag1.Cγ-/- mice displayed highly variable individual fates ranging from lack of
symptoms to severe disease requiring killing. Again, mild or asymptomatic infection correlated with partial virus control
whereas loss of control was associated with fatal outcome. So far, no differences in immune responses were observed
independent of the outcome leaving open the question of the control mechanism. In addition, adoptive transfer of naïve B
cells 1 day prior infection did not rescue the overall fate of Rag1-/- mice.
Conclusion: In TCR-/- mice B lymphocytes appear to provide a partial control of PVM infection likely by producing a virusneutralizing antibody response induced independently from T cell help. This partial control is sufficient to prevent severe
disease and fatal outcome. A protective capacity of adoptive transfer of B cell requires further evaluation, e.g. by varying
transplantation conditions.
Corresponding author:
Lisa Prager
[email protected]
Adaptive Immunity
P125
The human Cytomegalovirus glycoprotein pUL11 acts via CD45 to induce T cell IL-10 secretion
J. Zischke1, P. Mamareli2, R. Jacobs3, C. Falk4, T. Sparwasser2, T. F. Schulz1, P. Kay-Fedorov1
1Hanover
Medical School, Institute of Virology, Hanover, Germany
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
3Hanover Medical School, Department of Clinical Immunology and Rheumatology, Hanover, Germany
4Hanover Medical School, Institute of Transplant Immunology, IFB-Tx, , Hanover, Germany
2Twincore
Introduction: The complex changes wrought on the host"s immune system during an active HCMV infection are well
documented. Infected cells are masked and manipulated. Uninfected immune cells are also affected; the ability of peripheral
blood mononuclear cells (PBMC) to proliferate is reduced and the cytokine profile is altered. Levels of the anti-inflammatory
cytokine IL-10 increase during active infection. IL-10 inhibits the function of antigen presenting cells and the production of
proinflammatory cytokines, resulting in impaired T cell responses. Many different cell types can produce IL-10, with CD4 T
cells apparently the most important source in vivo. Few details of T cell IL-10 secretion during HCMV infection are known, but
IL-10 production from T cells is important for the establishment of high viral titres by murine cytomegalovirus.
Objectives: The mechanisms by which HCMV affects T cell IL-10 secretion are not understood. It is, however, known that
ligation of the lymphocyte phosphatase CD45 induces T cell IL-10 production. As we have shown that the HCMV
glycoprotein pUL11 interacts with CD45 we investigated the effects of pUL11 on IL-10 secretion by T cells and considered
the underlying changes to T cell signalling.
Material & Methods: Wild type and ΔUL11 HCMV and purified pUL11 were used to investigate changes in IL-10 secretion
and TCR signalling in primary PBMC and cell lines.
Results: Treatment of PBMC with pUL11 induces IL-10 producing T cells. Supernatants of pUL11 treated cells have antiinflammatory effects on untreated PBMC. CD45 can be a positive or negative regulator of TCR signal strength, depending on
its expression level, and pUL11 also has concentration dependent activating or inhibitory effects on T cell proliferation and on
the kinase function of the CD45 substrate Lck.
Conclusion: pUL11 is the first example of a viral protein targeting CD45 to induce T cells with anti-inflammatory properties.
It is also the first HCMV protein shown to induce IL-10 secretion in T cells. Understanding the mechanisms by which changes
in signal strength can influence T cell development and function may provide the basis for the development of novel antiviral
treatments and therapies against immune pathologies.
Corresponding author:
Penelope Kay-Fedorov
[email protected]
Adaptive Immunity
P126
The HSV-1 encoded immunoevasin ICP47 makes use of a highly conserved ProLeuLeu motif to arrest the
conformation of the transporter associated with antigen processing (TAP)
T. Matschulla1, R. Berry2, C. Gerke1, M. Döring3, J. Busch4, U. Kalinke3, F. Momburg5, H. Hengel1, A. Halenius1
1Albert
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
University, Clayton, Dept. of Biochemistry and Molecular Biology, Clayton, Australia
3Hanover Medical School, Hanover, Germany
4University of Duesseldorf, Duesseldorf, Germany
5German Cancer Research Center, Heidelberg, Germany
2Monash
The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum
(ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells.
The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a
high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of
ICP47 resides within the N-terminal region (residues 1-35).
When analyzing two C-terminally truncated ICP47-tagGFP fusion proteins (ICP47-N: 1-35 and ICP47-eN: 1-57) we found
that ICP47-N less efficiently blocked TAP dependent peptide transport compared to full-length ICP47 and ICP47-eN. Taking
advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of
TAP, we demonstrated that the short truncation mutant ICP47-N was not able to block US6 binding to TAP but full-length
ICP47 and ICP47-eN did. This suggested that the central part of ICP47 is required for complete TAP inhibition involving a
conformational arrest.
An ICP47 mutant which lacked the highly conserved ProLeuLeu motif (residues 50-52 mutated to AlaAlaAla) mimicked the
ICP47-N truncation mutant: block of MHC class I cell surface expression was reduced and, also, US6 bound to TAP in the
presence of the mutated ICP47. Therefore, the ProLeuLeu motif is essential for freezing of the TAP conformation. Moreover,
hierarchical functional interactions sites on TAP dependent on ProLeuLeu could be defined using a comprehensive set of
human-rat TAP1 and TAP2 chimeras, entirely consistent with the recently published high-resolution cryo-EM model of TAP
and ICP47 (Oldham et al., eLife 2016). Most interestingly, the ICP47 residues 50-52 adopt a hairpin-like conformation, the
apex of which sits alongside a region on TAP2 (the central helix 1) that forms the transmission interface for conformational
communication between cytosolic and transmembrane domains of TAP. Altogether, this data broadens our understanding of
the molecular mechanism underpinning TAP inhibition by ICP47, to include the ProLeuLeu sequence as a stabilizer that
tethers the TAP-ICP47 complex in an inward-facing conformation.
Corresponding author:
Anne Halenius
[email protected]
Adaptive Immunity
P127
A green fluorescent BAC-cloned human cytomegalovirus with an intact US-gene region and a self-excisable BAC
cassette for use in immunological research
K. Laib Sampaio1, Z. Wu2, N. Subramanian1, A. Weyell1, C. Sinzger1
1Ulm
University Medical Center, Institute of Virology, Ulm, Germany
Sloan Kettering Cancer Center, New York, United States
2Memorial
We had previously provided a bacterial artificial chromosome (BAC) clone of the human cytomegalovirus (HCMV) strain
TB40/E that combines the high titer production of lab strains with the extended cell tropism of recent isolates. However, this
virus is impaired in MHC class I down modulation because the viral genes US2-6 are deleted to accommodate the BAC
cassette. For immunological studies, a BAC clone of TB40/E that contains the full set of viral MHC modulators is desired.
Therefore, we aimed to generate a TB40/E-derived clone with a self-excisable BAC cassette, which contains the full-length
virus genome. As a starting point, we used a newly generated BAC clone TB40-BACKL7 that also carries the BAC cassette
in place of US2-6. At first, we relocated the BAC cassette into the UL73/UL74 gene region and modified it in a way that it will
be excised from the genome during reconstitution of the virus from the BAC-DNA. Next, the US2-6 region was genetically
restored, yielding the self-excisable full-length BAC clone TB40-BACKL7-SE. BAC-DNA was transfected into cell-cyclesynchronized fibroblasts, resulting in transfection efficiencies > 0.1 %. Foci of infected cells appeared within two weeks after
transfection, and cell-free HCMV stocks could be harvested from transfection cultures. As expected, the reconstituted virus
had excised the BAC cassette and did not contain any remnants of foreign DNA. It grew to high titers and displayed the
extended cell tropism, similar to the parental TB40/E virus. The analysis of MHC class I molecules on the surface of infected
cells confirmed a functional restoration of the US gene region, indicated by a more pronounced downregulation of MHC-I
surface molecules as compared to TB40-BACKL7 which lacks US2-6. In a final step, we inserted an EGFP-expression
cassette harboring a shortened form of the major-immediate-early promotor at an ectopic position downstream of US34A.
The resulting TB40-BACKL7-SE-EGFP had no growth defect and expressed EGFP at levels suitable for flow cytometric
analysis. Taken together, TB40-BACKL7-SE-EGFP may be a useful tool in immunological research, as it provides an intact
US region and facilitated detection of infected cells in FACS together with unrestricted possibilities for genetic engineering.
Corresponding author:
Kerstin Laib Sampaio
[email protected]
Adaptive Immunity
P128
Human cytomegalovirus-infected macrophages are refractory to immunoevasion mediated by the US2-11 genes and
elicit memory CD4+ and CD8+ T responses
C. Bayer1, C. Setz1, S. Varani2, J. van der Merwe3, Z. Wu1, J. Hegler1, T. Mertens1, G. Frascaroli1
1Ulm
University Medical Center, Institute of Virology, Ulm, Germany
of Bologna, Department of Diagnostic, Experimental and Speciality Medicine, Bologna, Italy
3Ulm University Medical Center, Institute of Molecular Virology, Ulm, Germany
2University
A large proportion of the human population harbours human cytomegalovirus (HCMV) and carries a remarkable amount of
HCMV-specific CD4+ and CD8+ T cells that may reach 20-50% of total T cells in the elderly. It is unclear how HCMV
provokes this large immune response, as HCMV is known for its unique capacity to inhibit antigen presentation and
recognition of infected cells. Among the other immunoevasion mechanisms, four viral glycoproteins encoded by the unique
short (US) regions US2, US3, US6 and US11 of HCMV downregulate major histocompatibility complex (MHC) class I and II
molecules and inhibit antigen presentation. Since macrophages (Mφ) support a low-level, persistent HCMV infection, we
hypothesized that these cells could be refractory to the US2-11 immunoevasins and preserve full antigen presentation
capability.
Monocyte-derived pro-inflammatory (M1-) and anti-inflammatory (M2-) Mφ were infected with wild type HCMV or a mutant
virus deleted of the four immunoevasins (RVTB40/E4∆US11) prior to investigation of MHC class I and II expression, antigen
presentation and induction of T-cell proliferation and activation.
Although expressed immediately after viral entry, US2-11 were not capable to induce MHC downregulation in HCMV-infected
Mφ. Mφ infected with wild type or RVTB40/E4∆US11 HCMV induced the expansion of autologous CD4+ and CD8+ T cells in
comparable extents. The proliferating T cells were terminally differentiated and, similarly to the natural HCMV-specific T cells,
belonged to the T effector memory compartment. Additionally, CD4+ and CD8+ T cells stimulated by HCMV-infected Mφ
showed markers of activation, by overexpressing CD69, producing interferon-γ and exhibiting cytolytic potential.
In conclusion, HCMV infection of Mφ triggers efficient antigen presentation, irrespectively on the expression of the
immunoevasive genes US2-11. Thus, our findings suggest that HCMV-infected Mφ can provide the necessary antigenic
boost in the host to maintain the large amounts of T cells committed to HCMV.
Corresponding author:
Carina Bayer
[email protected]
Adaptive Immunity
P129
Decreased immunocompetence in day-old chicks hampers effective immunization against highly pathogenic avian
influenza (HPAI)
J. Schmiedeke1, D. Hoffmann1, B. Hoffmann1, U. Blohm1
1Friedrich
Loeffler Institute, Institute of Immunology, Greifswald - Isle of Riems, Germany
Chicken meat and eggs are the best source of quality protein, and important vitamins and minerals. Especially also in
developing countries, poultry production is relatively inexpensive and widely available. The intense farming increases the risk
of disease spreading as birds are highly susceptible for several pathogens like avian influenza. To avoid economical losses,
immunization is performed routinely either in ovo as efficient mass vaccinations or immediately after hatching to minimize the
non-protected period. HPAI viruses as H5N1 poses a lethal threat to poultry and possesses a zoonotic potential. Hence,
major efforts are put into developing an efficient vaccination strategy.
Previous studies by our group demonstrated the efficient protection of adult chicken from a lethal challenge infection with
H5N1 by immunization using an H5N1 deletion mutant (H5Ndel), which lacked the neuraminidase as modified live vaccine.
In contrast, day-old chicks succumbed to H5Ndel immunization showing a typical pathology for HPAI. Thus, to adapt this
promising approach, further studies focussing on the immunocompetence development of chicken were performed.
Therefore, we analysed lymphoid tissues in 1 to 21 days old chicken regarding their cellular composition and localization
focussing on T cells by flow cytometry and immunohistochemistry. Special focus was set to T cell effector functions and
cytokine expression examined by previously established RT-qPCR.
CD8αβ T cell frequencies increased markedly in blood, spleen and cecal tonsil during the observed time period. Cellular
reorganization showing antigen presenting structures was detectable in the spleen at one week of age. Effector functions of
lymphocytes increased as well, shown by declining inhibitory properties. Especially increasing IL2 mRNA levels suggested
infiltration or proliferation of T cells in spleen, blood and some respiratory tissues that play an important role in the oculonasal
infection route after the first days of life. This data stresses the inability of juvenile chicken to mount an effective, T celldependent antiviral response and therefore questions the common practice of day-old chick vaccination. In order to protect
day-old chicks, effective immunization strategies and adjuvants need to be considered separately for each pathogen.
Corresponding author:
Julia Schmiedeke
[email protected]
Adaptive Immunity
P130
Canonical and non-canonical expression of a murine cytomegalovirus early protein CD8 T-cell epitope drives
memory inflation.
A. Fink1, A. Renzaho1, R. Holtappels1, M. J. Reddehase1, N. Lemmermann1
1University
Medical Center Mainz, Institute of Virology, Mainz, Germany
The ORF m164 belongs to the private genes of murine cytomegalovirus (mCMV) and codes for a 36.5kDa ER-resident type-I
glycoprotein expressed with Early (E) phase kinetics. We have previously identified a nonapeptide expressed by
m164/gp36.5 that is presented by the MHC-I allomorph H-2 Dd and induces an immunodominant CD8 T cell response during
acute mCMV infection in BALB/c (H-2d haplotype) mice. Along with the Immediate Early (IE) peptide IE1, the m164 peptide is
known to expand the CD8 T-cell pool during mCMV latency, a phenomenon known as memory inflation. This is thought to
indicate episodes of stochastic viral gene expression during latency driven by sporadic gene desilencing.
As the m164/gp36.5 protein is expressed in the E phase of infection, it was assumed that m164 peptide presentation does
not take place prior the E phase. Surprisingly, m164-specific CD8 T cells were able to recognize mCMV-infected fibroblasts
metabolically arrested in the IE-phase of infection, indicating an IE expression of the antigenic peptide. We were able to
characterize a 5kb IE mRNA spanning the epitope-encoding sequence in ORFm164, though being unrelated to the
abundantly expressed 2kb E mRNA of m164/gp36.5. Thus, m164 peptide presentation can result from IE and E mRNAs in
the mCMV-infected cell.
IE1 expression of epitopes was previously found to favor memory inflation. We therefore hypothesized that E phase and IE
phase mRNA species are differentially expressed during acute and latent mCMV infection, respectively. To address this, we
analyzed viral gene expression in the lungs of mCMV infected mice by transcript-specific RT-qPCR. In acutely infected lungs
we detected high numbers of E as well as IE mRNAs of m164 with a defined expression proportion. During latency, the gene
expression is, as expected, significantly lower but both m164 mRNAs were detectable. To our surprise, we did not detect any
preference for either mRNA species in the latently infected lung. We therefore conclude that memory inflation of m164
specific CD8 T cells is driven by stochastic expression of m164 IE as well as E mRNA.
Corresponding author:
Niels Lemmermann
[email protected]
Adaptive Immunity
P131
Ephemeral immune control of CMV infection in TCR transgenic mice recognizing a single viral epitope
L. Borkner1, F. Mbui1, Z. Chaudhry1, L. Cicin-Sain1
1Helmholtz
Center for Infection Research, Immune Aging and Chronic Infection, Braunschweig, Germany
Cytomegalovirus infection remains a significant challenge in immunocompromised and transplant patients, as well as in
individuals infected during pregnancy. The debilitating outcomes of CMV primary infection or reactivation from latency in
these populations, warrants accelerated research in CMV pathogenesis and therapeutic interventions.
The adaptive immune system, in particular the T cell compartment, plays an important role in virus control. Therefore,
recruitment, activation and maintenance of functional antigen-specific CD8 T cells appear to be sufficient in controlling CMV
growth.
To understand the minimal requirement for adoptive immunotherapy of CMV disease by CD8 T-cells, we have generated
recombinant murine CMVs expressing high and low avidity antigenic epitopes (SSIEFARL and KCSRNRQNL respectively) in
the context of an immediate early (IE2) or of an early (M45) gene. These mutant viruses were used to infect RAG2-/- mice
expressing the correspondent T-cell receptor. The single immune epitope provided strong immune protection in all tested
conditions, but immune protection and the inhibition of viral growth was maintained only for a brief period of time.
We postulate that the loss of immune protection might be attributed to either clonal exhaustion of epitope-specific CD8 T
cells or to immune escape of MCMV, through mutations of the epitope sequences recognized by antigen specific T- cells.
Preliminary analysis by PCR detected a polymorphism in the size of PCR products in some of the tested mice, arguing for a
loss of immune protection due to immune escape.
Ongoing work focuses on sequencing the viral genomes retrieved from infected mice at the locations corresponding to the
sites of epitope insertion and identification of single nucleotide polymorphisms. Furthermore, we will characterize the
phenotype and functionality of epitope-specific CD8 T cells over the course of infection.
We anticipate that our research may identify the minimal requirements for adoptive T-cell based immunotherapy against
cytomegalovirus.
Corresponding author:
Fredrick Mbui
[email protected]
Adaptive Immunity
P132
Fcγ receptor type I (CD64)-mediated abrogation of the capacity of dendritic cells to activate specific CD8 T cells by
IgG-opsonized Friend virus
Z. Banki1, D. Bichler1, R. Werner1, V. Oberhauser1, S. Verbeek2, D. von Laer1, U. Dittmer3, H. Stoiber1
1Medical
University of Innsbruck, Division of Virology, Innsbruck, Austria
University Medical Center , Leiden, Netherlands
3University of Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
2Leiden
Dendritic cells (DC) express Fcγ receptors (FcγR) for binding of antigens immune-complexed (IC) with IgG molecules. ICFcγR interactions have been demonstrated to modify activation and antigen-presenting functions of DCs. Utilizing Friend
virus, a mouse retrovirus model, here we investigated the effect of IgG-opsonization of retroviral particles on the infection of
DCs and subsequently the presentation of viral antigens by DCs for specific CD8 T cells. We found that IgG-opsonization
abrogates DCs infection and as a consequence significantly reduce the capacity of DCs to activate virus-specific CD8 T
cells. Effects of IgG-opsonization were mediated by the high affinity FcγR type I (CD64) expressed on DCs. Our results
suggests that different opsonization patterns on retroviral surface modulate antigen presenting functions of DCs, whereby in
contrast to complement, IgG reduces the capacity of DCs to activate CTL responses.
This study was supported by research projects of the FWF (I-2550) and of the DFG (Di714/17).
Corresponding author:
Zoltan Banki
[email protected]
Adaptive Immunity
P133
Combination of oncolytic virotherapy and DC-based immunotherapy for the treatment of melanoma
I. Koske1, Z. Banki1, I. Barnstorf1, C. Tripp2, P. Stoitzner2, N. Romani2, G. Wollmann1, J. Kimpel1, D. von Laer1
1Medical
2Medical
University Innsbruck, Division of Virology, Innsbruck, Austria
University Innsbruck, Department of Dermatology and Venereology, Innsbruck, Austria
VSV-GP, a novel chimeric Vesicular Stomatitis Virus (VSV) pseudotyped with the glycoprotein of the lymphocytic
choriomeningitis virus represents a promising oncolytic virus (OV) that preferentially targets and kills cancer cells. Release of
tumor antigens and activation of immune response by OV therapy might support dendritic cell (DC)-mediated anti-tumor
immunity.
Thus in our study we analyzed the efficacy and immune mechanisms of the combination of VSV-GP oncolytic virotherapy
with DC-based immunotherapy.
Combination of VSV-GP therapy and DC-based vaccination was investigated in the syngeneic subcutaneous B16-OVA
melanoma model. SIINFEKL-loaded CpG-activated DCs (DCVacc) and VSV-GP were applied intra- and peritumorally and
immune responses were analyzed in the spleen and tumor tissues.
The DCVacc/VSV-GP combination therapy resulted in a significantly improved survival compared to single treatments.
Surviving mice from the DCVacc/VSV-GP treated group showed a long lasting anti-tumor immunity against B16-OVA and
partial anti-tumor immunity against non-OVA B16 melanoma in rechallenge experiments. Analyzing specific cytotoxic T
lymphocyte (CTL) responses induced by DCVacc and VSV-GP single and combination treatments we found that both
DCVacc and DCVacc/VSV-GP induced comparable levels of OVA-specific CD8 T cell responses. In addition, a strong VSV
N peptide-specific CD8 T cell response was found upon VSV-GP and DCVacc/VSV-GP treatments. The improved
therapeutic effect by the DCVacc/VSV-GP combination treatment correlated with increased numbers of tumor infiltrating
lymphocytes (TIL) and elevated Tconv/Treg and CD8/Treg ratios seen also in non-treated collateral tumors. Furthermore,
depletion of CD8 T cells but not NK cells abrogated the therapeutic effect of DCVacc/VSV-GP.
Taken together, the combination of VSV-GP and DC-based immunotherapy might represent a promising therapeutic option
for the treatment of melanoma.
Corresponding author:
Iris Koske
[email protected]
Emerging Viruses
P134
Stratify and rescue Ebola in silico patients
G. Zhao1, S. Binder1, G. Pongratz2, M. Meyer-Hermann1,3
1Helmholtz
Centre for Infection Research, Department of Systems Immunology, Braunschweig, Germany
Hospital Duesseldorf, Funktionsbereich & Hiller Forschungszentrum für Rheumatologie, Dusseldorf, Germany
3Technical University of Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Braunschweig, Germany
2University
Question: We asked whether currently available human data from Ebola virus infections are sufficient in order to determine
fate decisive factors. As the answer is yes, we asked how to rescue patients classified as fatal cases.
Methods: We constructed a mathematical model of the cytokine response during Ebola virus infection and fitted the model to
all available data of fatal, survival and asymptomatic cases living in the same region and infected with the same strain of
virus (Zaire).
Results: We found that differential sensitivity of TNF-α induction to the virus is the key mechanism that can explain the
cytokine response data of the three groups of patients. Other parallel hypotheses, such as differential properties of the virus,
or differential sensitiviy of IFN-γ to the virus, were not consistent with the data. The model allowed to stratify patients based
on the blood TNF-α level at the time of presentation in the clinics: A patient is of high risk if his TNF-α level at this time is
lower than 40 pg/ml, and is likely a survivor at TNF-α level above 50 pg/ml. The model also suggested that antibody and IFNγ have similar viral clearance capacity. Antibodies are the key defense mechanisms in survivors while IFN-γ is the key
defense mechanism in fatalities. Importantly, the induction of IFN-γ in fatalities is 2 days later than antibodies induction in
survivors. The fatal cases can be rescued in silico by support of innate immunity together with an anti-inflammatory therapy.
Conclusion: Ebola patients can be stratified based on the blood TNF-α level at the day of presentation in the clinics into
survivors and fatal cases. It is possible to rescue the fatal cases in silico by immune-interventions that promote innate
immunity and suppress inflammation.
Corresponding author:
Gang Zhao
[email protected]
Emerging Viruses
P135
Genetically divergent Filoviruses in Chinese Rousettus and Eonycteris bats
X. L. Yang1, Y. Z. Zhang2, R. D. Jiang1, Z. L. Shi1
1Chinese
2Dali
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
University, Dali, China
Bats have been implicated as natural reservoirs for filoviruses based on serological or nucleotide evidence from 19 bat
species in 8 countries across Asia, Africa, and Europe. Previously, we discovered filovirus antibodies in several bat species
in China. Here we report genetically divergent novel filoviruses are circulating in the Rousettus and Eonycteris bats from
China. The 310-bp L-gene sequences exhibited 65–99% nucleotide (nt) identity among themselves and 61–78% nt identity
with known filoviruses. Phylogenetic analysis of these sequences suggests that at least 3 distinct groups of filovirus are
circulating in these bats. Q-PCR results showed these filoviruses were mainly located in the lung, with genome copy number
varying from 29 to 523,582/mg of tissue. Thus, these filoviruses may have the potential to be transmitted through the
respiratory tract. Co-infection with four different filoviruses was found in a single bat. ELISA and Western Blot showed the
antibodies reacting more strongly to EBOV NP than RESTV NP in some filovirus RNA negative bats. One of the viruses
named BtFilo9447 were tried to amplify the whole genome. The GP gene of BtFilo9447 shared 34-39% similarity on aa level
and 35-53% similarity on nt level with known filoviruses. Our results demonstrate that fruit bats may are important reservoirs
of filoviruses. Considering their feeding habitats, fruit bats are often in close contact with domestic animals and human
populations. It is therefore necessary to establish long-term and proactive surveillance of these viruses and related diseases.
Corresponding author:
Xing-Lou Yang
[email protected]
Emerging Viruses
P136
Zika virus production in different cellular systems
K. Himmelsbach1, D. Ploen1, E. Hildt1,2
1Paul-Ehlich
2German
Institute, Virology, Langen, Germany
Center for Infection Research (DZIF), Braunschweig, Germany
Zika Virus (ZIKV) was first recorded in 1947 in the Zika forest of Uganda. It is an 40 nm, enveloped (+)ssRNA Virus
belonging to the family of the Flaviviridae similar to dengue , yellow fever and West Nile virus. This arbo borne virus (Aedes
mosquitos) came into focus in the beginning of 2016, when the WHO declared the PHEC since it caused an epidemic in
Brazil. ZIKV infection is asymptomatic in 4 out of 5 cases. If infected during pregnancy ZIKAV can cause microcephaly in
newborns and there is evidence that it provokes Guillain-Barré syndrome.
Vero cells are commonly used for propagation and investigations concerning ZIKV for historical reasons. Resembling the
complete viral life cycle a cytopathic effect can be detected between 4-7 days post infection. In our hands we could reach
2x10^6 PFU/ml using Vero derived supernatant at an MOI=1. A comparison of the Uganda and the Polynesia genotypes
revealed a slight increase in the infectivity in case of Uganda. However Vero cells are from an African green monkey and
hence not a human derived system. In light of possible differences compared to human cell lines we investigated the
susceptibility of elven cell lines for Zika Virus infection. It turned out that the lung carcinoma A549 cells showed an even
higher susceptibility compared to Vero cells and the cytopathic effect was already detectable after 2-4 days post infection.
Also Huh7.5, Cos7 and 293T cells turned out to be highly susceptible for Zika infection, but not the CHO cells. Moreover the
neuronal cell lines N29 (mouse) and SHY5Y (human) showed a robust infection cycle. This is even more interesting in light
of the neurological symptoms caused by ZIKV. Since the first contact of humans and virus is normally a mosquito bite, also
the keratinocyte derived HaCaT cells were tested and turned out to support viral replication. The investigated cell lines
showed in parts strong differences in infectivity, viral replication and the release of infectious viral particles, which was
assayed by immunofluorescence analysis, Plaque assay and qPCR.
Based on these results robust platforms were established to produce high titers of infectious ZIKV and the results offer
various cell culture systems to study the viral life cycle or to test antivirals either in single cell culture systems or in a
comparative manner.
Corresponding author:
Kiyoshi Himmelsbach
[email protected]
Emerging Viruses
P137
Screening of new potential inhibitors for ZIKV infection
F. Elgner1, D. Ploen1, K. Himmelsbach1, T. Schirmeister2, A. Grünweller3, K. Lange-Grünweller3, W. Diederich3, E. Hildt1,4
1Paul
Ehrlich Institute, Virology, Langen, Germany
Medical Center Mainz, Mainz, Germany
3Philipps University Marburg, Marburg, Germany
4German Center for Infection Research (DZIF), Braunschweig, Germany
2University
The Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flaviviridae family which also harbors the yellow fever virus,
dengue virus (DENV) and hepatitis C virus. ZIKV has first been identified in 1947 in the Zika forest in Uganda where the
infection causes a mild febrile illness. Since 2007, the virus spreads from the African continent to French Polynesia and
South America. In the recent outbreak, ZIKV causes severe neurological phenotypes like microcephaly of infected newborns
and the Guillain-Barré syndrome. ZIKV is an enveloped virus harboring a positive-orientated RNA genome with a size of 10.8
kb that encodes for a single polyprotein precursor. The polyprotein is cleaved by host and viral proteases into the mature
structural and non-structural proteins. Until now, there is neither a protective vaccine nor a specific therapy available. To
identify new potential inhibitors, we tested different substances using A549 cells. The cells were infected with the ZIKV
strains French-Polynesia (PF13/251013-18) and Uganda (strain 976) (MOI = 0.1) and incubated with different substances
(CHIR99021, HWu180, Silvestrol, Simvastatin). The cells and corresponding supernatants were analyzed by qPCR to
determine the impact of each inhibitor on intra/extracellular ZIKV RNA. Changes in the release of infectious ZIKV particles
were detected by performing a plaque assay. In addition, the change in intracellular distribution of ZIKV proteins was
analyzed by immunofluorescence staining. In initial experiments, treatment with the GSK inhibitor CHIR99021 resulted in
decreased intracellular ZIKV RNA and a changed staining pattern of the ZIKV envelope protein, whereas the amount of
secreted viral RNA is unaffected; the same effect is observed after treatment with the HMG-CoA reductase inhibitor
Simvastatin. Treatment with Silvestrol resulted in a concentration-dependent decrease of both intra- and extracellular ZIKV
RNA. Moreover, treatment with the potential DENV protease inhibitor HWu180 decreased the intra- and extracellular
amounts of ZIKV RNA with a stronger effect on the French Polynesia strain. This data will help to reveal mechanisms of ZIKV
infectionand understand the different clinical outcomes to finally develop a ZIKV-specific therapy.
Corresponding author:
Fabian Elgner
[email protected]
Emerging Viruses
P138
Severe fever with thrombocytopenia syndrome – Transmission of SFTSV and migratory bird routes in East Asia and
SFTS in patients suspected of having scrub Typhus
K. H. Lee1
1Jeju
National University School of Medicine, Microbiology and Immunology, Jeju, South Korea
Severe fever with thrombocytopenia syndrome (SFTS) is tick-borne viral disease such as Crimean-Congo hemorrhagic fever
that was first suspected in China in 2009. The causative virus was isolated in 2009 and reported in 2011, and SFTSV
expanded its geographic distribution in 2012-2013, from China to South Korea and Japan. Most SFTSV infections occur
through Haemaphysalis longicornis, which acts as a transmission host between animals and humans. However, it is not
known if a genetic connection exists between the viruses in these regions and, if so, how SFTSV is transmitted across China,
South Korea, and Japan. We hypothesize that the SFTSV in South Korea share common phylogenetic origins with samples
from China and Japan. Further, we postulate that migratory birds, well-known carriers of the tick H. longicornis, are a
potential source of SFTSV transmission across countries. Most SFTSV infections occur through H. longicornis. However,
SFTSV infection can also occur between family members, and nosocomial transmission of SFTSV is also possible through
close contact with a patient. In this study, we first analyzed clinical, epidemiological, and laboratory data for SFTS patients
and family members of an index patient in Korea. The S segment of SFTSV was amplified from the sera of three patients,
and the S segment of SFTSV and IgG specific to SFTSV were detected in the serum from one family member; although this
individual had no history of exposure to H. longicornis, she frequently had close contact with the index patient. In Korea,
SFTSV infection among family members does not have to be reported, and we suggest that person-to-person transmission
of SFTSV among family members is possible in Korea. To determine prevalence of SFTS in South Korea, we examined
serum samples from patients with fever and insect bite history in scrub typhus endemic areas. Prevalence of this syndrome
among patients suspected of having scrub typhus was high (23.0%), suggesting possible co-infection.
References:
[1] Yeojun Yun et al., Phylogenetic Analysis of Severe Fever with Thrombocytopenia Syndrome Virus in South Korea and
Migratory Bird Routes between China, South Korea, and Japan. Am. J. Trop. Med. Hyg., 93(3), 2015, pp. 468-474.
[2] Jeong Rae Yoo et al., Family Cluster Analysis of Severe Fever with Thrombocytopenia Syndrome Virus Infection in
Korea. Am. J. Trop. Med. Hyg., 95(6), 2016, pp. 1351-1357.
[3] Yu Mi Wi et al., Severe Fever with Thrombocytopenia Syndrome in Patients Suspected of Having Scrub Typhus.
Emerging Infectious Diseases 22(11), 2016, pp. 1992-1995.
Corresponding author:
Keun Hwa Lee
[email protected]
Emerging Viruses
P139
Bafilomycin A1efficiently inhibits infection by ZIKV
C. Sabino1, D. Ploen1, K. Himmelsbach1, S. Akhras1, F. Elgner1, E. Hildt1,2
1Paul
Ehrlich Institute, Virology, Langen, Germany
Center for Infection Research (DZIF), Braunschweig, Germany
2German
The Zika virus (ZIKV) infection has spread widely, mainly in Africa, South and Central America and South Pacific. This
mosquito-borne virus had its first appearance on a Rhesus macaque, in the Zika forest of Uganda, in 1947. Like other
flaviviruses, the ZIKV has a single-stranded positive RNA genome that encodes a polyprotein. This polyprotein is processed
by viral and host proteases into three structural proteins and seven non-structural proteins. Besides the known asymptomatic
or mild symptomatic outcome in individuals, there were reports of neurological complications, such as Guillain-Barré
syndrome (GBS) and newborns with microcephaly. Thus, understanding the life cycle of the virus and discovering ways to
reduce or even eradicate the infection of ZIKV has become urgent.
Activation of autophagy upon flavivirus infection has been reported in several studies (Ren et al., 2016). To investigate the
impact of ZIKV on autophagy and its relevance for ZIKV life cycle, we analyzed the effect of Bafilomycin A1 on ZIKV-infected
cells. Bafilomycin A1 is an inhibitor of vacuolar type H+-ATPase (Yoshimori et al., 1991) that is crucial for the acidification of
organelles including Golgi, endosomes and phagosomes in addition to lysosomes. Therefore, Bafilomycin A1 leads to a
higher pH which prevents the releasing of the virus genome into the cell i.e.by stopping fusion of the virus membrane with
organelles membranes (Hunt et al., 2010).
Human alveolar epithelial cells (A549) were infected with French Polynesia and Uganda strains and were harvested at 24, 48
and 72 hpi (hours post-infection). The treatment was applied 2 hours before and after infection, designated by treatment preinfection and treatment post-infection, respectively. With this experiment, we were able to demonstrate that 10nM of
Bafilomycin A1 was able to diminish the infection effectively as evidenced by RT-qPCR, immunofluorescence, western-blot
and plaque assay. The results show that, by using this modulator, it only takes 24hpi to decrease significantly the amount of
infected cells for treatment post-infection. If Bafilomycin is present before infection (pre-infection) 24hpi there are almost no
infected cells.
In conclusion, these data indicate that inhibition of endosomal acidification efficiently impairs establishment of ZIKV infection.
Corresponding author:
Catarina Sabino
[email protected]
Emerging Viruses
P140
Isolation and characterization of Zika virus imported to China and development of SYBR green one-step real-time
RT-PCR for virus detection.
C. Deng1, S. Liu1, M. Xu1, B. Zhang1
1Chinese
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
We successfully isolated infectious ZIKV in a C6/36 cell culture system from the serum of an imported ZIKV case with a very
low viral load. This ZIKV isolate has registered in China Centre for General Virus Culture Collection (CCGVCC (Accession
number: IVCAS 6.6110). Genome sequencing and alignment analysis indicated that cell culture with C6/36 cells could
reduce the possibility of non-synonymous substitutions in the ZIKV genome during the process of virus isolation. In addition,
the findings of this study shed light on the infectious features and genomic variability of ZIKV.
A SYBR Green based one-step real-time RT-PCR assay was developed for rapid detection of ZIKV. Our results revealed
that the real-time assay is highly specific and sensitive in detection of ZIKV in cell samples. Importantly, the replication of
ZIKV at different time points in infected cells could be rapidly monitored by the real-time RT-PCR assay. Specifically, the
real-time RT-PCR showed acceptable performance in measurement of infectious ZIKV RNA. This assay could detect ZIKV at
a titer as low as 1 PFU/mL. The real-time RT-PCR assay could be a useful tool for further virology surveillance and diagnosis
of ZIKV.
Figure 1: Phenotypic characterization and specific substitutions in the ZIKV SZ-WIV01 strain. (A) Immuno fluores-cence
assay (IFA) of Vero and C6/36 cells infected with the third passage of SZ-WIV01 at 24 h post-infection. (B)Plaque
morphology in BHK-21 and Vero cells infected with the third passage of SZ-WIV01 at 96 h post-infection. (C)Five specific
non-synonymous substitutions in the Chinese imported strains (SZ-WIV01, SZ01, ZJ03) from Samoa.
Figure 2: The quantification of infectious ZIKV and detection limit of the real-time RT-PCR assay. (A) The growth curves of
ZIKV on Vero cells. The virus titer of each time was quantified by plaque assay (black columns). The copy numbers of
genomic RNA at each time was quantified by real-time RT-PCR (black line). (B) Regression analysis between viral titers and
RNA genome copy numbers. (C) Standard curve constructed by ZIKV viral RNA extracted from 10-fold diluted WT ZIKV
stock with known titer. (D) Amplification plots of different dilutions of the real-time RT-PCR assay for detection limit.
Corresponding author:
Chenglin Deng
[email protected]
Figure 1
Figure 2
Emerging Viruses
P141
Investigating the host tropism of bovine hepaciviruses
C. Baechlein1,2, D. Meyer1, A. L. Baron1, S. Peine3, N. Fischer4,5, P. Becher1,2
1University
of Veterinary Medicine Hanover, Institute of Virology, Hanover, Germany
Centre for Infection Research (DZIF), Partner Site Hanover-Braunschweig, Germany
3University Medical Center Hamburg-Eppendorf, Department of Transfusion Medicine, Hamburg, Germany
4University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany
5German Centre for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Germany
2German
The emergence of novel viruses and variants of viruses poses a significant risk to human health. Most of so called emerging
Infections originate from animals, from where a spill-over to humans might be driven by viral evolution leading to changes of
viral properties.
Recently, hepaciviruses have been detected in numerous animal species including bats, rodents and horses. Moreover, we
identified a novel HCV-like virus in bovine serum samples which was termed Bovine hepacivirus (BovHepV) [1]. The virus
was found to replicate to high amounts in cattle, but the host tropism was not investigated so far. However, this could be of
particular importance. The so-called non-primate hepaciviruses have been initially identified in canine respiratory samples
but were later regularly confirmed to infect horses implying cross-species transmission. Furthermore, BovHepV could be
involved in the pathogenesis of liver disease with so far unknown etiology, both in animal species as well as potentially in
humans.
To address this question, we developed serological assays to detect BovHepV specific antibodies in serum samples of
different animal species and humans. Truncated BovHepV E2 glycoprotein was expressed in Leishmania tarentolae and was
used to set up an indirect antibody ELISA. With this, serum samples from cattle (n=307), pigs (n=393), sheep (n=188) and
goats (n=126) have been investigated so far. As expected, the highest seroprevalence was found in cattle (58.5%), but also
pigs and sheep exhibited serum antibodies against BovHepV E2. Moreover, serum samples from human blood donors were
investigated for the presence of virus specific antibodies. Out of 200 serum samples, n=20 (10%) were found to elicit a week
E2 antigen specific immune response. Current work is assessing the specificity of the observed antigen response by
applying luciferase precipitation assays (confirmation assay) based on E2 and non-structural NS3 proteins.
This work was financially supported by the German Federal Ministry of Food and Agriculture (BMEL), grant number
2816HS002.
References:
[1] Baechlein, Fischer, et al. 2015. J Virol. 89(14):7007-15.
Corresponding author:
Christine Baechlein
[email protected]
Emerging Viruses
P142
Transmission of Rift Valley fever virus from viremic lambs to Culex pipiens mosquitoes native to The Netherlands
R. Vloet1, C. Vogels1, C. Koenraadt1, G. Pijlman1, L. Van Keulen1, P. Wichgers Schreur1, J. Kortekaas1
1Wageningen
Bioveterinary Research, Virology, Lelystad, Netherlands
Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus of the genus Phlebovirus that causes severe disease in
ruminants and occasionally humans. The disease is endemic to the African continent, the Arabian Peninsula and several
islands located off the coast of Southeast Africa. Globalization, climate change and their impact on the distribution of
mosquito vectors raise concerns about future incursions into currently unaffected areas including Europe. We have
previously demonstrated that lambs native to the Netherlands are highly susceptible to RVFV. To further assess the risk of a
future RVFV incursion into the Netherlands, we here report the vector competence of the most abundant and widespread
mosquito species in the country; Culex (Cx.) pipiens. Artificial feeding experiments demonstrated that RVFV readily infects
laboratory-reared Cx. pipiens mosquitoes, followed by dissemination to the salivary glands. Infection and dissemination rates
were positively influenced by virus dose, temperature and time after feeding. To assess the likelihood of transmission in the
field, mosquitoes were allowed to feed on lambs during different stages of viremia. Remarkably, efficient transmission of the
virus occurred within a timeframe of little more than 24 hrs, a finding that sheds new light onto the epidemiology of RVF.
Corresponding author:
Rianka Vloet
[email protected]
Emerging Viruses
P143
The Zika virus toolbox-development of an animal model and molecular assays for an emerging Flavivirus
D. Anderson1, O. Sessions1, S. Mani1, T. Chawla1, B. Ho Wenkae1, J. H. Tan1, X. M. Ong1, U. Sangumathi Kamaraj1, L. F.
Wang1, V. von Messling1,2
1Duke-NUS
2Paul
Medical School, Programme in Emerging Infectious Diseases, Singapore, Singapore
Ehrlich Institute, Veterinary Medicine, Langen, Germany
Introduction: Zika virus (ZIKV) belongs to the family Flaviviridae, which includes globally relevant pathogens such as
dengue, yellow fever and Japanese encephalitis. Although originally isolated in 1947, ZIKV is considered an emerging
pathogen due to the recent wider spread and the more severe complications, such as neurological disorders that have been
observed during recent outbreaks in the South Pacific and Latin America.
Objectives: Animal models are essential for understanding ZIKV transmission and pathogenesis, and for evaluating
candidate vaccines and therapeutics. The objective was to determine the susceptibility and pathogenesis immunocompetent
monkeys to ZIKV. In addition, we aimed to develop both molecular and serological assays for the detection and
characterization of ZIKV.
Material & Methods: Cynomolgus macaques (Macaca fascicularis) were inoculated with ZIKV clinical isolates belonging to
the French Polynesia lineage (ZIKV-Brazil, ZIKV-ILM and Zika-SG) and disease pathogenesis examined.
Next Generation Sequencing (NGS) combined with targeted enrichment of viral genomes can greatly enhance the
discrepancy in the virus to host RNA ratio. We designed a hybridization-based enrichment assay to capture nucleic acid from
NGS libraries prepared from ZIKV RNA.
The immunoassay LIPS (Luciferase Immunoprecipitation system) was used to monitor exposure to ZIKV. LIPS quantitatively
detects the amount of antibody bound to luciferase tagged antigens. ZIKV Envelope (E) and Non Structural 1 (NS1) proteins
were used as they generate high antibody responses during ZIKV infection.
Results: ZIKV pathogenesis in cynomolgus macaques is similar to pathogenesis in humans. We were able to capture and
sequence the complete ZIKV genome. A comparison of unenriched and enriched NGS libraries yielded 0.7% and 85.7%
ZIKV-specific sequence reads, respectively; a 119.6x enrichment of viral genetic material. We have successfully expressed
ZIKV E and NS1 proteins and screened ZIKV positive patient samples using LIPS.
Conclusion: During an outbreak, accurate virus characterization aids in diagnosis, risk factor investigation and potentially
control measure implementation. These tools will be valuable for detection of ZIKV, evaluating vaccines and therapeutics as
well as understanding disease pathogenesis.
Corresponding author:
Danielle Anderson
[email protected]
Emerging Viruses
P144
Porcine Epidemic Diarrhea – on the hunt for its molecular pathogenesis
C. Kristen-Burmann1, R. Ehmann1, M. Ackermann2, V. Thiel3, G. Tekes1
1Justus
Liebig University Giessen, Institute of Virology, Giessen, Germany
of Zurich, Institute of Virology, Zurich, Switzerland
3University of Bern, Institute of Virology and Immunology, Vetsuisse Faculty, Bern, Switzerland
2University
Introduction: Porcine epidemic diarrhea (PED) represents an important disease of swine worldwide. It is caused by porcine
epidemic diarrhea virus (PEDV), member of the family Coronaviridae genus Alphacoronavirus. While viruses with low
pathogenicity usually lead to harmless infections, the highly virulent strains induce severe disease in newborn piglets with
high mortality rates, causing high economic losses for the swine industry. After severe outbreaks in Europe during the 1980s
and 1990s, PEDV became endemic in Asia. Lately, besides low virulent strains, highly virulent PEDVs were detected for the
first time in the USA and in some European countries. To prevent further expansion of the virus and minimize the economic
losses caused by highly virulent variants, enhanced focus on PEDV research is required.
Objectives: Our aim is to identify viral genes and gene products that contribute to the development of the severe form of the
disease using well-defined recombinant viruses generated by reverse genetics. Furthermore, we intend to identify biological
markers for differentiation between high- and low-pathogenic forms.
Material & Methods: Accordingly, we established a reverse genetic system for a field PEDV isolate and a DNA copy of the
PEDV field strain Minnesota (MN) was introduced into vaccinia virus genome.
Results & Conclusion: Our first experiments showed that the infectious clone based on the original databank sequence did
not allow the rescue of PEDVs. However, exchange of two nucleotides in the 5' UTR in combination with three amino acids in
the S protein enabled recovery of recombinant PEDVs. In order to assess the involvement of the S gene and ORF 3 in PED
pathogenesis, we generated a panel of recombinant viruses using our recently developed system. Ongoing animal
experiments with newborn piglets will reveal the impact of genetic changes in the S and 3 genes on disease severity.
Corresponding author:
Claudia Kristen-Burmann
[email protected]
Emerging Viruses
P145
Recent emergence of a novel porcine pestivirus – Interference with classical swine fever diagnosis?
A. Postel1, D. Meyer1, A. Petrov1, M. Wendt2, P. Becher1
1University
2University
of Veterinary Medicine Hanover, Institute of Virology, Hanover, Germany
of Veterinary Medicine Hanover, Clinic for Swine, Small Ruminants, Forensic Medicine and Ambulatory Service, Hanover, Germany
Pestiviruses are highly variable RNA viruses belonging to the family Flaviviridae and comprising pathogens of economically
important animal diseases like Classical Swine Fever (CSF). A genetically very distinct pestivirus, designated atypical
porcine pestivirus (APPV), was recently discovered in healthy pigs and an association with congenital tremor in newborn
piglets was found. This clinical syndrome is also observed after intrauterine infection with CSF virus (CSFV) and therefore
both infections need to be differentiated by laboratory investigations.
In this study, the virological and the serological differentiation between APPV and CSFV were addressed. Furthermore, it
was the aim to shed light on the dynamic of APPV infection and the induced antibody response.
Serum samples of pigs were screened by two newly developed qRT-PCRs being APPV-specific and broadly reactive
(PanPesti-PCR). APPV genome positive sera were tested in three quantitative real-time RT-PCRs (qRT-PCR) specific for
CSF. For serological investigations an APPV-specific ELISA was developed. In addition, three commercially available
Pestivirus ELISAs were applied. Successful APPV isolation on porcine kidney cells allowed immunostaining with ELISApositive field sera and to test the specificity of established monoclonal antibodies. The newly developed assays were applied
to investigate serially taken samples from piglets of a clinically affected pig herd.
Apart from the developed PanPesti qRT-PCR, no cross-reactivity between CSFV and APPV genome detection was observed
when applying methods which are routinely used for CSFV diagnosis. First epidemiological investigations revealed the
specificity of the APPV-ELISA and provide evidence for a high seroprevalence in different regions of Germany. Piglets
(n=20) from one herd facing problems with congenital tremor showed significant differences in time course of genome loads,
seroconversion and antibody kinetics.
Taken together, there is no evidence that the relative high APPV prevalence observed in Germany interferes with laboratory
tests applied in routine CSF diagnosis. First analyses of field sera revealed significant differences in viremia and antibody
kinetics depending on the time point of APPV infection.
Corresponding author:
Alexander Postel
[email protected]
Emerging Viruses
P146
First outbreak and co-circulation of three different Usutu virus strains in Eastern Germany
M. Sieg1, V. Schmidt2, U. Ziegler3, M. Keller3, D. Höper3, K. Heenemann1, A. Rückner1, H. Nieper4, A. Muluneh4, M.
Groschup3, T. Vahlenkamp1
1University
of Leipzig, Institute of Virology, Leipzig, Germany
of Leipzig, Clinic for Birds and Reptiles, Leipzig, Germany
3Friedrich Loeffler Institute, Greifswald - Isle of Riems, Germany
4Landesuntersuchungsanstalt für das Gesundheits- und Veterinärwesen Sachsen, Dresden, Germany
2University
Usutu virus (USUV) is a mosquito-borne flavivirus accounting for large-scale deaths in resident bird populations. Here we
show the first introduction of USUV to Eastern Germany resulting in massive death of birds, particularly blackbirds (Turdus
merula).
We found that three diverse USUV lineages (Europe 3, Africa 2, Africa 3-like) circulated simultaneously. Moreover we
detected Usutu virus in Culex pipiens in a region where no dead birds were reported, strengthening the need for mosquito
monitoring to uncover the spread of arboviruses. Further, phylogenetic analyses revealed that mutations accumulated in
particular in the NS3 region within short time periods. In addition, comparison of whole genome sequences showed that
isolates of the cluster Africa 3-like are changing at an extraordinarily high mutation rate, suggesting accelerated genetic
changes of USUV with potential emerging threats for veterinary and public health.
Corresponding author:
Michael Sieg
[email protected]
Emerging Viruses
P147
Polyanions as the backbone for broad-spectrum antiviral agents
F. Schandock1, C. Frich Riber1, A. Röcker1, P. Gajda1, K. Borup Løvchall1, F. Kreppel1, A. N. Zelikin1, J. Münch1
1Ulm
University Medical Center, Institute of Molecular Virology, Ulm, Germany
Introduction: Viral pathogens constitute an enormous socio-economic burden. Against most human pathogenic viruses
including ZIKV no specific antiviral therapy exists. In contrast to broad spectrum antibiotics, viruses are dealt with on the "one
bug – one drug" basis, but the field is growing that "broad spectrum antiviral agents" are going to make the highest impact
and change the ways that viral pathogens are being dealt with. A sub-class of broadly active antiviral agents is based on
polymers, high molar mass compounds assembled using repeating building blocks. Since the 1960"s, anionic polymers such
as heparin are known as efficient inhibitors of viral infections, but their strong anti-coagulative properties prevented their
clinical development as antivirals.
Objectives: Here, we sought to develop polyanions with less or no anti-coagulative and broad antiviral activity against
important viral pathogens including HIV-1, HSV-2, influenza, lyssa, rabies, lassa and adenovirus as well as (re)emerging
Ebola, Marburg, SARS and ZIKV.
Material & Methods: 14 synthetic polymers with chemically diverse set of anionic functionalities were prepared by
Reversible Addition Fragmentation chain Transfer. The antiviral potential against ZIKV was tested by using a colorimetric
MTT assay, cell-based immunodetection assay and confocal microscopy. Antiviral effect against HIV-1 and HSV-2 was
tested in reporter cell lines. Inhibition of influenza, lyssa, rabies, lassa, Ebola, Marburg and SARS virus entry was assessed
using luciferase encoding lentiviral pseudotypes. Antiviral activity against adenovirus was tested by using an EGFP reporter
virus and flow cytometry.
Results: Blood coagulation assay revealed 6 polyanions with marginal or no anti-coagulant activity. Structure-activity
analysis revealed no polymer could inhibit all of the viruses tested in this work. However, for each of the enveloped viruses,
there was at least one polymer that acted as a strong antiviral agent. Overall, hydrophobicity of polyanions was identified as
a significant contributor to the antiviral activity.
Conclusion: We identified chemically well-defined polymers as novel inhibitors of a variety of enveloped viruses, including
major human pathogens against which no antiviral therapy exists. Some of these polymers hold promise for clinical
development as they have no anti-coagulant activity.
Corresponding author:
Franziska Schandock
[email protected]
Emerging Viruses
P148
Occurrence of Shrew- and Mole-Borne Hantaviruses in Germany
B. Klempa1,2, L. Radosa1, P. T. Witkowski1, P. Köppen-Rung 1, M. Ličková2, A. Stang3, L. Striešková4, T. Szemeš4, S.
Essbauer5, R. G. Ulrich6, D. H. Krüger 1
1Charité
Medical School, Institute of Virology, Berlin, Germany
Research Center, Slovak Academy of Sciences, Institute of Virology, Bratislava, Slovakia
3Ruhr-University, Department of Molecular and Medical Virology, Bochum, Germany
4Comenius University, Department of Molecular Biology, Bratislava, Slovakia
5Bundeswehr Institute of Microbiology, Department Virology & Rickettsiology, Munich, Germany
6Friedrich Loeffler Institute, Institute of Novel and Emerging Infectious Diseases, Greifswald - Isle of Riems, Germany
2Biomedical
Since 2007, more than 20 new hantaviruses associated with insectivores were described worldwide. New hosts as bats,
shrews, and moles completely changed our view on hantavirus ecology, formerly believed to be only rodent-borne viruses.
Our extensive molecular screening of insectivores revealed the presence of two shrew-borne hantaviruses in Germany.
Newfound Seewis virus (associated with Eurasian common shrew, Sorex araneus) and Asikkala virus (associated with
Eurasian pygmy shrew, Sorex minutus) showed strong geographic clustering as well as high genetic divergency.
Objective of this study was to elucidate whether not only shrew- but also mole-borne hantaviruses occur in Germany.
Mole samples were tested by genus-reactive nested RT-PCR targeting the large (L) genomic segment. Positive samples
were then subjected to further sequencing efforts including RT-PCR-based Sanger and next-generation-sequencing (NGS)
using Illumina Miseq and NextSeq500 technologies.
The study led to detection and genetic characterization of the mole-borne Nova virus (NVAV). Sequence comparisons with
other NVAV strains showed a high degree of sequence diversity. Phylogenetic analyses confirmed that the German strains
form a separate clade within the monophyletic group of NVAV sequences.
Opportunistic testing of a single specimen of Talpa europaea mole found dead in the vicinity of Wandlitz village near Berlin
showed that moles can carry another hantavirus, tentatively called Wandlitz virus. After numerous RT-PCR- and NGS-based
sequencing efforts failed, sufficient sequence information was obtained from the tissue sample through a combination of
particle-associated nucleic acids (PAN) purification, unspecific pre-amplification, and Illumina Nextseq500 technology. The
virus is highly divergent from NVAV and more related to shrew-borne hantaviruses. Most recent analyses indicated that the
virus is closely related to the Bruges virus identified in Belgium at the same time.
Our studies showed the presence and revealed first genomic sequence data of two shrew-borne and two mole-borne
hantaviruses in Germany. Further steps will aim for evaluation of the public health relevance of these new insectivore-borne
viruses.
Corresponding author:
Boris Klempa
[email protected]
Emerging Viruses
P149
The molecular tweezer CLR01 inhibits Zika and other sexually transmitted viruses
A. Röcker1, J. Müller1, M. Harms1, J. Münch1, F. Schandock1, E. Sanchez-Garcia2, K. Bravo-Rodriguez2, E. Dietzel3, A.
Kupke3, S. Becker3, J. Schmidt-Chanasit4, O. Vapalahti5, T. Schrader6
1Ulm
University Medical Center, Molecular Virology, Ulm, Germany
Planck Institute of Coal Research, Mülheim an der Ruhr, Germany
3Philipps University Marburg, Institute of Virology, Marburg, Germany
4Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany
5University of Helsinki and Helsinki University Hospital, Departments of Virology and Immunology, and Veterinary Biosciences, Helsinki, Finland
6University of Duisburg-Essen, Faculty of Chemistry, Essen, Germany
2Max
Introduction: We have previously shown that the molecular tweezer, CLR01, binds to and destroys enveloped viruses such
as HIV-1, HSV-2, HCMV, and HCV. Its antiviral activity is mediated through a specific interaction of CLR01 with lipid raft rich
regions in the viral membrane without causing cytotoxic effects. The antiviral activity is not abrogated in the presence of
semen suggesting that CLR01 has prospects for microbicide development. Microbicides are antiviral compounds that can be
applied inside the vagina or rectum and could protect against HIV and other sexually transmitted infections.
Objectives: In this study, we wanted to investigate the effect of CLR01 on Ebola and Zika virus (ZIKV), two (re)-emerging
viruses responsible for recent global health problems.
Material & Methods: Computational modeling was performed to study interaction and destruction of CLR01 with model
membranes. The effect of CLR01 and an inactive CLR03 control on replication-competent Ebola virus was assessed by
plaque reduction assay. Antiviral activity of CLR01 against ZIKV was studied using lab-adapted and recent clinical isolates by
confocal microscopy, flow cytometry and cell-based immunodetection assays in relevant target cells.
Results: The simulations indicate that CLR01 is able to form inclusion complexes with lipid head groups of membrane
components DOPC and SM, resulting in a destabilization of the viral membrane. We found that CLR01 inhibits infection of
replication-competent Ebola virus and pseudotyped lentiviral particles of Ebola, Marburg, Rabies and SARS virus. Moreover,
CLR01 inhibits ZIKV prototype strain MR766 infection in a dose-dependent manner. Further analyses revealed that CLR01
destroys the ZIKV envelope time-dependently, with complete loss of infectivity after 10 min of virion exposure. The tweezer
also inactivated ZIKV epidemic strains FB_GWUH_2016 and PRVABC-59 and prevented infection of human foreskin, cervix
and colon cells. Finally we show that CLR01 remains antivirally active in the presence of semen.
Conclusion: CLR01 is a broad-based inhibitor of enveloped viruses and retains antiviral activity in the presence of human
semen. Thus, CLR01 has prospects to be developed as microbicide to limit or block the spread of viruses transmitted by
sexual intercourse such as HIV, ZIKV or Ebola.
Corresponding author:
Annika Röcker
[email protected]
Emerging Viruses
P150
Semen inhibits Zika Virus infection
M. Harms1, J. Müller1, S. Joas1, M. Hayn1, A. Dietz2, J. von Einem2, A. Schubert2, D. Michel2, O. Vapalahti3, J. Shorter4, J.
Schmidt-Chanasit5, F. Ebner6, J. Münch1
1Ulm
University Medical Center, Institute of Molecular Virology, Ulm, Germany
University Medical Center, Institute of Virology, Ulm, Germany
3University of Helsinki and Helsinki University Hospital, Departments of Virology and Immunology, and Veterinary Biosciences, Helsinki, Finland
4Perelman School of Medicine at the University of Pennsylvania, Cell and Molecular Biology Graduate Group, Philadelphia, United States
5Bernhard Nocht Institute of Tropical Medicine, Virology, Hamburg, Germany
6Ulm University Medical Center, Department of of Obstetrics and Gynecology, Ulm, Germany
2Ulm
Introduction: Zika virus (ZIKV) is transmitted to humans primarily via mosquitoes. However, several countries reported
cases of sexual transmission of ZIKV, including male-to-female, female-to-male, and male-to-male transmissions. In infected
patients, ZIKV is detectable in semen at 107-108 RNA copies/ml and remained detectable for up to 6 months after onset of
symptoms. Despite these high viral loads, rates of sexual ZIKV transmission seem to be low.
Objectives: Semen is a complex body fluid containing factors that may affect ZIKV infectivity. Here we set out to explore the
effects of whole semen on ZIKV infection. Furthermore we test the susceptibility of cells and tissues derived from the
anogenital tract to clarify whether they support ZIKV infection and may serve as entry portals of the virus.
Material & Methods: The effect of semen on ZIKV infection was detected using plaque reduction assays, confocal
microscopy, flow cytometry and cell-based ZIKV immune-detection assays. All assays were performed under conditions
avoiding semen induced cytotoxicity.
Results: We found that just 1-5 % of pooled human semen and seminal plasma inhibit infection of Vero cells with the ZIKV
MR766 and recent clinical isolates. This potent anti-ZIKV activity of semen was confirmed using individual fresh human
ejaculates and was also observed when human target cells were infected. In addition we found that cells derived from
foreskin, cervix, and colon as well as vaginal and endometrial tissues support productive ZIKV infection. However, infection
of these potential entry portals for sexually transmitted ZIKV was again efficiently inhibited by semen.
Conclusion: Our findings demonstrate that semen potently inhibits ZIKV infection, providing a plausible explanation for the
low frequency of sexual ZIKV transmission, despite exceedingly high viral titers in semen.
Corresponding author:
Mirja Harms
[email protected]
Emerging Viruses
P151
VrAP – Full length de novo genome assembly of unknown RNA viruses
M. Fricke1, F. Zirkel2, C. Drosten2, S. Junglen2, M. Marz1
1Friedrich
Schiller University Jena, Faculty of Mathematics and Computer Science, Jena, Germany
of Bonn Medical Center, Institute of Virology, Bann, Germany
2University
Introtduction: Within the last decade a lot of new viral genomes have been published. Genome assemblers specifically
designed for viral genomes are rare. Although viral RNA genomes are relatively short, a de novo full viral genome assembly
of small sequenced reads is a challenging task due to the high level of genetic diversity and a low or uneven coverage of
viral reads compared to host reads. We present a de novo genome assembler for RNA viruses called VrAP (Viral Assembly
Pipeline). Our pipeline classifies the contigs to distinguish host from viral sequences by annotation and ORF density scores.
We tested VrAP on real datasets generated with different sequencing technologies. We identified new viruses representing
new strains, species and even genera and families.
Objectives: We present VrAP, a tool to assemble complete viral RNA genomes, followed by contig annotation to distinguish
host and viral contigs. Furthermore, we provide wet lab guidelines to produce optimal input data.
Methods: VrAP consists of four basic steps:
(1) Host genome filter
(2) Read correction
(3) Genome assembly and super contig construction
(4) Genome identification and annotation
Results: We tested VrAP with different input samples from Aedes albopictus mosquitoes, sequenced either with Illumina,
454 or Ion Torrent. To simulate the discovery of novel strains, species, genera or families, the sequence information of the
considered viruses were excluded from the reference database. We assembled and identified all included viral genomes.
Except of two assemblies, we covered the complete viral genomes/segments in a single contig. Strikingly, with our new ORF
density method we can identify viruses without any sequence homology to known references.
Conclusion: With VrAP and our wet lab guidelines we provide an easy and cheap workflow to assemble full viral genomes
out of small read libraries and with different sequencing techniques, which outperforms commonly used assemblers and
assemblers specifically designed for viral data. Our pipeline reduces the high amount of manual work and time for genome
assembly, super contig construction and annotation to a single run of VrAP.
Corresponding author:
Markus Fricke
[email protected]
Emerging Viruses
P152
Encephalitis and ganglionitis in domestic sheep caused by astroviruses
F. Pfaff1, K. Schlottau1, S. Scholes2, A. Courtenay3, B. Hoffmann1, D. Höper1, M. Beer1
1Friedrich
Loeffler Institute, Institute of Diagnostic Virology, Greifswald - Isle of Riems, Germany
Consulting Veterinary Services, Penicuik, United Kingdom
3University of Liverpool, Neston, United Kingdom
2SAC
Astroviruses are frequently associated with diarrhea and gastroenteritis in young individuals and have been described for a
multitude of mammals (genus Mamastrovirus) and birds (genus Avastrovirus). Recently, mamastroviruses have been
repeatedly associated with neurological diseases in humans, minks, and cattle. Here we describe for the first time two cases
of severe neurological disorders in domestic sheep, caused by a novel mamastrovirus. In June 2013, a four-year-old Welsh
Mountain ewe and in March 2014 a ten-day-old lamb of the same breed and the same flock presented progressive
neurological signs including depressed sensorium, tremor, and unusual behaviour. Neuropathological examination of the
brain and spinal cord detected non-suppurative polioencephalomyelitis and dorsal root ganglionitis, characteristic of
neurotropic viral infections. The presence of dorsal root ganglionitis, and immunohistochemical analyses of brain material
strongly militated against Louping ill, caused by Louping ill virus infection which occurs commonly in upland areas of the
British Isles. Using high-throughput sequencing and metagenomic analysis of tissue samples from both animals, we
identified a novel ovine astrovirus (OvAstV). Viral genome was detected in different parts of the central nervous system by
specific RT-qPCR. Although both cases presented nine months apart, viruses from both cases had nearly identical genomes
differing in only five from a total of 6454 nucleotide positions. Phylogenetic analysis revealed a close relation of these
OvAstV to neurotropic bovine astroviruses (BoAstV) and an enteric OvAstV. In conclusion, these are the first reported cases
of astrovirus infection in domestic sheep associated with encephalitis and ganglionitis. Besides arthropod-borne viruses,
such as Louping ill virus, OvAstV needs to be considered in differential diagnosis of encephalitis and ganglionitis in domestic
sheep. The close phylogenetic relationship of neurotropic BoAstV and OvAstV may indicate a possible transmission between
small ruminants and cattle.
Corresponding author:
Florian Pfaff
[email protected]
Emerging Viruses
P153
If you seek thou shalt find – Optimised sampling efforts and screening assays to assess the diversity and ecology
of coronaviruses in South African bats
W. Preiser1, N. L. Ithete1, N. Cronjé1, T. Suliman1
1Stellenbosch
University / NHLS, Medical Virology, Cape Town, South Africa
Bats are thought to be reservoir hosts for all alpha- and beta-coronaviruses (α-, β-CoV). Following the emergence of SARS
in 2002/03 and the subsequent identification of Rhinolophus sinicus as the likely ancestral SARS-CoV source, a wide
diversity of bat CoV has been described worldwide.
Our research aims to better understand CoV diversity and ecology in South African bats. In addition to general
"opportunistic" surveillance, we are conducting species-specific investigations, through close transdisciplinary collaborations
with ecologists and zoologists, concentrating on Neoromicia capensis and Rhinolophus spp. These include longitudinal
studies of bat colonies to determine shedding patterns and diversity of viruses present.
Since 2011, over 800 individual bats belonging to 24 different species have been sampled along rainfall and altitudinal
gradients across different biomes (Fynbos, Forest, Nama Karoo, Grassland, and Savanna). Sample types include faecal
pellets, saliva and urine swabs, and when voucher specimens are sacrificed for museum collections, also blood and organs.
We previously contributed 12 of 15 bat α-CoV sequences reported from South Africa, along with one β-CoV sequence from
N. capensis, named NeoCoV. Phylogenetic inference shows that NeoCoV belongs to the same viral species as the recently
emerged MERS-CoV, responsible for the ongoing outbreak in the Arabian Peninsula.
An improved screening protocol greatly increased the overall CoV detection rate from approximately 9% to 15%. Additional
CoV sequences have been identified in various bat species (N. capensis, N. nanus, R. clivosus, R. capensis, Pipistrellus
hesperidus, Miniopterus natalensis). The majority of these are α-CoVs, with approximately 20% prevalence in N. capensis.
Of particular interest are the detection of SARS-CoV-related β-CoV in R. capensis and R. clivosus and of NeoCoV-related βCoV sequences in P. hesperidus. Preliminary analyses of the partial RdRp, nucleocapsid and spike gene fragments suggest
that these viruses share a common ancestor with Neo-CoV.
Screening and virus characterisation efforts are ongoing. Through an interdisciplinary approach we hope to achieve a better
understanding of CoV and of virus-host ecology in South African bats.
Corresponding author:
Wolfgang Preiser
[email protected]
Emerging Viruses
P154
Transmission of Zika virus by mosquitoes from Central Europe
A. Heitmann1,2, J. Stephanie1,3, R. Lühken1, M. Leggewie1, M. Badusche1, B. Pluskota4, N. Becker4, O. Vapalahti5, J.
Schmidt-Chanasit1, E. Tannich1
1BNITM,
Molecular Parasitology, Hamburg, Germany
Hamburg, Germany
3BNITM, Hamburg, Germany
4Institute of Dipterology, Speyer, Germany
5University, Helsinki, Finland
2institute,
The emerging outbreak of the Arbovirus Zika virus (ZIKV) in south and central America reinforce the interest in vector
competence of different mosquito species for ZIKV. It is well accepted that Aedes aegypti is the most important vector in
south and central america. In order to assesses the risk of spread of ZIKV in temperate climate zones, it is important to
investigate the vector potential of other mosquito species. German populations of Culex pipiens pipiens biotype pipiens,
Culex pipiens pipiens biotype molestus, Culex torrentium and Aedes albopictus were experimentally infected with ZIKV,
using Aedes aegypti and Italian Aedes albopictus as positive controls. Infection of mosquitoes was done by artificial
bloodmeal with the asian genotype ZIKV strain, isolated during the outbreak (ZIKV_FB-GWUH-2016). Mosquitoes were
incubated at 27°C or 18°C and analysed 14 and 21 days post infection. We analyzed the infection rate by real-time qPCR
and the transmission rate by observing the cytopathic effect. None of the Culex taxa showed vector competence for ZIKV
either at 27°C or at 18°C. In contrast, Aedes albopictus were susceptible for ZIKV but only at 27 °C, with transmission rates
similar to an Aedes aegypti lab colony tested in parallel.
Corresponding author:
Anna Heitmann
[email protected]
Emerging Viruses
P155
Phylogeny of TBE virus in Austria and Central Europe
M. Bestehorn1, G. Dobler2, L. Chitimia-Dobler2, K. Stiasny3, F. X. Heinz3
1University
of Hohenheim, Institute of Zoology, Parasitology, Stuttgart, Germany
Institute of Microbiology, Munich, Germany
3Medical University of Vienna, Department of Virology, Vienna, Austria
2Bundeswehr,
Tick-borne encephalitis virus (TBEV) is the most important tick-borne virus in Europe and Asia. Recent studies show that the
origin of TBEV lies in the Siberian region, from where it spread into Central Europe more than 300 years ago. New data
show, that areas with human TBE cases moved from Eastern Austria to the federal states of Tyrol and Vorarlberg (Heinz et
al. 2015). To understand the spreading of TBEV in Austria and in Central Europe, E genes of TBEV strains from recently
emerged endemic Austrian areas were sequenced and compared to E sequences from former Austrian isolates available in
data bases. A phylo-geographic analysis was conducted forming a relation of genetics and geographic places of occurrence.
The resulting data show that the isolates from the 1970s and 1980s cluster in phylogenetic arbitrary genotypes with viruses
from Germany, Czech Republic, Slovak Republic, Slovenia, Switzerland and Russia. These results clearly show that the
recently emerged TBE endemic areas in Austria are not formed by continuous spread of TBEV strains from existing natural
foci along natural landscape aisles (river valleys of Ziller and Inn), but seem to be recently introduced from other regions in
Central Europe. The mode of introduction is yet unclear, however recent data from Germany suggest that migratory birds
may play an important role in the recent spread of TBEV in Europe and emergence in new regions.
Corresponding author:
Malena Bestehorn
[email protected]
Emerging Viruses
P156
First detection and phylogenetic characterization of a TBE virus strain isolated from Dermacentor reticulatus in
Northern Saxony, Germany
M. Bestehorn1, L. Chitimia-Dobler2, M. Pfeffer3, G. Dobler2
1University
of Hohenheim, Institute of Zoology, Parasitology, Stuttgart, Germany
Institute of Microbiology, Munich, Germany
3University of Leipzig, Institute of Animal Hygiene Public Veterinary Health, Leipzig, Germany
2Bundeswehr,
Tick-borne encephalitis (TBE) is the most important viral CNS infection in Central Europe. TBE virus is a member of the
family Flaviviridae and is known to be transmitted by the tick species Ixodes (I.) ricinus in Central Europe. We report the first
isolation and phylogenetic characterization of TBE virus of the Western subtype from Dermacentor (D.) reticulatus ticks in the
district of Northern Saxony, Federal State of Saxony in Germany.
So far the district of Northern Saxony has been assumed to be TBE free. In September 2016 a human TBE case was
reported from there and the location of the acquired tick-bite was a forest close to the city of Torgau. 930 ticks, of which 816
were identified as adult D. reticulatus and 114 as I. ricinus, were gained by flagging. The ticks were processed in pools of 5
adults or 10 nymphs, according to species and gender. Pools were processed according to standard procedures of our lab.
The RNA was screened for TBEV, using the real-time RT-PCR by Schwaiger & Casisnotti.
Three pools of D. reticulatus and one pool of I. ricinus ticks were found TBEV positive. The minimal infection rate was
determined as 0.27 % for D. reticulatus adult ticks and as 1.14 % for I. ricinus. The molecular characterization revealed a
TBE virus strain, which belongs to the Western (European) subtype. The phylogenetic analysis of the E genes, which were
directly amplified from the positive tick pools, revealed the closest genetic relationship to a TBE virus strain from
neighbouring Poland, isolated from I. ricinus in the 1970s. Some minor differences between the E genes of the D. reticulatus
strains and the I. ricinus strain were detected, which have to be analysed in detail.
We report the first detection of a TBE virus strain in D. reticulatus in Germany. The close phylogenetic relationship to a Polish
TBE strain indicates a limited spread of the respective strain from Poland to Germany, possibly via birds or terrestrial
animals. This virus strain offers the ideal opportunity to further characterise its phenotypic and pathogenetic pattern to
elucidate the influence of the arthropod vector on the characteristics of TBE viruses for the first time.
Corresponding author:
Malena Bestehorn
[email protected]
Emerging Viruses
P157
Molecular epidemiology of the Dengue virus involved in Dengue disease cases registered in Mozambique between
2014-2015
I. Mahumane Gundane1, F. Barreto dos Santos2, F. de Bruycker Nogueir2, E. Samo Gudo 1
1Instituto
Nacional de Saude, Maputo, Mozambique
Cruz Institute, Rio de Janeiro, Brazil
2Oswaldo
Dengue, the most important and prevalent arthropod-borne viral disease is an infectious, febrile illness transmitted to
humans and other hosts by biting of Aedes mosquitoes. Four (DENV 1-4) antigenically distinct serotypes of the virus can
cause dengue with or without warning signs, and severe dengue. About 2.5 billion people worldwide live in countries at risk
for dengue occurrence. Mozambique is among the 100 countries at risk for dengue outbreak emergency and has historical
reports of the disease by serotype 3 between 1984 and 1985 (first report of DENV-3 in Africa). About 30 years after the first
reported cases, new dengue cases have been reported since 2014 in Cabo Delgado and Nampula (Northern Provinces of
Mozambique). In order to perform the molecular characterization of the DENV isolated during 2014-2015 in Mozambique, we
selected 42 positive specimens confirmed by RT-PCR. From these, 6 samples were submitted to sequencing, based on the
240pb fragment corresponding to the E / NS1 gene junction using BigDye Terminator Cycle Sequencing Ready Reaction kit
v.3.1 and DNA analyzer (ABI 3730) and the phylogenetic tree was constructed using the Neighbor-joining method and TN93
model. DENV-2 was the only one identified DENV seroptype. The genetic analysis showed that the Mozambican sequences
fell into the Cosmopolitan Genotype which were closely related to sequences from Tanzania (2014 DENV-2 outbreak) and
China (2010 and 2014 DENV-2 outbreaks). Our findings suggest an interplay of the viruses among those countries, thus
highlight the need of epidemiological studies including molecular analyzes of viruses once these constitute a valuable tool to
monitor both introduction, evolution and spread of viruses as well as to predict epidemiological consequences during
epidemic and inter-epidemic periods.
Corresponding author:
Isabel Mahumane Gundane
[email protected]
Emerging Viruses
P158
Development of a high-throughput colorimetric Zika Virus infection assay
J. Müller1, M. Harms1, A. Schubert2, B. Mayer3, S. Jansen4,5, J. P. Herbeuval6, D. Michel2, T. Mertens2, O. Vapalahti7, J.
Schmidt-Chanasit4,5, J. Münch1
1Ulm
University Medical Center, Insititute of Molecular Virology, Ulm, Germany
University Medical Center, Institute of Virology, Ulm, Germany
3Ulm University Medical Center, Institute of Epidemiology and Medical Biometry, Ulm, Germany
4Bernhard Nocht Institute of Tropical Medicine, Virology, Hamburg, Germany
5German Centre for Infection Research (DZIF), Partner Site Hamburg-Luebeck-Borstel, Hamburg, Germany
6CNRS UMR-8601, Université Paris Descartes, CICB, Paris, France
7Departments of Virology and Immunology, and Veterinary Biosciences, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
2Ulm
Zika virus (ZIKV) is an emerging pathogen that causes congenital infections, which may result in birth defects such as
microcephaly. Currently, no approved treatment or vaccination is available. ZIKV can be readily detected in cell culture where
virally infected cells are stained by specific antibodies.
As ZIKV regularly causes a cytopathic effect, we were wondering whether this viral property can be used to quantitatively
determine viral infectivity.
We here describe the use of an 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide-(MTT)-based cell viability
assay that allows to determine ZIKV-induced cell death.
We show that this colorimetric assay quantifies ZIKV infection over a broad range of viral dilutions in both monkey and
human cells. It allows convenient high-throughput screening of antiviral compounds or compound libraries but also doseresponse studies with defined drugs or interferons. Moreover, the MTT assay allows a rapid and exact determination of ZIKV
neutralizing antibody titers without the need for a microscopic quantification of plaques as it is normally done in Plaque
reduction neutralization test (PRNT) assays. This MTT-based ZIKV detection assay can be evaluated by naked eye or
computational methods, has a broad linear range, does not require large equipment or costly reagents, and thus represents
an interesting alternative to antibody-based ZIKV detection assays, which is of particular advantage in resource-poor
environments.
We propose to use this simple, fast and cheap method for quantification of ZIKV neutralizing antibodies and testing of
antiviral compounds.
Corresponding author:
Janis Müller
[email protected]
Emerging Viruses
P159
Prevalence of recently discovered human polyomaviruses (HPyVs) in children with gastroenteritis in Accra, Ghana
S. Silling1, B. Mensah2, J. Bonney2, H. Pfister1, U. Wieland1, J. Fobil2
1University
2University
of Cologne, Institute of Virology, Cologne, Germany
of Ghana, Accra, Ghana
Question: Gastrointestinal disease is a major health problem worldwide especially for small children in developing countries.
Besides the known enteric pathogens the role of emerging viruses such as human polyomaviruses (HPyVs), especially
Malawian polyomavirus (MWPyV) and St. Louis polyomavirus (STLPyV), should be investigated.
Methods: 505 fecal specimens were collected from hospitalized children 5 years of age or less with primary diagnosis of
acute gastroenteritis in Accra, Ghana. DNA was extracted and checked for inhibition of the PCR reaction by ß-globin PCR.
Of 317 ß-globin-positive samples type-specific PCRs were performed for the detection of MWPyV, STLPyV, HPyV12 and
NJPyV-2013.
Results: In the children, MWPyV was prevalent at 1.89% and STLPyV was prevalent at 3.47%. HPyV12 or NJPyV-2013 was
not detected.Three out of 6 MWPyV-positive and 4 out of 11 STLPyV-positive stool specimens were co-infected with other
gastrointestinal pathogens. Single infection with either of the two HPyVs occurred exclusively in children under 2 years of
age.
Conclusion: These findings suggest, that STLPyV and MWPyV might be associated with pediatric infectious gastroenteritis
especially in small children.
Corresponding author:
Steffi Silling
[email protected]
Emerging Viruses
P160
Discovery and isolation of a novel pestivirus species in pigs in Austria
B. Lamp1, L. Schwarz2, S. Högler3, C. Riedel1, L. Sinn1, H. Weissenböck3, A. Ladinig2, T. Rümenapf1
1Medical
University of Vienna, Institute of Virology, Vienna, Austria
for Swine, Vetmeduni, Vienna, Austria
3Medical University of Vienna, Institute of Pathology, Vienna, Austria
2Clinic
A novel pestivirus species was discovered in an Austrian piglet producing farm during virological examinations of congenital
tremor cases. Piglets revealing a severe hypomyelination of the white substance in cerebellum and spinal cord were
negatively tested for Atypical porcine pestivirus (APPV) and classical pestiviruses. Employing a recently developed panpestivirus PCR, a so far unknown pestivirus genome fragment could be amplified. This virus, provisionally termed "Linda
virus", could readily be propagated in cultured porcine cells. A full genomic sequence was determined using primer walking
together with Sanger sequencing and RACE-PCRs. Pairwise alignment of Linda virus sequences with other pestiviruses
revealed a nucleotide identity of less than 68% and an amino acid identity of less than 70% to its closest relative,
Bungowannah virus. Furthermore, we identified an E2 specific antibody cross-reactive against Linda virus, which was used
to confirm the presence of Linda virus in the CNS of affected piglets at the site of lesions. In conclusion, we describe a
putative novel pestivirus species distinct from recently discovered APPVs, which most likely also causes congenital tremor in
domesticated swine.
Corresponding author:
Benjamin Lamp
[email protected]
Emerging Viruses
P161
Find the right sample – Using non-invasive samples for the diagnosis of the emerging and reemerging viruses
M. Niedrig1, P. Patel2, A. Abd El Wahed 3, R. Schädler1, S. Yactayo4
1Robert
Koch Institute, Berlin, Germany
MOLBIOL Syntheselabor GmbH, Berlin, Germany
3Georg-August-University, Division of Microbiology and Animal Hygiene, Göttingen, Germany
4World Health Organization, Control of Epidemic Diseases (CED), Geneva, Switzerland
2TIB
The emergence of several viral infections during the last decades like Dengue, West Nile, SARS, Chikungunya, MERS-CoV,
Ebola, Zika and Yellow Fever raised many questions on speed and reliability of laboratory diagnostic tests for identification of
suspected cases. All important questions arising with an emerging infection like source and route of transmission, infectivity
of patients, recommendation of safety measures for health care personal and distribution in an animal reservoir or other
vectors require the performance of laboratory diagnosis assays. To investigate the patients infections status, most often a
blood sample is taken not only for the analysis of blood parameters but also for pathogen detection mostly by PCR or for
testing the presence of specific antibodies. However, sampling of blood requires medically trained personal and comprises
some risks for the patient as well as for the health care personal, in particular with highly infectious haemorrhagic fever like
Ebola. Therefore, the sampling by non-invasive methods (e.g. saliva and/ or urine) might be a very valuable alternative for
investigating a diseased patient. Usability of saliva and urine for diagnostic testing has been reported for many viral
infections, however, it is often not performed in the broad routine diagnostic. To analyse the usefulness of alternative noninvasive samples for diagnostic of infectious viral diseases mentioned above. we reviewed the use of saliva and urine for the
diagnosis of recently emerging viruses in comparison to conventional blood sampling. Since the extensive evaluation of
different samples for human patients is often not performed, also investigations of animal models or vectors are considered.
Therefore, we performed an electronic literature review by PubMed for alternative sampling for SARS, West Nile, Dengue,
MERS-CoV, Ebola, Zika and Yellow Fever. The search resulted in 711 papers of possible relevance, of which we included
128 in the study. Our systematic analysis shows that there should be put more emphasis on non-invasive sampling to
disclose more easy alternatives for collecting human samples for clinical and routine investigations.
Corresponding author:
Matthias Niedrig
[email protected]
Emerging Viruses
P162
Severe Fever with Thrombocytopenia Syndrome Virus Gn/Gc polyprotein processing is critical for viral infectivity
and requires an internal Gc signal peptide
T. Plegge1, H. Hofmann-Winkler1, M. Spiegel1, S. Pöhlmann1
1German
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
Introduction & objective: Bunyavirus Gn and Gc proteins are incorporated into the viral envelope and facilitate viral entry
into target cells. The Gn and Gc proteins are produced as a single precursor protein, Gn/Gc, and depend on cleavage by
host cell proteases for formation of mature Gn and Gc proteins. The severe fever with thrombocytopenia syndrome virus
(SFTSV), an emerging bunyavirus, causes severe disease with case-fatality rates of up to 30 %.
Objective: Here, we investigated the maturation and intracellular trafficking processes of the SFTSV-Gn/Gc precursor
protein into the mature Gn and Gc proteins.
Material & Methods: We employed Western blot analysis, protease inhibitors and mutant glycoproteins to investigate Gn/Gc
processing. Furthermore, we used immunofluorescence and confocal microscopy for the analysis of subcellular localization
of Gn and Gc. Rhabdoviral vectors harboring wt or mutant SFTSV glycoproteins were employed to analyze Gn and Gcdriven host cell entry.
Results: We found that expression of both SFTSV Gn and Gc is required for infectivity of rhabdoviral vectors. Gn and Gc
were both incorporated into rhabdoviral particles even when expressed from separate open reading frames and confocal
microscopy showed that Gn facilitated the transport of Gc from the endoplasmic reticulum into the Golgi apparatus.
Furthermore, we found that SFTSV-Gn/Gc maturation does not require activity of the proprotein convertases furin and SKI-1.
Instead, deletion of a predicted Gc signal peptide in the context of the Gn/Gc precursor blocked the generation of mature Gn
and Gc and abrogated Gn and Gc-dependent transduction.
Conclusion: Our findings suggest that the SFTSV-Gn/Gc precursor is cleaved downstream of an internal Gc signal peptide
by signal peptidase and that cleavage is essential for viral infectivity. A similar strategy is employed by Rift Valley fever and
Uukuniemi virus, like SFTSV members of the genus phlebovirus, suggesting that diverse phleboviruses exploit signal
peptidase for glycoprotein maturation. Whether additional processing of mature Gn and Gc by other host cell proteases is
required for viral entry remains to be determined.
Corresponding author:
Teresa Plegge
[email protected]
Emerging Viruses
P163
Inhibition of subtilisin kexin isoenzyme-1 (SKI-1) and signal peptide peptidase (SPP) interferes with Ebola virus
glycoprotein-driven host cell entry
T. Plegge1, M. González Hernández1, M. Spiegel1, S. Pöhlmann1
1
German Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
Introduction: Ebola virus (EBOV) and Lassa virus (LASV) can cause hemorrhagic fevers against which neither approved
treatments nor vaccines are available. EBOV and LASV harbor a single viral glycoprotein in their envelopes, EBOV-GP and
LASV-GPC respectively, which orchestrates virus entry into target cells. Both LASV-GPC and EBOV-GP are synthesized as
inactive precursors and depend on priming by a host cell protease to transit into an active form. LASV-GPC is processed by
subtilisin kexin isoenzyme-1 (SKI-1), an enzyme known to be required for cholesterol synthesis and sorting of lysosomal
proteins. EBOV can be primed by the cysteine proteases cathepsin B/L upon uptake of virions into host cell endosomes.
However, the proteases involved in EBOV-GP priming are not fully understood.
Objective: Here, we analyzed whether SKI-1 and signal peptide peptidase (SPP), an enzyme required for processing of
bunyavirus glycoproteins, are involved in EBOV-GP and LASV-GPC priming.
Material & Methods: We employed rhabdoviral vectors to analyze LASV-GPC- and EBOV-GP-driven entry and its inhibition
by the SKI-1 inhibitor PF-429242 and the SPP inhibitor (Z-LL)2 ketone.
Results: The incubation of cells producing LASV-GPC-bearing VSV particles with SKI-1 inhibitor markedly reduced particle
infectivity, as expected, while the effect on infectivity of EBOV-GP-bearing particles was modest. When the inhibitor was
added to target cells before addition of VSV particles, entry driven by both LASV-GPC and EBOV-GP was efficiently
reduced. Blockade of LASV-GPC-dependent entry was rescued by addition of cholesterol, indicating that interference with
cholesterol metabolism accounted for the blockade of LASV-GPC-driven entry by SKI-1 inhibitor. In contrast, no rescue was
observed for EBOV-GP. Finally, the SPP inhibitor blocked EBOV-GP- but not LASV-GPC-dependent entry, and time-ofaddition experiments revealed that entry was inhibited at an early stage.
Conclusion: Our results suggest that EBOV-GP maturation in infected cells is independent of SKI-1 and SPP activity, while
EBOV-GP-driven host cell entry depends at least partially on these proteases. Further studies are required to elucidate
whether they directly act on EBOV-GP or process cellular proteins required for EBOV entry.
Corresponding author:
Teresa Plegge
[email protected]
Emerging Viruses
P164
Ebola virus glycoprotein requires an intact receptor binding domain for tetherin antagonism
C. Brinkmann1, I. Nehlmeier1, K. Walendy-Gnirß1, J. Nehls2, M. González Hernández1, M. Hoffmann1, X. Qiu3, A. Takada4, S.
Pöhlmann1
1German
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
3Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Canada
4Hokkaido University Research Center for Zoonosis Control, Division of Global Epidemiology, Sapporo, Japan
2University
Introduction: The interferon-inducible antiviral host cell factor tetherin can block the release of many enveloped viruses from
infected cells. However, several viruses evolved proteins that remove tetherin from the site of viral budding and thereby
ensure efficient viral spread in tetherin-positive cells. The glycoprotein (GP) of Ebola virus (EBOV), a highly pathogenic
filovirus, mediates host cell entry and counteracts tetherin by a so far poorly understood mechanism. Moreover, it is unclear
whether tetherin counteraction is conserved between diverse filovirus GPs.
Objectives: We aimed to clarify whether tetherin antagonism is conversed between diverse filovirus GPs and to map
determinants in EBOV-GP that control tetherin antagonism.
Material & Methods: Virus-like particle assays based on HIV-1 Gag and EBOV VP40 were used to examine blockade of
particle release by tetherin and tetherin antagonism by GP. Mutagenic analysis of GP was performed to identify domains and
single amino acid residues required for tetherin counteraction.
Results: We show that besides ebolavirus and marburgvirus GPs also the GP of Lloviu virus, the only member of the genus
cuevavirus with the family filoviridae, counteracts tetherin, indicating that tetherin antagonism is a conserved feature of
filovirus GPs. Moreover, we present evidence that adequate processing of N-linked glycans is a prerequisite for tetherin
counteraction by EBOV-GP but not Vpu. Moreover, mutagenic analysis revealed that the mucin-like domain and the furin
cleavage site in EBOV-GP were largely dispensable while an intact receptor binding domain (RBD) was required for tetherin
counteraction. Finally, we identified a GP-specific antibody that interfered with GP-dependent tetherin antagonism,
suggesting that tetherin counteraction might occur at the cell surface.
Conclusion: Our findings show that tetherin counteraction is conserved between filovirus GPs, requires an intact RBD and
can be blocked by antibodies. It will be interesting to determine whether antibodies from EBOV infected patients interfere
with GP-dependent tetherin antagonism.
Corresponding author:
Stefan Pöhlmann
[email protected]
Emerging Viruses
P165
Characterization of Ebola virus neutralizing antibodies from human survivors of the 2014-2016 West African Ebola
epidemic
C. Hahn1, S. K. Fehling1, V. Krähling1, R. Thom2, Y. Hall2, O. Dolnik1, M. W. Carroll2, S. Becker1, T. Strecker1
1Philipps
2Public
University Marburg, Institute of Virology, Marburg, Germany
Health England, Salisbury, United Kingdom
Introduction: Passive immunotherapy with high-titer immune sera has been used successfully for treatment or prevention of
various bacterial and viral infections. Convalescent serum and plasma from patients who have recovered from Ebola virus
disease (EVD) is considered as a promising therapeutic strategy to treat acutely infected Ebola patients. Therefore, it is
important to assess the levels of Ebola virus (EBOV)-specific antibody titers and neutralizing antibody activities in individual
convalescent samples.
Objectives: We assessed the levels of total anti-EBOV IgG and neutralizing antibody response of the plasma of 120 EVD
survivors from Guinea using whole-virion ELISA and a Zaire EBOV neutralization assay, which have been used in recent
EBOV vaccine studies. Furthermore, we tested the suitability of a recombinant replication-competent VSV expressing EBOV
glycoprotein GP (rVSVΔG-EBOV-GP) to detect neutralizing antibody activities to EBOV in BSL-2 containments.
Methods: With Ethical approval from the Guinean authorities we collected blood samples from 120 Ebola survivors. Samples
were taken from volunteers 3 to 14 months following recovery from EVD. Each convalescent sample was analyzed by wholevirion ELISA and virus neutralization assay using either infectious EBOV or rVSVΔG-EBOV-GP.
Results: Neutralizing EBOV-specific antibodies were detected and remained stable over the sampling time-frame. There
was a significant range in neutralization titer, <4 to >1000, which was not related to time since recovery in the 1 year range
that we assessed. Furthermore, even after 14 months post recovery there does not appear to be a significant reduction in
neutralization titer. Comparison of the neutralization antibody activity revealed a good correlation of test results between the
two neutralization assays as well as with the whole-virion ELISA.
Conclusion: Plasma can be obtained from convalescent patients for therapy for a long period after recovery. However, prescreening is essential to select sera with a high titer. Our described rVSVΔG-EBOV-GP surrogate assay has potential
applications in vaccine and convalescent plasma therapy studies.
Corresponding author:
Thomas Strecker
[email protected]
Trafficking
P166
Cell-to-cell spread of dengue viral RNA via C189-bound vacuoles between mosquito cells
C. C. Cheng1, W. J. Chen1, C. F. Yang1
1Chang
Gung University, Graduate Institute of Biomedical Sciences, Taoyuan City, Taiwan
Question: Dengue virus is a mosquito-borne virus transmitted by Aedes aegypti and Aedes albopictus. As a result, the virus
must replicate in mosquito cells and disseminate within the infected mosquito. We have previously observed the dengue
virus may infect neighboring cells through C189 (a tetraspanin)-enriched membrane-bound vacuoles (C189-VCs) in a mode
of cell-to-cell transmission. It remains unclear whether the virus may directly transfer viral RNA from one cell to the other or
not, making it more efficient in virus spread between cells.
Methods: Sucrose gradient centrifugation was used to collect fractions containing C189-VCs which were referred for RTPCR and Western blot to detect viral RNA and proteins. Further, C189-VCs with HA-tag were isolated from the lysate of
transfected cells via immunoisolation with specific magnetic beads. In order to observe intercellular trafficking of C189-VCs, a
co-culture of infected and uninfected cells (transfected with a GFP-expressing vector) was also applied to detect the
existence of viral negative-sense RNA from the recipient cells.
Results: In the present study, we revealed that C189-VCs fractions collected from sucrose gradient centrifugation are
abundant of viral RNA, both positive and negative sense. Moreover, viral RNA was clearly shown in C189-VCs harvested by
immunoisolation with specific magnetic beads. It suggested that C189-VCs actually carry free negative-sense viral RNAs
which can be transmitted to neighboring cells. In the study using co-culture assay with or without 20mM NH4Cl treatment (for
inhibiting virus replication in cells), C189-VCs were also observed to play a role of viral RNAs transfer from one cell to the
other.
Conclusion: The result of this study demonstrated that Dengue virus and its RNA may spread between cells via C189-VCs
as a vehicle. This phenomenon provides insight to elucidate how ingested dengue virus can disseminate so efficiently in the
mosquito.
Corresponding author:
Chih-Chieh Cheng
[email protected]
Trafficking
P167
The intra-cellular cholesterol transport inhibitor U18666A blocks the release of HCV particles
F. Elgner1, R. Medvedev1, H. Ren1, D. Ploen1, K. Boller1, E. Hildt1,2
1Paul
Ehrlich Institute, Virology, Langen, Germany
Center for Infection Research (DZIF), Braunschweig, Germany
2German
Hepatitis C virus (HCV) particles are described as lipoviroparticles (LVPs) which are released by infected cells similar to
VLDLs. However, the mechanism of both VLDL and HCV LVP secretion is still poorly understood. Classical secretion via the
canonical ERGIC pathway as well as endosome-dependent secretion have been proposed in the context of HCV. Recently,
at least in vitro, also the role of exosomes in the transmission of HCV has been reported. The relevance of the endosomal
and autophagosomal pathways for the HCV particle secretion was highlighted in previous studies of our group in which we
revealed the cellular proteins Syntaxin-17, Syntaxin-4, α-Taxilin and TIP47 to be involved in this process. The aim of this
study was to further investigate the role of the endosomal and autophagosomal pathways in HCV LVP secretion.
For this purpose, several full-length HCV constructs coding for mCherry- and EGFP-labeled viral proteins were generated
and characterized. These constructs are able to replicate in transfected cells and assemble viral particles which are properly
released. The fusion proteins are synthesized, and fluorescence is detectable. These constructs can be used as a tool to
investigate the life cycle and the release pathway of HCV.
To further investigate the relevance of late endosomes/MVBs for the viral morphogenesis, we used the MVB inhibitor
U18666A in a protocol which does not inhibit the viral replication. We observed a significant inhibition of the HCV LVP
release in both Huh7.5 cells and primary human hepatocytes. Intact particles accumulate intracellularly together with lipids
and apoE in large structures, positive for the markers LAMP2, p62, CD63 and Rab7 which were identified as lipid-rich
multilamellar bodies by TEM. Moreover, the density of the retained LVPs is comparable with the density of releasecompetent LVPs; we aim at highlighting the post-assembly step. The accumulation of HCV particles in endosomal structures
was confirmed by subcellular fractionation. The overexpression of transdominant negative mutants of ESCRT proteins lead
to a comparable phenotype, highlighting the relevance of the endosomal pathway in the release of HCV.
This study helps to understand the mechanism of how HCV LVPs are MVB-dependently released and characterizes the
action of the MVB inhibitor U18666A in more detail.
Corresponding author:
Fabian Elgner
[email protected]
Trafficking
P168
Murine cytomegalovirus recruits the retromer complex to promote NEMO degradation by autophagy
E. Muscolino1, R. Brost1, M. Rizzato1, E. Krause1, S. Loroch2, V. Juranic Lisnic3, W. Brune1
1Heinrich
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
- Institute of Analytical Sciences, Dortmund, Germany
3University of Rijeka, Histology and Embryology/ Center for Proteomics, Rijeka, Croatia
2ISAS
Question: Autophagy is a key homeostatic process in eukaryotic cells that results in lysosomal degradation and recycling of
unnecessary or dysfunctional cellular components. Viruses can misuse autophagy for their own benefit. We have previously
shown that the murine cytomegalovirus (MCMV) protein M45 blocks proinflammatory NF-κB signalling by interacting with the
regulatory subunit of the IKK complex, NEMO, and redirecting it to autophagosomes. However, the molecular mechanism of
autophagy induction and selective NEMO degradation by M45 remained to be determined.
Methods: M45 interacting proteins were identified by affinity purification and mass spectrometry and verified by coimmunoprecipitation. NEMO and VPS26B expression and localization upon MCMV infection were analysed respectively by
immunoblotting and immunofluorescence. TBC1D5 knockout cells were obtained by CRISPR/Cas9 mutagenesis.
Results: Here we show that NEMO degradation occurs in two steps. First, M45 interacting with NEMO causes its
accumulation in the detergent insoluble fraction, most likely as misfolded protein aggregates. A short region with the Cterminal part of M45 was identified as responsible for this process. In the second step, insoluble NEMO is targeted to
autophagosomes for degradation in lysosomes. In a SILAC screen for M45 interaction partners we identified three
components of the retromer complex. This complex is part of the endosomal protein sorting machinery and can associate
with late endosomes and autophagosomes. A strong interaction between M45 and the retromer component Vps26B was
confirmed by co-immunoprecipitation. Similar to NEMO, Vps26B is directed to autophagosomes and degraded in an M45dependent manner during viral infection. We further show that Vps26B colocalizes with NEMO in MCMV-infected cells and
that M45-induced NEMO degradation is inhibited in Vps26B-deficient cells. As it is known that Vps26B can interact with the
key autophagy marker LC3-II via the adaptor protein TBC1D5, we tested whether TBC1D5 is involved in M45-mediated
NEMO degradation. Indeed, NEMO degradation was strongly reduced in MCMV-infected TBC1D5 knockout cells.
Conclusion: Collectively these data suggest that M45 recruits the retromer component Vps26B to promote the degradation
of insoluble NEMO aggregates via the adaptor protein TBC1D5.
Corresponding author:
Elena Muscolino
[email protected]
Trafficking
P169
Intracellular transport of the Marburg virus glycoprotein
S. Halwe1, S. Becker1, O. Dolnik1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Marburg virus (MARV), belonging to the family Filoviridae, is the causative agent of severe hemorrhagic fevers with lethality
rates of up to 90 %. The matrix protein VP40 and the glycoprotein GP represent the major components of MARV envelope
whose formation takes place at the plasma membrane. Regarding delivery of both proteins to the cell surface the current
model delineates trafficking of MARV VP40 via endosomal compartments and transport of MARV GP via the classical
secretory pathway involving endoplasmic reticulum, Golgi apparatus and trans-Golgi-network. Recombinant expression of
both proteins, GP and VP40, leads to an accumulation of GP in VP40-rich clusters in the cell periphery which are supposed
to act as budding-sites for filamentous VLPs. However, neither the exact transport pathways nor potential host factors
regulating trafficking of GP and VP40 to these clusters are known.
In this study, we show by confocal immunofluorescence microscopy that in non-polarized HuH7 cells, MARV GP co-localizes
with marker proteins of the recycling endosome (RE) including Rab8, Rab11, Rab13, transferrin receptor and the v-SNARE
VAMP3 in the perinuclear area as well as in small vesicles. Moreover, transferrin uptake assays revealed a high degree of
co-localization between GP and fluorescently labeled transferrin at 30 minutes after uptake suggesting an intersection of
trafficking pathways used by newly synthesized GP and endocytosed transferrin in RE. Furthermore, overexpression of a
cytoplasmic VAMP3 mutant, which is supposed to inhibit VAMP3-mediated fusion events, leads to a significant reduction of
GP surface expression. Upon co-expression of GP and VP40, RE-associated cellular factors, but not those localizing to
early/late endosomes, accumulate together with GP in VP40-rich clusters at the plasma membrane.
Taken together, these results suggest that MARV GP traverses the RE before reaching the plasma membrane. Furthermore,
peripheral MARV VP40 clusters seem to be connected to RE-associated transport pathways and act as target destination for
MARV GP.
Corresponding author:
Sandro Halwe
[email protected]
Trafficking
P170
Composition of a transport of p8 complex (TOPC) to understand transfer of the HTLV-1 p8 protein to target cells
N. Donhauser1, S. Millen1, M. Jeninga1, E. Socher2, J. Hofmann3, H. Sticht2, A. K. Thoma-Kress1
1Friedrich
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
Alexander University Erlangen Nuremberg, Division of Bioinformatics, Institute of Biochemistry, Erlangen, Germany
3Friedrich Alexander University Erlangen Nuremberg, Division of Biochemistry, Department of Biology, Erlangen, Germany
2Friedrich
The Human T-cell leukemia virus type 1 (HTLV-1)-encoded accessory protein p8 induces cellular conduits, which are thought
to facilitate transfer of p8 to target cells and virus transmission. Using bioinformatics, we previously identified vasodilatorstimulated phosphoprotein (VASP) as a novel interaction partner of p8, which is crucial for transfer of p8 to target T-cells.
VASP prevents elongating actin-filaments from capping, thereby promoting filament elongation, but VASP is not an active
motor protein. In this study, we asked whether additional host factors interact with p8 and p8:VASP complexes and whether
a transport of p8-complex (TOPC) is composed. Bioinformatics predicted putative interactions of p8 with Wiscott-Aldrich
syndrome protein (WASP) and with the related Neuronal WASP (N-WASP). [ak1] Both proteins are involved in initiating the
growth of actin-filaments. Co-immunoprecipitations confirmed that p8 interacts with WASP, but not with N-WASP. Since
WASP:VASP complexes have been described in lymphocytes and the interaction of p8 and WASP was more and more
diminished in the presence of increasing amounts of the detergent N-Octylglucoside, our data suggest an indirect interaction
of p8 and WASP via VASP. Co-immunoprecipitations confirmed the existence of a WASP:VASP:p8 complex in Jurkat Tcells. Use of liquid chromatography–mass spectrometry (LC-MS/MS) upon precipitation of p8 in 293T cells revealed that a
motor protein, Myosin-X, is an additional interaction partner of p8. Co-immunoprecipitations and immunofluorescence
analysis confirmed a p8:Myosin-X interaction. Strikingly, Myosin-X is known to promote formation of filopodia by transport of
cargo like VASP to the tips of filopodia. Since we observed a rapid transfer of GFP-labelled Myosin-X and of p8 to cocultured target T-cells, our current working model proposes that p8 hijacks the Myosin-X:VASP complex to be transferred to
target T-cells. Taken together, our work provides novel insights into the molecular mechanisms of p8 transfer to target Tcells.
Corresponding author:
Andrea K. Thoma-Kress
[email protected]
Virus Vectors and Gene Therapy
P171
Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells
Z. Dong1, M. Pan1
1Southwest
University, State Key Laboratory of Silkworm Genome Biology, Chongqing, China
Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent,
novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly
developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9)
technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the
CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as
a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus
effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which
minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing
the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible
CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future.
Corresponding author:
ZhanQi Dong
[email protected]
Virus Vectors and Gene Therapy
P172
Specific and stable repression of HIV provirus expression in vitro by chimeric DNA methyltransferase
H. Zhu1, J. Liu1
1State
Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China
HIV-1 provirus cannot be eradicated completely by current treatment. Thus, innovative strategy has been proposed to silence
the HIV virus permanently and stay in peace with them. Targeted DNA methylation provides potential applications in gene
silencing. Here, we describe a chimeric DNA methyltransferase (ZF-Dnmt1) composed of designed zinc-finger proteins and
DNA methyltransferase Dnmt1. We show that transient transfection of this chimeric protein lead to efficient repression of
HIV-1 provirus expression through increasing the methylation of CpG islands around 5-LTR in HIV-1 infected and latently
infected cells. Furthermore, the repression of HIV-1 expression caused by ZF2-Dnmt1 is stable and heritable over cell
generations. Besides, no apparent toxicity of ZF2-Dnm1 is found on cell proliferation or cell cycle progression. Taken
together, our results demonstrate the potential of this novel approach for anti-HIV-1 therapy.
Corresponding author:
Huanzhang Zhu
[email protected]
Figure 1
Figure 2
Virus Vectors and Gene Therapy
P173
Development of novel cornaviral vector
A. Naskalska1, A. Dąbrowska2, P. Nowak2, K. Pyrć1,2
1Jagiellonian
2Jagiellonian
University, Malopolska Center of Biotechnology, Krakow, Poland
University, Biochemistry, Biophysics and Biotechnology, Krakow, Poland
Background: Virus like particles (VLPs) are structures composed of viral proteins but devoid of genetic material that
efficiently transduce multiple human cells. Possibility of biotechnological modification of VLPs, such as encapsidation of
heterologous moieties or introducing foreign epitopes on their surface, renders VLPs versatile delivery platforms or tools of
immunization.
The aim of this project was to obtain VLP from human coronavirus NL63 (HCoV–NL63) - an enveloped positive stranded
RNA coronavirus causing mild respiratory infections. The advantage of HCoV-NL63 based vector is the narrow tropism of the
parental virus which infects only ciliated cells of human airway epithelium resulting in high specificity of potential VLPs.
Methods & Results: First, HCoV-NL63 VLPs were produced using baculovirus-insect cells expression system. Electron
microscopy imaging confirmed successful assembly of virus like structures. Subsequently, several modifications of proteins
forming VLPs were introduced, such as tagging or incorporating heterologous proteins, in order to investigate their plasticity.
Florescent microscopy examination showed that HA-tagged but not GFP-tagged HCoV-NL63 VLPs are assembly competent.
Furthermore, it was found that VLPs support substitution of HCoV-NL63 membrane protein by influenza matrix 1 protein.
Next, HCoV-NL63 VLPs purification method was optimized and the obtained material was assessed by Dynamic Light
Scattering technique. Finally, cells naturally permissive to HCoV-NL63 infection were incubated with obtained VLPs and
Laser Scanning Confocal Microscopy analysis showed that HCoV-NL63 VLPs are functional in terms of cell transducing
capacities[Fig.1].
Conclusion: In conclusion, different variants of functional, non-replicative and tissue specific HCoV-NL63 based vector were
obtained and characterized. Further investigation will asses HCoV- NL63 VLP mediated transferring of therapeutics to
human alveolar eptihelium (in vitro model) and immune response eliction (in vivo model).
Figure 1: HCoV-NL63 VLPs transduction in LLC-Mk2 cells. Left: cells incubated with VLPs, right: control cells. VLPs stained
in green, actin in red, nuclei in blue.
Corresponding author:
Antonina Naskalska
[email protected]
Figure 1
Virus Vectors and Gene Therapy
P174
Generation of recombinant Newcastle disease virus expressing surface glycoproteins of Peste des petits ruminants
virus
M. Murr1, B. Hoffmann2, T. C. Mettenleiter1, A. Römer-Oberdörfer1
1Friedrich
2Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
Loeffler Institute, Institute of Diagnostic Virology, Greifswald - Isle of Riems, Germany
Peste des petits ruminants (PPR) is a highly contagious disease in small ruminants as sheep and goats caused by Peste des
petits ruminants virus (PPRV), a member of the genus Morbillivirus in the family Paramyxoviridae. PPR becomes more
important because of the eradication of rinderpest and the abolition of vaccination against it, which conveyed a cross
protection against PPRV.
The surface glycoproteins hemagglutinin (H) and fusion protein (F) are considered to be the most important viral proteins to
induce a protective immune response in the host since neutralizing antibodies generated during infection are directed against
H and F.
In order to differentiate between infected and vaccinated animals, recombinant marker vaccines (DIVA vaccines) are useful.
Therefore, we generated two recombinant Newcastle disease viruses (NDV) by reverse genetics expressing either the PPRV
H or F protein. The vector backbone is derived from the lentogenic NDV strain Clone 30. The genes coding for PPRV strain
Kurdistan H and F protein were inserted into the intergenic region between the NDV F and HN encoding open reading
frames, resulting in rNDVGuPPRVFGK and rNDVGuPPRVHGK. Both viruses replicate well in embryonated specific
pathogen free (SPF) chicken eggs. Furthermore, productive replication in two cell lines of goats and sheeps was detected,
indicating a possible suitability for a live vaccine virus in the host animals. Expression of PPRV F and H was detected by
western blot analyses. However, a further enhancement of the expression is desirable and will be tried by manipulation of the
viral genome. A quantitative PCR to detect the viral genome was established and can be used for analyses of animal
experiments in the future. Since PPR is present in Africa, Asia and the Middle East, thermostability of a vaccine virus is an
important feature and was determined for generated vectored viruses with 37 °C for 15 days, and 42 °C for four days.
Corresponding author:
Magdalena Murr
[email protected]
Virus Vectors and Gene Therapy
P175
Expression of viral immunogens by novel pestivirus replicons
A. Dalmann1, I. Reimann1, M. Beer1
1Friedrich
LoefflereInstitute, Greifswald - Isle of Riems, Germany
Replicons, autonomously replicating defective RNA genomes that are not able to generate infectious particles, are useful
tools for the expression of foreign antigens. This could be used in vitro, but also in vivo. Emerging infectious diseases are
e.g. occurring at an increased frequency worldwide, and further expression technologies might be useful to respond rapidly
to emerging viruses.
The main objective of our study was to develop novel pestivirus replicons as an expression system. Here, the atypical
pestivirus Bungowannah virus (BV) is used as backbone due to its broad in vitro host range, which is unique for pestiviruses.
Coding sequences, non-essential for RNA replication, were removed from these replicons, creating up to 5 kb of space for
insertion of heterologous sequences.
In our study, we describe the construction and characterization of BV replicons with single or multiple deletions within the
structural proteins, and the nonstructural proteins Npro, p7 and NS2. We could show, that BV replicons are able to efficiently
replicate in vitro, and nanoluciferase (Nluc) expressing constructs were generated to compare the replication efficiency of the
different BV replicons. Our data also show, that constructs with a complete deletion of the genome region encoding the
structural proteins C, Erns E1 and E2 (BV_∆C-E2) are the most efficient replicons. Interestingly, the construct BV_∆Erns
with a partial deletion within the envelope protein Erns replicated on a lower level, but a first cycle of replicating virus progeny
was generated which could not be further passaged (defective in second cycle, DISC), allowing protein expression for more
than one cycle. As a first proof of principle, we generated e.g. the replicon BV_∆Erns2xGcN, which allowed the expression of
an immunogenic domain of Schmallenberg virus glycoprotein Gc (GcN). Further studies for in vitro and in vivo
characterization were started.
In conclusion, a novel Bungowannah virus based replicon system with unique features was established and tested.
Corresponding author:
Anja Dalmann
[email protected]
Virus Vectors and Gene Therapy
P176
Fibrils that enhance virus infection interact with cell membrane protrusions
S. Rode1, M. Hayn1, A. Röcker1, S. Sieste2,3, C. Meier2, D. Markx4, F. Kirchhoff1,5, P. Walther6, M. Fändrich4, T. Weil2,3,5, J.
Münch1,5
1Ulm
University Medical Center, Institute of Molecular Virology, Ulm, Germany
University, Institute of Organic Chemistry III, Ulm, Germany
3Max Planck Institute of Polymer Research, Mainz, Germany
4Ulm University, Institute of Protein Biochemistry, Ulm, Germany
5Ulm University, Ulm-Peptide Pharmaceuticals, Ulm, Germany
6Ulm University, Central Facility for Electron Microscopy, Ulm, Germany
2Ulm
Introduction: Positively charged peptide nanofibrils (PNF) increase HIV-1 infection and lentiviral vector transduction. The
best characterized naturally occurring PNF is SEVI (semen-derived enhancer of viral infection) present in semen. The best
studied engineered PNF derived from 12mer peptide EF-C. The mechanism underlying transduction enhancement depends
on the polycationic surface of the PNF which binds to the negatively charged membranes of viruses and cells thereby
increasing virion attachment and fusion. However, the exact mechanism e.g. how fibril/virus complexes interact with the cell
surface is unclear.
Objectives: To analyze interactions of SEVI and EF-C PNF with the cell surface.
Material & Methods: We performed electron microscopy of cells exposed to SEVI and EF-C PNF. To study kinetics of PNF
binding to cells we generated and characterized PNF covalently labelled with two fluorescent ATTO dyes using fluorescence
spectroscopy, Zeta potential measurements, confocal microscopy and flow cytometry.
Results: We found that both fibrils were not homogenously distributed at the cell membrane. SEVI and EF-C PNF
specifically interacted with cellular protrusions that seem to capture the fibers while a significant fraction of the fibrils was still
detectable at the cell surface after 3 days. This suggests that EF-C PNF are not completely endocytosed and may remain
associated with the cell membrane. Moreover, we found that PNF induced invaginations of cytoplasm membrane. For life cell
imaging, we covalently coupled EF-C peptide with fluorescent labels. Minute amounts of labelled peptide were mixed with an
excess amount of native peptide. Fibrils with morphological similarity to parenteral EF-C PNF were formed. These PNF were
fluorescent and enhanced retroviral infection as efficiently as unlabeled fibrils. Compared to classical stains of amyloid fibrils
(e.g. Thioflavin T), covalently labeled PNF were more stable and allowed a more sensitive detection in cell culture.
Conclusion: PNF that increase viral infection preferentially interact with cellular protrusions and induce invagination of the
cytoplasma membrane. The newly generated covalently labelled EF-C PNF will allow life cell imaging for closer analysis the
underlying mechanisms.
Corresponding author:
Sascha Rode
[email protected]
Virus Vectors and Gene Therapy
P177
LCMV-GP pseudotyped oncolytic vesicular Stomatitis virus for the treatment of solid tumors
L. M. Schreiber1, C. Rodriguez Urbiola1, J. Kimpel1, P. Erlmann1, H. Zwierzina2, D. von Laer1, G. Wollmann1
1Medical
University of Innsbruck, Division of Virology, Innsbruck, Austria
Cancer Research Institute, Translational Cancer Research, Innsbruck, Austria
2Tyrolean
Lung and prostate cancer account for 35% of new cancer cases and deaths in men each year. Lung cancer alone claims
26% of cancer deaths in women each year. Oncolytic viruses (OV) that preferentially replicate in and kill tumor cells are a
potent novel treatment option for cancer patients after failure of standard therapies. Through cell lysis, OV set free tumor
antigens, which in combination with the OV adjuvant effect, unleashes a strong anti-tumor immune response. Our group
previously reported that oncolytic vesicular stomatitis virus (VSV) pseudotyped with the LCMV glycoprotein (VSV-GP) is a
promising, highly efficient and safe oncolytic virus. Here, we propose the use of the oncolytic VSV-GP for the treatment of
lung and prostate cancer.
We analyzed oncolytic efficiency of VSV-GP and IFN-I induced antiviral response in a panel of cell lines. VSV-GP was further
tested in vivo in diverse xengoraft and syngeneic mouse models and in different metastasis models.
VSV-GP exhibited high oncolytic efficiency in vitro, efficiently killing the majority of lung and prostate cancer cell lines tested.
However, IFN-I response was active in some cell lines, which might lead to shortcomings in virotherapeutic treatment. In
vivo, VSV-GP showed therapeutic effect, which ranged from tumor growth delay to complete tumor regression in diverse lung
and prostate cancer subcutaneous (s.c.) xenograft models. VSV-GP was able to delay tumor growth and increase survival
rate also in an s.c. syngeneic model for PCa. IFN-I receptor (IFNAR) knock-out cells were generated and used to investigate
the influence of IFN-I signaling in the therapeutic outcome. In IFNAR-/- tumors, virus replication increased significantly,
though therapeutic outcome improved only marginally, suggesting that other factors may be involved in resistance to VSVGP therapy. Furthermore, we could show tumor-to-tumor spread of VSV-GP and tumor remission of bone metastasis of PCa
when administered systemically.
VSV-GP is a novel and safe therapeutic for the treatment of solid tumors. We have shown successful treatment of both local
and metastatic tumors in mouse models. Further study of the interaction of the triad VSV-GP – immune system – tumor is
essential to identify resistance factors and improve therapeutic outcome.
Corresponding author:
Liesa-Marie Schreiber
[email protected]
Structure and Assembly
P178
Influence of a newly identified late domain motif in the Ebola virus matrix protein on particle release
L. Wendt1,2, L. Kämper2, M. L. Schmidt2, T. Hoenen2
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
2Friedrich
Ebola virus (EBOV), which belongs to the family of Filoviridae, causes severe hemorrhagic fevers in humans. New infectious
EBOV particles are released by budding from the host cell; a process which is driven by the viral matrix protein VP40. To this
end VP40 encodes two overlapping late domain motifs at amino acid position 7 to 13 (PTAPPEY), which are also crucial for
the formation of virus-like particles (VLPs). However, neither of these late domain motifs is absolutely essential for viral
replication in cell culture, indicating that alternative budding mechanisms must also exist. Recently, a potential third late
domain motif (YPx(6)I) was identified at amino acid position 18 to 26 of VP40. To analyze its importance in viral budding and
Ebola virus particle infectivity, we used a transcription and replication-competent virus-like particle (trVLP) system. This
system is based on a tetracistronic minigenome that encodes a reporter, as well as VP40, the EBOV nucleocapsidassociated protein VP24, and the viral glycoprotein GP1,2. Coexpression of this minigenome together with the viral proteins
driving genome replication and transcription leads to the production of these minigenome-encoded proteins, and thus the
formation of trVLPs, which incorporate minigenomes and can infect target cells. The advantage of this system is that the
minigenome-encoded viral proteins are expressed in a regulated fashion rather than overexpressed. Thus, the study of viral
morphogenesis, budding, and entry can be modeled in a more authentic way compared to conventional VLP assays. Using
this system we show that the new potential late domain motif (YPx(6)I) does not appear to contribute to trVLP infectivity and
budding. This finding has significance for our understanding of the budding process of EBOV, and also for the identification
and prioritization of molecular targets for the development of novel therapeutics against these viruses.
Corresponding author:
Lisa Wendt
[email protected]
Structure and Assembly
P179
African henipavirus G protein contains a functional endocytosis motif
L. Behner1, A. Maisner1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
Introduction: Though henipavirus-related virus sequences were often found in African bats, live virus could not be isolated
so far. Consequently, the potential of these African henipaviruses to replicate and spread in humans or livestock cannot be
explored by infection studies, but can only be evaluated by studying the biological activity of individual viral genes. Since
efficient binding of henipavirus G proteins to cellular ephrin receptors and functional support of fusion-competent F proteins
are crucial prerequisites for productive henipavirus replication in any host cell, we compared the biological activities of two
surface glycoproteins G and F of the prototype African bat henipavirus GH-M74a (GH) with those of human pathogenic
Nipah virus (NiV). The failure of GH glycoproteins to induce syncytia formation in standard henipavirus-permissive cells was
mainly due to the fusion-defective GH-F protein. In contrast to F, GH-G was shown to be functional, as it was able to support
some limited cell-cell fusion upon co-expression with NiV-F. This revealed that GH-G possesses fusion-helper function,
though to a more limited extent compared to fully functional NiV-G.
Objectives: Aim of this investigation was to evaluate the functional importance of a potential endocytosis motif in the
cytoplasmic tail of GH-G for its surface expression and fusion-helper function.
Material & Methods: GH-G wildtype and mutant proteins were transiently expressed and characterized regarding total and
surface expression (western blots, surface biotinylation assay), endocytosis (antibody uptake and biotin endocytosis assay)
and fusion-helper activity (heterotypic fusion assay with NiV-F).
Results: When we mutated the YFGL endocytosis motif in the GH-G cytoplasmic tail by exchanging the critical Y to A, we
found that the G protein was no longer internalized. Although, the surface expression of the mutant was slightly increased,
fusion-helper function upon co-expression with NiV-F was not improved.
Conclusion: We revealed that GH-G possess a functional cytoplasmic endocytosis motif that downregulated its surface
expression to some extent. Although an endocytosis-negative mutant showed a slightly enhanced surface expression, the
limited fusion-helper activity of the prototypic African henipavirus G protein was not increased.
Corresponding author:
Laura Behner
[email protected]
Structure and Assembly
P180
Characterization of interaction domains in the large tegument protein pUL36 of herpes simplex virus
D. Otoo1, A. Pholman1, L. Ivanova1, K. Döhner1, T. Krey1, B. Sodeik1
1Hanover
Medical School, Institute of Virology, Hanover, Germany
Introduction: The open reading frame UL36 of herpes simplex virus (HSV) encodes for the large inner tegument protein
pUL36 of 3164 amino acid that is conserved across all herpesviruses. HSV-1-pUL36 and its homologs serve as multivalent
cross‐linkers between the capsid and the tegument.
Objective: Our aim was to analyze the capsid binding potential of several fragments of the C-terminal third/half of HSV-1pUL36.
Methods: Based on secondary structure predictions, we aligned conserved regions of HSV-1-pUL36 with its corresponding
orthologues of 11 other herpesviruses across the 3 herpes subfamilies. We cloned the full-length pUL36 and 12 subfragments with a GFP tag at their C-terminus without intersecting any predicted alpha-helices or beta-sheets, and transfected
these constructs prior to infection with mCherry-tagged parental virus, HSV-1(17+)Lox-CheVP26, or a mutant lacking pUL36,
HSV-1(17+)Lox-CheVP26-ΔUL36.
Results & Conclusion: Using the confocal fluorescence microscopy, we observed that the full-length pUL36 and 2 of the 12
pUL36 sub-fragments colocalized with nuclear and cytoplasmic capsids of both the parental and the ΔUL36 mutant virus.
With the exception of the full-length pUL36, all 12 sub-fragments did not colocalise with organelles of secondary
envelopment. HSV-1(17+)Lox-CheVP26-ΔUL36 is impaired in capsid envelopment and cell egress, but upon transfection of
pUL36 full-length, there was virus spread to neighbouring cells. Elucidating the functions of pUL36 and defining its molecular
interaction interfaces with capsids may provide novel strategies for antiviral therapy.
Corresponding author:
Dorcas Otoo
[email protected]
Structure and Assembly
P181
Mapping of disulfide bonds in the small hepatitis B virus envelope protein S
N. Gerstenberg1, F. Zörndlein1, S. Suffner1, V. Bruss1
1Helmholtz
Centre Munich, Institute of Virology, Neuherberg, Germany
The 226 aa long small envelope protein S of the hepatitis B virus is synthezised as an integral membrane protein with 4
transmembrane regions (N and C terminus are ER luminal). It contains 14 cystein residues forming intra- and intermolecular
disulfide bridges between S chains. Expression of S in eucaryotic cells causes, like in infected hepatocytes, the secretion of
spherical subviral particles (SVP) with a diameter of 20 nm consisting of approximately 100 S chains and lipid.
We try to map disulfide bonds in S by characterizing a C-terminally HA-tagged S protein (S-HA) and cysteine to serine point
mutants of S-HA after transient transfection of Huh7 cells with respect to SVP formation (Western blot of cell culture lysates
and supernatants), cystin linked dimer formation (unreduced PAGE and Western blot), number of free –SH groups
(derivatization with 5 kDa methoxypolyethylene glycol maleimide and determination of the molecular weight of products), and
number of oxidized cystein residues.
Secreted SVP formed by wildtype (WT) S-HA consisted mainly of cystin-linked dimers, only low amounts of higher oligomers
were detected. Between 5 and 8 free –SH groups could be found in this material. The number of oxidized cysteines was
between 5 and 7. Blocking free –SH groups with N-ethylmaleimid (NEM) drastically reduced the HBsAg reactivity of this
material.
A mutant (C107) containing only 5 cysteine residues (all 4 cysteines in the cytoplasmic loop between transmembrane region
(TM) 1 and 2 which are absolutely required for SVP formation as well as the luminal cysteine 107 shortly after TM2) was
competent for SVP secretion and formed mainly disulfide bridged dimers. Mal-PEG labeling showed one main oxidized
cysteine residue in C107. This finding is consistent with a model where cystein residue 107 forms a disulfide bridge with
cystein residue 107 of the dimeric partner.
Corresponding author:
Nadine Gerstenberg
[email protected]
Structure and Assembly
P182
Dimerization efficiency of the canine distemper virus matrix protein dictates membrane budding activity
F. Bringolf1, M. Herren1, J. P. Langedijk2, A. Zurbriggen1, P. Plattet1
1University
2Janssen
of Bern, Division of Experimental Clinical Research, Bern, Switzerland
Infectious Diseases and Vaccines, Leiden, Netherlands
Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane.
Although mechanistic details remain largely unknown, structural evidences strongly suggest that the budding process is
associated with the formation of an oligomeric M-lattice beneath the plasma membrane, with M-dimers as oligomeric units.
Aiming at providing functional data, we here investigated the role of the canine distemper virus (CDV) M-protein"s selfassembly and budding capacity. We first designed three alanine-scan block mutants (6A) locating within three different
strategic oligomeric positions according to a structural homology model. The first, 6A-SVFSAN138 stretches from the core of the
dimer interface at position 138 to the heart of the monomeric structure at position 133. The two others, 6A-ARPEEL90 and 6AIEKMGL246 locate within two distal α-helices at the surface of the dimeric structure, which are potentially involved in dimer-dimer
contacts. We additionally focused on the core of the dimeric interface by solely mutating the asparagine 138 (N 138) into
several non-conservative amino acids.
Cellular localization combined with dimerization and co-immunoprecipitation assays, performed under various denaturating
conditions, revealed that the self-assembly pattern of three 6A-mutants deeply differed from the one displayed by wt M. The
mutants were furthermore intracellular transport-incompetent, thereby preventing CDV nucleocapsid proteins" relocation to
the cell periphery as well as production of virus-like particles (VLPs). Conversely, all N138 M-mutants remained capable of
self-assembly, although to various extend, which correlated with proper accumulation and redistribution of nucleocapsid
proteins at the plasma membrane. However, membrane deformation and VLP assays unveiled different efficiencies in
inducing membrane remodeling and productive budding directly correlating with the dimerization or/and expression profiles.
Overall, our data provided mechanistic evidence that the efficiency of CDV M-dimerization/oligomerization governs the cell
periphery accumulation as well as the membrane budding activity.
Corresponding author:
Fanny Bringolf
[email protected]
Structure and Assembly
P183
Regulation of vesicle-mediated nuclear egress of herpesvirus capsids
B. Klupp1, T. Hellberg1, S. Rönfeldt1, W. Fuchs1, T. C. Mettenleiter1
1
Friedrich Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
Newly assembled herpesvirus capsids exit the nucleus by a vesicle mediated transport through the nuclear envelope. Vesicle
formation at the inner nuclear membrane (INM) is orchestrated by the viral nuclear egress complex (NEC) composed of the
conserved membrane-anchored pUL34 and its interaction partner pUL31. The recently solved crystal structures of NECs
from different herpesviruses revealed molecular details of this unique complex. Both proteins possess a globular fold with
pUL34 forming a pedestal for pUL31 at the INM. The pUL31 N-terminus forms a hook-like extension clamping the core of
pUL34.
Despite detailed structural data, how NEC assembly and disassembly is regulated remains enigmatic. A regulatory role was
attributed to the highly flexible N-terminus of pUL31 which was omitted for the crystal structures. For pseudorabies virus
(PrV), an alphaherpesvirus of pigs, this part comprises 25 amino acids (aa), including a bipartite nuclear localization signal
(NLS) with three stretches of basic residues. Complete deletion of this part (pUL31-∆NLS) resulted in a predominant
cytoplasmic localization attributed to a nuclear export signal (NES) present in the C-terminus. pUL31-∆NLS was
nonfunctional and exerted a dominant negative (DN) effect on PrV-wild type replication, which might be due to the
mislocalization of the complex partner pUL34.
To test for function of key residues involved in NEC assembly and disassembly, truncations as well as site-specific mutations
were introduced into PrV pUL31 and the resulting mutant proteins were tested for localization, vesicle formation after
coexpression with pUL34, and functional complementation of the PrV UL31 deletion mutant.
Our results show that half of the pUL31 N-terminus including the NLS is dispensable for function. A single stretch of basic
residues is sufficient to allow for nuclear egress. Mutation of the NES in pUL31-∆NLS abrogated the DN effect indicating that
the NES is functional and reroutes the complex from the INM. Putative phosphorylation sites in this N-terminal domain, which
are predicted to influence complex disassembly, seem not to play a role.
Thus, our data indicate a complex interplay between import and export signals in pUL31 regulating protein localization,
complex formation and disassembly during herpesvirus nuclear egress.
Corresponding author:
Barbara Klupp
[email protected]
Structure and Assembly
P184
Analyzing self-assembly of Ebola virus nucleoprotein with native mass spectrometry
J. Kopicki1, J. Heidemann1, C. Muñoz-Fontela2, C. Uetrecht1,3
1Heinrich
Pette Institute, Dynamics of Viral Structures, Hamburg, Germany
Pette Institute, Emerging Viruses, Hamburg, Germany
3European XFEL GmbH, Schenefeld, Germany
2Heinrich
Ebola virus (EBOV) forms helical filaments containing the nucleoprotein (NP) as essential component. NP 1-450 includes
domains for self-assembly and RNA-binding. Therein, a motif (NP313-321) is present that is very conserved among Filoviridae.
The study aims to identify the motifs function, its role in self-assembly, besides the effect of RNA and EBOV proteins on this
process. By creating NP313-321 mutants it can be determined if the motif is crucial for the assembly, and hence for infectivity.
The strong conservation among Filoviridae gives occasion to do comparative experiments to examine if the idea is applicable
to other viruses.
NP is expressed in E. coli. Proteins are purified by affinity chromatography and size-exclusion chromatography (SEC).
Expression and purification are evaluated by SDS-PAGE and Western Blot (WB). Native mass spectrometry (MS) is used to
identify NP monomers and assemblies, furthermore to run interaction studies with binding partners. Assemblies are observed
by electron microscopy (EM).
Purity of NP was verified by SDS-PAGE and WB. Presence of nucleic acids indicated by UV absorbance at 260 nm was
decreased by strong nuclease treatment linked with sonication. During SEC retention time of NP-containing fractions was
lower than expected, suggesting existence of oligomers with larger hydrodynamic radius than monomers. NP samples
viewed by EM showed occurrence of ring particles (Ø40 nm). Monomeric NP was detected by MS in buffer with low ionic
strength. Peaks representing a pentameric formation were dominant in a large range of salt concentrations.
Presence of nucleic acids was persistent during our experiments indicating binding of E. coli RNA. NP contains domains for
self-interactions that control the process of assembly. SEC and EM results show that a specific oligomer, a ring-like shape,
seems to be an intermediate structure during the formation of full NC. The stoichiometric composition of NP that was found to
be most prevalent in MS measurements was a pentamer. It was detected at physiological salt concentration, whereas
monomer only could be observed in buffers with very low ionic strength, indicating that lower salt concentrations prevent selfassembly, therefore the proteins function. Next, assembly and importance of the conserved residues will be studied in more
detail.
Corresponding author:
Janine Kopicki
[email protected]
Structure and Assembly
P185
Filamentous/chained morphology of enveloped virus particles – Defects of budding or electron microscopy
artefacts?
L. Kordyukova1, R. Mintaev1, S. Abramchuk2, S. Markushin3
1Lomonosov
Moscow State University, Belozersky Institute of Physico-Chemical Biology, Moscow, Russian Federation
Moscow State University, Chemical Dpt, Moscow, Russian Federation
3Mechnikov Institute of Vaccine and Sera, Moscow, Russian Federation
2Lomonosov
The enveloped virus virions morphology is for long considered to reflect the normal or disturbed virus budding. Recently
several papers have stressed the morphology of Influenza virus virions might change during the virus sample preparation for
the electron microscopy analysis. We now probed several protocols to concentrate the influenza virus virions from the
allantoic fluid to retain their native morphology before the "quick" electron microscopy analysis (negative staining with
phosphotungstic acid). Among them were the glutaric aldehyde fixation of the virions within the allantoic fluid before the
ultracentrifugation or the Amicon filtration of the cleared allantoic fluid (the ultracentrifugation step was skipped). As a control
the standard procedure of the virus purification through the 20% sucrose cushion during the ultracentrifugation was applied.
The A/WSN/33 (H1N1) recombinant influenza virus obtained by the reverse genetics technique was compared to the
A/WSN/33 (H1N1) laboratory virus strain. Unexpectedly, while the laboratory strain virus was mostly spherical or slightly
elongated (albeit up to 5% of virus particles were irregular) the recombinant virus preparation included up to 30% of
filamentous virions or long chains of spherical/elongated particles. Since the influenza virus morphology is mostly depends
on the matrix M1 protein structure we have sequenced the M gene in both preparations. There were two amino acid changes
detected in the recombinant virus M1 compared to the laboratory strain as well as the influenza virus database. These are
serine 126 to cysteine and isoleucine 219 to valine substitutions. The "reverse" mutations were introduced into the M gene by
the site directed mutagenesis, and the recombinant virus was further analyzed by the developed electron microscopy
protocols. Unfortunately, no visible morphology improvements were found. About the same portion of filamentous or chained
virions were detected. We conclude that the standard protocol of the influenza virus preparation for negative staining does
not seriously disturb the virions morphology. However, the question remains why the recombinant WSN virus has such
disturbed morphology, and is it correct to study the enveloped virus budding using the recombinant viruses.
Corresponding author:
Larisa Kordyukova
[email protected]
Tumor Viruses
P186
HPV8 increases the spare respiratory capacity of keratinocyte through ATP5B
S. Heuser1, J. Seeger2, G. P. Marcuzzi1, S. Majewski3, H. Kashkar2, B. Akgül1
1University
of Cologne, Institute of Virology, Cologne, Germany
of Cologne, Institute of Medical Microbiology, Immunology and Hygiene, Cologne, Germany
3Medical University of Warsaw, Department of Dermatology and Venereology, Warsaw, Poland
2University
Introduction: Members of the genus Betapapillomavirus (e.g. HPV8) can facilitate the development of cutaneous squamous
cell carcinoma in immunosuppressed organ transplant recipients and in individuals suffering from epidermodysplasia
verruciformis (EV). We demonstrated that the HPV8-E7 oncoprotein disturbs the normal differentiation profile of
keratinocytes, which results in enhanced cell proliferation and invasion. Recent studies also highlighted that metabolic
reprogramming through mitochondrial dysfunction is required to enable tumorigenesis. We therefore hypothesised that
HPV8-E7 may interact with the regulation of oxidative-phosphorylation, which supports oncogenic functions.
Material & Methods: Seahorse MitoStress and GlycoStress Assay; Affymetrix genechip cDNA microarray; Co-IP/mass
spectrometric analysis (Co-IP/MS); yeast-two-hybrid (Y2H) studies; immunofluorescence staining; Western blotting.
Results: Co-IP/MS and Y2H experiments led to the identification of the beta-subunit of the mitochondrial H+-ATP-synthase
(ATP5B) as putative E7 interacting factor in both assays. This interaction was confirmed in Co-IP experiments and
cytoplasmic co-localisation was shown in keratinocytes expressing Flag-tagged E7. ATP5B protein levels were unchanged in
E7-positive cells cultured in low-calcium media, but were upregulated in keratinocytes grown in DMEM/F12/FCS-based
media. Whereas ATP5B levels were low in normal human skin, strong pan-epithelial signals were found in skin cancer of EV
patients. Several ATP5 and NADH:ubiquinone-oxidoreductase subunits were also targeted by E7 at transcriptional level. To
clarify whether these interactions affect mitochondrial function, the oxygen-consumption-rate and glycolysis were determined.
E7-positive cells grown in low-calcium or DMEM/F12/FCS-based media did not show significant changes in glycolysis. E7
cells grown in serum-containing media, however, showed higher basal respiration, ATP production and showed an increase
in spare respiratory capacity. SiRNA mediated knockdown of ATP5B abolished the E7 mediated increase in the spare
respiratory capacity.
Conclusion: We identified that HPV8-E7 expression results in mitochondrial respiratory chain dysfunction, which might be
relevant during viral life cycle and oncogenesis.
Corresponding author:
Baki Akgül
[email protected]
Tumor Viruses
P187
Host telomerease RNA can complement the functions of viral telomerase RNA in Marek"s disease virus induced
transformation
A. Kheimar1,2, B. B. Kaufer2
1Institute
2Freie
of Virology, Veterinary Medicine, Berlin, Germany
Berlin University, Institute of Virology, Berlin, Germany
Introduction: Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes deadly lymphoma in chickens.
MDV encodes a viral telomerase RNA that has an enhances telomerase activity and plays a crucial role in MDV
pathogenesis and tumor formation. Despite its high sequence identity of 88% with the chicken telomerease RNA (chTR) it
remains unknown if the vTR evolved to enhance MDV virulence and transformation.
Objective: Until now, it remains unknown if overexpression of cellular TRs can contribute to cellular transformation.
Therefore, we used the MDV tumor model as a natural virus-host system to evaluate the effect of the overexpression of a
cellular telomerase RNA.
Material & Methods: To determine if the cellular chTR can aid in cellular transformation, we initially deleted vTR (vΔvTR)
and subsequently inserted chTR into the vTR locus (vchTR) in RB-1B, a very virulent MDV strain. We quantified the
expression of vTR and chTR by qRT-PCR, determined the replication properties and the pathogenesis and tumor formation
in vivo.
Results: qRT-PCR revealed that chTR in the (vchTR) virus was highly expressed at levels comparable to vTR in the wild
type (wt) virus. Neither vTR deletion nor chTR replacement affected virus replication in vitro and in vivo. Tumor development
was severely impaired in the absence of vTR, while chTR restored tumor formation almost to wt levels.
Conclusion: Our data provides the first evidence that cellular TRs possess tumor-promoting functions using this natural
model for virus-induced tumor formation.
Corresponding author:
Ahmed Kheimar
[email protected]
Tumor Viruses
P188
IL-6 signaling induces a unique STAT3/IRF1 pathway in HPV-transformed cells sensitizing them to
chemotherapeutic drugs
B. Walch-Rückheim1, J. Pahne-Zeppenfeld2, K. Bastuck1, J. Fischbach1, C. Wickenhauser3, L. C. Horn4, R. Büttner5, P.
Mallmann6, R. M. Bohle7, R. Ströder8, E. F. Solomayer8, S. Smola1
1Saarland
University, Institute of Virology, Homburg, Germany
of Cologne, Center for Molecular Medicine and Institute of Virology, Cologne, Germany
3Martin Luther University Halle-Wittenberg, Institute of Pathology, Halle/Saale, Germany
4University of Leipzig, Institute of Pathology, Leipzig, Germany
5University of Cologne, Institute of Pathology, Cologne, Germany
6University of Cologne, Department of Gynecology and Obstetrics, Cologne, Germany
7Saarland University, Institute of Pathology, Homburg, Germany
8Saarland University, Department of Gynecology and Obstetrics, Homburg, Germany
2University
Cervical cancer still represents one of the most common causes of cancer-related death in women worldwide. Invasive
cancer develops from persistent high-risk human papillomavirus (HPV) infection through well-defined stages of cervical
intraepithelial neoplasia (CIN1-3). This process takes years or decades and it is assumed that further changes within the
(pre)neoplastic cells and their microenvironment critically influence the course of disease. Cervical cancer therapy is still a
major clinical challenge. Neoadjuvant chemotherapy regimens can markedly improve cervical cancer outcome in a subset of
patients, while other patients show poor responses but may encounter severe adverse effects. Thus, there is a strong need
for predictive biomarkers to improve clinical management of cervical cancer patients. Here we show that cervical cancer cells
show a unique response to the cytokine IL-6. In contrast to most other tumors, where IL-6/STAT3-signalling mediates
resistance to cell death, HPV-positive cancer cells are sensitized to chemotherapeutic drugs after activation of their IL-6
signaling pathway. By applying transcriptome profiling we identified the pro-apoptotic factor IRF1 as a target gene in IL-6stimulated cervical cancer cells. We also show that the STAT3-inducible mediator IRF1 was required for cell death
enhancement. Clinically most relevant, our data demonstrate that patients with high IRF1 expression in cancer biopsies prior
to therapy displayed the strongest responses to chemotherapeutic drugs. In further analysis we investigated the impact of
the HPV oncoproteins E6 and E7 as well as the impact of viral transformation on this peculiar response.
In summary, our study has identified a key role of the STAT3/IRF1 pathway for chemosensitization in HPV-transformed cells.
Our results suggest that pretherapeutic IRF1 expression should be evaluated as a novel predictive biomarker for
neoadjuvant chemotherapy responses in patients with HPV-driven cancers.
Corresponding author:
Sigrun Smola
[email protected]
Tumor Viruses
P189
The ORF012 gene of the oncogenic Marek's disease virus Type 1 encodes a novel SR-like phosphoprotein essential
for virus growth
T. Schippers1, N. Osterrieder1
1Free
University Berlin, Institute of Virology, Berlin, Germany
Marek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by
generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those
of related herpesviruses has eased functional characterization of many MDV genes. However, MDV carries a number of
unique open reading frames (ORFs) that have not yet been investigated regarding their coding potentials and the functions
of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme
left end of the MDV unique long region. Using reverse transcriptase PCR, we showed that ORF011 and ORF012 are not
individual genes but form a single gene through mRNA splicing of a small intron, resulting in the novel ORF012. We
generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth
defect in vitro and could not be passaged in cultured cells, suggesting an essential role for the ORF012 product in virus
replication. Further studies revealed that protein 012 (p012) localized to the nucleus in transfected and infected cells, and we
identified by site-directed mutagenesis and green fluorescent protein (GFP) reporter fusion assays a nuclear localization
signal (NLS) that was mapped to a 23-amino-acid sequence at the protein's C terminus. Nuclear export was blocked by
leptomycin B, suggesting a potential role for p012 as a nucleocytoplasmic shuttling protein. In addition, p012 is
phosphorylated at multiple residues within and proximal to the NLS, a modification that regulates its subcellular distribution.
Specifically, we could demonstrate that a serine-arginine (SR) rich protein domain in p012 is phosphorylated by the cellular
kinase SRPK-1 which usually modifies cellular SR proteins, a family of nuclear phosphoproteins mainly involved in splicing
and other functions within the mRNA processing network. Therefore, we are currently investigating whether MDV p012 could
constitute a novel viral "SR-like" protein with similar functions to its cellular counterparts.
Corresponding author:
Timo Schippers
[email protected]
Tumor Viruses
P190
Spotting the effect on HTLV-1 transmission – COL4A1 and COL4A2 are novel targets of the Tax-1 oncoprotein
S. Millen1, C. Gross1, M. Mann1, K. Überla1, A. K. Thoma-Kress1
1Friedrich
Alexander University Erlangen Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany
Human T-cell leukemia virus type 1 (HTLV-1) infects CD4+ T-cells preferentially via cell-to-cell transmission requiring not only
reorganization of the cytoskeleton but also expression of the viral key-player and oncoprotein Tax. Collagens are not only
part of the basal membrane but also represent an important component of the viral biofilm, which depicts a fundamental
route of virus transmission. Thus, we asked, (1) if and what type of collagens are expressed in HTLV-1-infected T-cells, (2)
whether these collagens are (up)regulated by Tax, and (3) which effect such an upregulation exerts on virus transmission.
Making use of microarrays, RT-PCR, qPCR, western Blot, luciferase assays and immunofluorescence analysis, we found
that Col4α1 (COL4A1) and Col4α2 (COL4A2) are the only collagens to be upregulated in the presence of Tax in three
independent systems. Being transcribed from a shared and common bidirectional promoter, COL4A1 and COL4A2 are
translated into individual α-chains that finally assemble to heterotrimers (α1α1α2), underlying several posttranslational
modification steps. Intriguingly, both COL4A1 and COL4A2 transcripts and Collagen4 protein (COL4) were upregulated in
HTLV-1-positive T-cell lines. However, COL4 protein was only detectable in HTLV-1-infected cell lines that produce virions,
suggesting that COL4 either contributes to viral transmission or plays its major role upon cellular transformation.
Mechanistically, we found that Tax induces COL4A1 and COL4A2: (1) Repression of Tax in a Tax-transformed T-cell line led
to a significant reduction of COL4A1/A2. (2) Overexpression of Tax in Jurkat T-cells led to an induction of COL4A1/A2. This
finding was further supported by luciferase-based promoter studies indicating that Tax activates the COL4A2 and, to a less
extent, the COL4A1 promoter. (3) COL4A1/A2 increased in a time-dependent manner in a Tax-inducible T-cell line. Making
use of short hairpin RNAs and the CRISPR/Cas9 technology, we generated COL4A1/A2 knock-down and knock-out cells,
which will help to unravel the impact of these two structural proteins on HTLV-1 transmission. Taken together, we identified
COL4A1 and COL4A2 as novel cellular targets of Tax, which might foster viral transmission.
Corresponding author:
Sebastian Millen
[email protected]
Tumor Viruses
P191
Establishment of an outbred latency model with the cottontail rabbit papillomavirus in New Zealand White rabbits
allows measuring the effect of UV and immunosuppression on latent skin infections
J. Xi1, M. Schneider1, J. Haedicke-Jarboui1, A. Iftner1, F. Stubenrauch1, T. Iftner1
1University
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany Germany
Cutaneous Human Papillomaviruses (cuHPVs) cause the most prevalent HPV infections in the skin of the general
population. The ability of cuHPVs to persist in the absence of disease, i.e latency, has long being suspected. In humans,
administration of immunosuppressive drugs such as cyclosporine A (CsA) to organ transplant recipients (OTRs) has been
proposed to reactivate latent infections leading to apparent tumors. In this study, we employed the cottontail rabbit
papillomavirus (CRPV) model to investigate whether latent cuHPVs infections are controlled by the immune system and/or
UV radiation. We observed that similar to infections in men a median level of 13 copies per 1000 cells viral DNA can be
detected at 88% (198/223) of the latently infected skin sites up to 14 months p.i.. When latent skin samples were explanted
in cell culture, viral copy numbers dramatically increased. In addition we observed a minor elevation of viral genome copy
numbers in latently infected skin sites upon immunosuppression, but no increased tumor formation. Surprisingly, the
combination of immunosuppression and UVA +UVB radiation showed no significant difference compared to
immunosuppression alone. In conclusion, we established an outbred CRPV/rabbit model for the study of latent infections with
cutaneous papillomaviruses which allows investigating mechanisms controlling the latent state.
Corresponding author:
Jin Xi
[email protected]
Tumor Viruses
P192
Identification and characterization of phosphorylation sites of the human papillomavirus 31 E2 and E8^E2 proteins
T. Iftner1, S. van der Poel1, M. Dreer1, A. Velic2, B. Macek2, F. Stubenrauch1
1University
2Eberhard
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
Karls University, Proteome Center Tübingen, Tübingen, Germany
The HPV E2 protein is involved in viral transcription, replication and nuclear retention of viral genomes. In addition, HPV
encode the E8^E2 protein, which represses viral replication. E8^E2 is composed of the E8 part fused to the C-terminal half of
E2 which consists of the hinge and the DNA binding/dimerization domain. E2 proteins can be phosphorylated in the hinge
region and this has been linked to protein stability, DNA-binding activity and chromosomal attachment. However, the
phosphorylation status of E8^E2 is unknown.
Mass spectrometry analyses of HPV31 E2 and E8^E2 proteins immunoprecipitated from transfected HeLa cells revealed
three phosphorylated serines in the hinge region of the E8^E2 protein and two of these sites were also identified in E2. PhosTag analyses indicated that a large fraction of E8^E2 and only a minor fraction of E2 is phosphorylated. Mutation analysis
revealed that S78 is the major phosphorylation site of E8^E2. Reporter assays show that E8^E2 S78A has an impaired
repression function whereas E8^E2 S78E retains repression activity. In contrast, the corresponding E2 mutants do not show
a strong phenotype. Western blot and immunofluorescence analyses indicate that the E8^E2 S78A protein localizes to the
nucleus and is present in similar amounts as the wild-type protein. Surprisingly, HPV31 genomes with mutations of serine-78
are maintained as extrachromosomal elements at copy numbers similar to wt genomes in monolayer cells. Analyses of the
behavior of mutant genomes in differentiated keratinocytes are ongoing.
In summary, our data suggest that serines in the hinge of E8^E2 are phosphorylated and this influences transcriptional
repression activity.
Corresponding author:
Thomas Iftner
[email protected]
Tumor Viruses
P193
A role for the KSHV non-structural membrane protein K15 in virus reactivation
B. Abere1,2, N. Samarina1,2, E. Hage1,2, T. F. Schulz1,2
1Hanover
2German
Medical School, Institute of Virology, Hanover, Germany
Centre for Infection Research (DZIF), Braunschweig, Germany
Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the highly vascularized tumor Kaposi's sarcoma
(KS). We are looking for new therapeutic targets to inhibit KSHV replication and its pathogenesis. The KSHV K15 protein
induces the expression of genes involved in inflammation and angiogenesis. Inflammatory and angiogenic factors expressed
during inflammation or upon KSHV infection itself are known to induce the activation of the KSHV lytic cycle. Therefore, we
sought to investigate whether K15 could play a role in KSHV reactivation. For this, we used a recombinant KSHV genome in
the Bacterial artificial chromosome 36 backbone (KSHV-Bac36) from which we deleted the gene encoding K15 (KSHVBac36ΔK15) and siRNA mediated knock down of K15 from the infected cells. Deletion of the K15 gene from the viral
genome or its depletion by siRNA mediated knockdown lead to reduced virus reactivation as evidenced by the decreased
expression levels of KSHV lytic proteins as well as reduced infectious virus release. Further investigation of cellular signaling
pathways activated during KSHV reactivation revealed that deletion of K15 from the viral genome compromised the ability of
KSHV to activate PLCγ1, Akt1 and Erk1/2 upon induction of the lytic cycle.
As a proof of principle, the isolated PLCγ2 cSH2 domain, previously reported (Gramolelli et.al, 2015) as a dominant negative
inhibitor of the K15-PLCγ1 interaction, was used to block KSHV reactivation. Moreover, we used an in vitro kinase assay and
Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) to establish a screen of compounds for potential
inhibition of this interaction and K15-mediated pathogenesis. We found that K15 is phosphorylated in the context of viral
infection both during latency and upon reactivation and a similar phosphorylation of K15, in vitro, results in a stronger binding
to PLCγ1. Additionaly, we tested whether the isolated PLCγ1 cSH2 domain could also block K15 binding to PLCγ1 in order to
use it as a positive controle during the screening assay.
Altogether, our data suggest that the KSHV K15 protein plays an important role during virus reactivation and targeting the
recruitment of PLCγ1 to the K15 could provide a new avenue to inhibit KSHV reactivation and its pathogenesis.
Corresponding author:
Bizunesh Abere
[email protected]
Antiviral Therapy and Resistance
P195
Evaluating the significance of serum chemokines as prognostic biomarkers in Pegylated IFN-α-2a /ribavirin-treated
chronic hepatitis C patients using Luminex xMAP technology
M. H. Hamed1, P. Pushparaj1, S. Bazarah1, S. Karim1, K. Alghamdy1, I. Qadri1
1King
Abdul Aziz University, Biological Sciences, Jeddah, Saudi Arabia
Introduction: Hepatitis C viral (HCV) infection inflicts a major clinical burden globally. Pegylated IFN-α-2a (PEG-IFN-α-2a)
with ribavirin (RIB) therapy induces an array of chemokines to tackle the HCV infection; however, some of the HCV patients
develop resistance to PEG-IFN/RIB therapy.
Objectives: To assess the role of chemokines induced in response to PEG-IFN-α-2a with ribavirin (PEG-IFN/RIB) therapy,
as prognostic biomarkers in chronic HCV patients.
Material & Methods: A total of 20 patients with HCV infection and ten healthy controls were enrolled in this study. Based on
the therapeutic response to PEG-IFN/RIB, HCV patients were further categorized into two groups, namely, HCV-Responder
(HCV-R), and HCV-non-responder (HCV-NR). We analyzed some of the key chemokines such as IP-10, MIG, MCP-1,
EOTAXIN, RANTES, IL-8, MIP-1a, and MIP-1b in eighteen HCV patients. These chemokines were precisely examined by a
magnetic bead-based multiplex immunoassay approach based on Luminex X-MAP multiplex technology, using a MAGPIX
instrument (Luminex Corporation, USA).
Results: A significant elevation of ALT and AST enzymes were observed in HCV-NR. Besides, the PEG-IFN/RIB therapy
both MIG and MCP-1 in HCV-NR patients were significantly induced. PEG-IFN/RIB therapy significantly increased the levels
of chemokines, such as IL-8, IP-10, EOTAXIN, MIG, RANTES, and MIP-1β, in HCV-R. The highest increase in MIG and
MCP-1 levels was observed at the end of the therapy in HCV-NR (p=0.0018 and p=0.0453, respectively),) related to HCV-R
(p=0.0393 and p=0.2092, (respectively). IL-8 (p=0.003) was significantly increased in HCV-R compared to control; however,
there was no significant difference in IL-8 levels between HCV-R and HCV-NR. Furthermore, IL-8, IP-10, EOTAXIN, MIG,
RANTES, MIP-1β levels were increased in HCV-R, indicating the chemokine response to PEG-IFN/RIB therapy.
Conclusion: MCP-1 and MIG are known to recruit monocytes and T cells into the areas of inflammation. Hence, MCP-1 and
MIG could be the potential biomarkers in HCV-NR and might be associated with the development of liver fibrosis, liver failure,
and hepatocellular carcinoma.
Corresponding author:
M. Haroon Hamed
[email protected]
Antiviral Therapy and Resistance
P196
Polyvalent 2D entry inhibitors for Pseudorabies Virus and African Swine Fever Virus
J. Rahn1, B. Ziem2, I. Donskyi2, K. Silberreis3, L. Cuellar2, J. Dernedde3, G. Keil1, T. C. Mettenleiter1, R. Haag2
1Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
University Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany
3Charité Medical School, Institute of Laboratory Medicine, Berlin, Germany
2Free
Introduction: African Swine Fever Virus (ASFV) is one of the most dangerous viruses for pigs, since the infected animals
usually succumb rapidly. The virus is endemic in Africa, but also spread via Caucasian countries into the Russian Federation
and the Eastern border of the EU. Since there is no vaccine or antiviral drug available to curtail the infection, control
strategies including novel inhibitors are urgently needed.
Another highly relevant virus infection in pigs is Aujeszky´s disease caused by the alphaherpesvirus Pseudorabies virus
(PrV).
Objectives: In this study, we report on a simple mimicry of heparan sulfate surfaces by the synthesis of two polyvalent entry
inhibitors by using a [2+1] cycloaddition and their biological evaluation.
Material & Methods: In general, the designed inhibitors are a combination of the bioactive polyglycerol sulfate and a twodimensional graphene sheet.
Results: Due to the overall polyanionic character, the synthesized 2D architectures are able to bind enveloped virus particles
during the adhesion process and thereby exhibit strong inhibitory effects as demonstrated for ASFV and PrV. In this context,
both 2D entry inhibitors could achieve equal or better inhibition efficacies than the common standards enrofloxacin and
heparin.
Conclusion: Overall, the developed polyvalent entry inhibitors are non-toxic and efficient nanoarchitectures that interact with
various types of enveloped viruses. Thereby, they prevent viral adhesion to the host cell, especially targeting viruses which
rely on a heparan sulfate-facilitated entry mechanism.
Corresponding author:
Jessica Rahn
[email protected]
Antiviral Therapy and Resistance
P197
A new antiviral approach – Inhibition of the RNA helicase eIF4A by the natural compound Silvestrol
A. Grünweller1, F. W. Schulte1, N. Biedenkopf2, K. Lange-Grünweller1, C. Müller3, M. Schlitzer1, J. Ziebuhr3, S. Becker2, R. K.
Hartmann1
1Philipps
University Marburg, Institute of Pharmaceutical Chemistry, Marburg, Germany
University Marburg, Institute of Virology, Marburg, Germany
3Justus Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
2Philipps
In view of the recent Ebola virus (EBOV) outbreak in West Africa, the identification of potent antiviral drugs with a broadspectrum activity is urgently required. Here we analysed the natural compound silvestrol, a potent and selective inhibitor of
the DEAD-box RNA helicase eIF4A, for its antiviral activity. This enzyme is a promising antitumor target because its helicase
activity is required for 5´-cap-dependent translation initiation of several protooncogenes with highly structured and GC-rich
5´UTRs. Interestingly, such capped 5´UTRs with extensive RNA secondary structures can also be found in mRNAs from
viruses with (-)-RNA or (+)-RNA genomes.
We observed a strong antiviral effect of silvestrol in Ebola-infected Huh-7 cells and in primary human macrophages at low
nM concentrations [1]. The expression of Ebola proteins like VP40 or the highly abundant nucleocapsid protein NP were
strongly reduced, whereas essential housekeeping proteins were only slightly affected by silvestrol. This is in line with the
observed low toxicity of silvestrol in primary cells. Moreover, potent antiviral activity of silvestrol was also seen in MRC5 cells
(human embryonic lung fibroblasts) infected with the Coronaviruses HCoV-229E or MERS, both of which have a (+)-RNA
genome. Interestingly, the 5´-cap independent Rhinovirus 1A that harbours an IRES in its 5´UTR was not affected by
silvestrol.
Since eIF4A is a host protein, it is very unlikely that viruses may develop resistance e.g. by escape mutations after eIF4A
inhibition, thus making this helicase an interesting new antiviral target. Nevertheless, due to its structural complexity, the
chemical synthesis of silvestrol is very difficult. Therefore we screened chemical data bases to identify molecules with similar
but less complex structural features and comparable properties as silvestrol, with the goal to develop new medically relevant
antiviral lead structures.
References:
[1] Biedenkopf N, Lange-Grünweller K, Schulte FW, Weißer A, Müller C, Becker D, Becker S, Hartmann RK and Grünweller
A.* The natural compound Silvestrol is a potent inhibitor of Ebola virus replication. Antiviral Research 137:76-81, (2016).
Corresponding author:
Arnold Grünweller
[email protected]
Antiviral Therapy and Resistance
P198
Neuraminidase inhibitor effectivity varies with influenza virus subtype
S. Duwe1,2, J. Milde1, T. C. Harder3, B. Schweiger1
1Robert
Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
Koch Institute, Division of Influenza and Other Respiratory Viruses, National Reference Centre for Influenza, Berlin, Germany
3Friedrich Loeffler Institute, Institute of Diagnostic Virology, Greifswald - Isle of Riems, Germany
2Robert
Background: Clinical efficacy of neuraminidase (NA) inhibitors to influenza viruses is type- and subtype- specific and has
been discussed controversially. Nevertheless, they are the only compounds currently effective against influenza virus
infections. In Europe, only oseltamivir and zanamivir are approved for treatment and prevention of influenza by the European
Medicines Agency (EMA). In view of the pandemic potential of avian influenza viruses, eg A(H5N1), A(H5N8), A(H7N9), and
the fact that human influenza viruses may cause severe and lethal courses of illness, there is an urgent need for effective
antiviral drugs. Therefore, the study focused on the susceptibility of influenza A viruses to oseltamivir, zanamivir, peramivir,
and laninamivir, that are currently available commercially.
Methods: Susceptibility of human and avian influenza A viruses to NA inhibitors was tested by using a validated fluorometric
NA inhibition assay, followed by calculating the 50% inhibitory concentration (IC50) of the respective inhibitor. Influenza A
viruses were selected to represent NA group-1 (N1, N4, N5, N8), and group-2 (N2, N3, N6, N7, N9) viruses as well as
viruses of low and high pathogenicity (subtypes H5 and H7).
Results: All tested viruses showed in vitro susceptibility to the NA inhibitors tested. Influenza A NAs belonging to group-1
were more sensitive to zanamivir than to oseltamivir, whereas group-2 NAs were more sensitive to oseltamivir than to
zanamivir. Laninamivir inhibited influenza A viruses more efficiently than oseltamivir and zanamivir and even oseltamivirresistant viruses. Peramivir was the most potent in vitro inhibitor of all tested influenza A group-1 and group-2 viruses.
Conclusion: Influenza virus infection is a serious and frequently underestimated disease. Due to resistance development
and limitation of approved compounds, there is an urgent need of effective and reliable medicines for treatment and
prevention of influenza. The recently in Japan, South Korea, and the USA approved NA inhibitor peramivir and the in Japan
licensed laninamivir might to be potent drugs with intravenous application or nasal inhalation. Based on experiences from
treatment of influenza virus infections in Asia and the USA, approval of these two NA inhibitors for Europe should be
considered by the EMA.
Corresponding author:
Susanne Duwe
[email protected]
Antiviral Therapy and Resistance
P199
Influence of the hormone vitamin D3 (Cholecalciferol) and its metabolites on the in vitro replication of Hepatitis B
virus infection
K. Singethan1, W. Dammermann2,3, M. Feuerherd1, C. Kühne3, J. Schulze zur Wiesch3, U. Protzer1, S. Lüth2,3
1Technical
University Munich and Helmholtz Centre Munich, Institute of Virology, Munich, Germany
Medical School, Center for Internal Medicine II, Brandenburg an der Havel, Germany
3University Medical Center Hamburg Eppendorf, Hamburg, Germany
2Brandenburg
Several smaller studies regarding the treatment of chronic hepatitis C showed, that the addition of vitamin D3
(cholecalciferol, calciol) to the conventional peg Interferon-alpha-2b/Ribavirin therapy leads to higher SVR rates in untreated
patients infected with HCV genotype 1 (Abu-Mouch et al., 2011). Similar results could be observed for HCV genotype 2 and
3 infections. The molecular mechanism behind this phenomenon is still not fully understood, although several publications
revealed that the metabolite 25(OH)-vitamin D3 (Calcidiol) inhibits HCV replication by activating IFN-stimulated genes (ISG)
(Gal-Tanamy et al., 2011; Matsumura et al., 2012).
Notably, the role of vitamin D3 in HBV infection is still unclear. Here we show the influence of vitamin D3 and its metabolites
25(OH)- and 1a,25(OH)2-vitamin D3 on HBV replication and infection. Vitamin D3 and/or its metabolites inhibit HBV
replication. Like in HCV replication the induction of type 1 interferon as a direct consequence of 25(OH)-vitamin D3 treatment
led to a reduction of HBV replication. HBeAg-secretion, HBV rcDNA and cccDNA were significantly reduced after treatment
with 25 (OH)-vitamin D3. The viability assays showed no overall toxicity in HepaRG cells. 25(OH)-vitamin D3, but not Vitamin
D3 and 1a,25(OH)2-Vitamin D3 reduced HBeAg secretion at concentrations of 10 µM significantly. HBV rcDNA and cccDNA
levels decreased at concentrations of 100 µM, while Interferon-alpha transcription rates significantly increased 72h after
treatment with 25(OH)-vitamin D3. It is currently under investigation if this leads to the induction of ISG.
These data suggest that the combination of HBV therapy with vitamin D3 might be beneficial and supportive for patients with
chronic hepatitis B.
Corresponding author:
Katrin Singethan
[email protected]
Antiviral Therapy and Resistance
P200
Generation and characterization of HCMV mutants with point mutations in the UL27 gene with regard to antiviral
drug resistance
M. Lippmann1, T. Weitbrecht1, M. Widmann1, E. Imrich1, K. Hamprecht1, G. Jahn1, K. Göhring1
1University
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
Introduction: In immunocompromised patients after stem-cell or organ transplantation HCMV can cause life-threatening end
organ disease resulting in dependence on antiviral therapy. Resistance of HCMV to antiviral drugs such as Ganciclovir
(GCV), Foscarnet (PFA) and Cidofovir (CDV) remains a grave problem and is typically associated with mutations in either the
viral UL54-gene and/or the viral UL97-gene. In some cases analysis of HCMV-isolates from stem-cell transplant recipients in
our diagnostics department showed resistance to GCV even though no mutations in the UL97 and/or the UL54 gene were
found. A potential connection between resistance to Maribavir (MBV) and mutations in the UL27-gene was shown in recent
studies (Chou et al., 2004; Hakki et al., 2011) suggesting other possible origins of antiviral resistance.
Objectives: We identified mutations in the UL27 gene which are present in the abovementioned HCMV-isolates and
investigate their effect on antiviral resistance to even GCV, PFA, CDV and MBV.
Methods: The full-length UL27- gene sequence of 11 samples was compared to that of two reference strains. To generate
HCMV-mutants containing the desired mutations each point mutation was introduced into the AD169-derived BAC pHB5UL32-EGFP (kindly provided by Christian Sinzger, Ulm) using en-passant mutagenesis (Tischer et al., 2010). Viral
reconstitution after transfection of BAC-DNA into HFF cell culture was detected by plaque formation and green fluorescence
signal. Successfully reconstituted mutants were characterized with regard to their growth-kinetics and their resistance level to
antiviral agents using a co-culture plaque-reduction-assay.
Results: 11 UL27-mutations consisting of 8 amino acid exchanges (A20D, Q14K, L133I, S173I,A184V, P307L, V310A,
A558D) and 3 reading frame shifts caused by either insertion (InsT10) or deletion (DelG1564, DelT1589) were identified.
From all of them recombinant virus strains were generated and were completely characterized regarding their resistance
profile and their replication kinetics.
Conclusion: We generated and characterized 11 recombinant virus strains with defined point mutations in the UL27 gene
found in patient isolates. This work may contribute to a better understanding of HCMV drug resistance in transplant
recipients.
Corresponding author:
Katharina Göhring
[email protected]
Antiviral Therapy and Resistance
P201
Generation and characterization of newly detected mutations in respiratory syncytial virus fusion protein F
concerning resistance to palivizumab
T. Weitbrecht1, K. Hamprecht1, G. Jahn1, K. Göhring1
1University
Hospital Tübingen, Institute of Medical Virology and Epidemiology Epidemiology of Viral Diseases, Tübingen, Germany
Background: Respiratory syncytial virus (RSV) (with 2 subgroups A and B) is known as one of the most important
respiratory tract pathogen in young children under 2 years of age. Especially, RSV infection in premature infants and children
with cardiac and respiratory problems can lead to severe lower respiratory tract illness such as bronchiolitis and pneumonia
with long-lasting hospitalization. Since its discovery in 1956, RSV therapy options remain unsatisfactory with only two
licensed pharmaceutical substances. Ribavirin, a synthetic nucleoside analogue, was approved by FDA in treatment of
severe RSV infection in hospitalized children but it has serious side effects at the doses needed to eliminate RSV.
Palivizumab, a monoclonal antibody against the RSV fusion protein F, offers passive immunity in prophylactic treatment for
high-risk infants. RSV mutants that exhibit resistance to palivizumab have been shown to have amino acid (AA) changes in
the antigenic site II (AA 262-275) on the F protein. Due to the intensive cost of this prophylactic therapy, it is important to
know whether mutation related to resistance against palivizumab is triggered during the therapy and whether mutation in
other regions of the F protein can induce resistance to palivizumab.
Methods: The F protein gene sequences of 8 patient isolates (all RSV A) were compared with that of three reference strains:
line 19, A2, and Long strains. We introduced identified mutations into the pSynkRSV-line19F BAC (Hotard et al. 2012) using
"en-passant" mutagenesis (Tischer et al. 2006). Reconstructed BAC was then transfected into BSR-T7/5 cells (kindly
provided by K. Conzelmann, Munich) for virus rescue. Recombinant virus from BSR-T7/5 was used to infect Vero cells in
order to propagate recovered virus for further characterization step (e.g. virus growth and susceptibility testing against
palivizumab).
Results: We identified 7 point mutations and 6 polymorphisms. "En-passant" mutagenesis was successfully conducted for 2
of 7 point mutations. Further steps in our experiment procedures are still ongoing.
Conclusion: We generate and characterize recombinant virus with identified mutations in patient isolates. This work may
contribute a new reverse genetics method in RSV research and promote a better understanding of resistance to palivizumab.
Corresponding author:
Trinh Weitbrecht
[email protected]
Antiviral Therapy and Resistance
P202
HIV-1 drug resistance development in women following Option B+ for PMTCT
P. Machnowska1,2, A. Hauser1, K. Meixenberger1, B. Altmann1, N. Bannert1, P. Busingye3, J. Rubaihayo4, G. HarmsZwingenberger2, S. Theuring2
1Robert
Koch Institute, Division of HIV and Other Retroviruses, Berlin, Germany
Medical School, Institute of Tropical Medicine and International Health, Berlin, Germany
3Holy Family Virika Hospital, Fort Portal, Uganda
4Mountains of the Moon University, Department of Public Health, Fort Portal, Uganda
2Charité
Introduction: Since 2012, WHO guidelines for the prevention of mother-to-child transmission (PMTCT) of HIV-1 in resourcelimited settings recommend the initiation of lifelong antiretroviral combination therapy for all pregnant HIV-1 positive women
independent of CD4 count and WHO clinical stage (Option B+). However, long-term outcomes regarding development of
drug resistance are lacking until now.
Objectives: Between January and December 2013, 124 pregnant HIV-1 positive women were enrolled within antenatal care
services in Fort Portal, Uganda. 59/124 (47.6%) women were followed-up until 12 and/or 18 months postpartum (ppm) and
were included into the drug resistance mutation (DRM) analysis.
Methods: An RT-PCR covering resistance associated positions in the protease and reverse transcriptase HIV-1 genomic
region was performed. PCR-positive 12 and/or 18 ppm samples and their respective baseline samples were analysed by
next generation sequencing (NGS) regarding HIV-1 drug resistant variants including minorities, defined as DRMs detected by
NGS below the Sanger-like cut-off of 20%.
Results: DRMs were detected in 3/59 (5.1%) women at 12 and/or 18 ppm. The NNRTI-associated DRM K103N was present
in all three women. Two women displayed dual-class resistance against all recommended first-line regimen drugs. Minor
variants of DRMs were detected in one woman at 12 ppm and one woman at 18 ppm (NNRTI-associated DRMs A98G 4.4%,
V108I 6.0% and P225H 13.6%).
Conclusion: Our findings provide first evidence that the WHO-recommended Option B+ for PMTCT is effective in a cohort of
Ugandan HIV-1 positive pregnant women with regard to the low selection rate of DRMs compared to previous PMTCT
regimens.
Corresponding author:
Patrycja Machnowska
[email protected]
Antiviral Therapy and Resistance
P203
Resistance testing of clinical varicella-zoster virus isolates collected over three decades
A. Hoffmann1, K. Döring1, N. T. Seeger1, M. Bühler1, A. Sauerbrei1
1University
Hospital Jena, Institute of Virology and Antiviral Therapy, Jena, Germany
Introduction: Varicella-zoster virus (VZV) is a ubiquitous human α-herpesvirus that causes varicella as primary infection and
zoster after reactivation of latent virus. Especially in immunocompromised patients, long-time treatment of zoster with
acyclovir (ACV) can lead to the emergence of resistant VZV. Based on the mode of action, resistance is mostly caused by
mutations in the ORF36, encoding the viral thymidine kinase (TK) and rarely in the ORF28 encoding the DNA polymerase
(pol). Sequence analyses in diagnostics can be used to provide a quick assessment of the viral drug susceptibility.
Objectives: Our study aimed on the characterization of novel amino acid (aa) substitutions in the VZV TK and pol to expand
the existing pool of information on genetic polymorphisms.
Material & Methods: A total of 158 clinical VZV strains, collected from 1984 to 2014, were analyzed genotypically for nonsynonymous mutations in the TK and pol gene by DNA amplification and sequencing. Viral strains containing novel or
unclear aa substitutions were tested phenotypically for drug sensitivity using plaque reduction assay. On this account, strains
with alterations in the TK were tested against ACV, penciclovir and brivudin (BVDU), and strains with altered pol were tested
against ACV, BVDU, and foscarnet.
Results: The genotypic results revealed that in the 158 strains examined, an aa substitution rate of 0.025 and 0.220 per
strain could be determined for TK and pol, respectively. Overall, four substitutions with hitherto unknown significance were
detected in the TK and 14 in the pol. The subsequent phenotypic assay revealed that all concerning viral strains were
sensitive against the antiviral drugs tested. The determined 50% effective concentrations were comparable to that of the
sensitive VZV reference strain vOka.
Conclusion: Phenotypic tests for the assessment of VZV drug susceptibility are time consuming and associated with a lot
experimental effort. Comprehensive databases on phenotypic effects of TK and pol polymorphisms could improve the
interpretation of genotypic data. Mutations in VZV TK or pol conferring natural gene polymorphism are rare events. The
characterization of 18 novel polymorphisms in TK and pol can help to provide a better identification of resistance mutations.
Corresponding author:
Anja Hoffmann
[email protected]
Antiviral Therapy and Resistance
P204
A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and
inhibits entry into fibroblasts and endothelial cells
C. Stegmann1, D. Hochdorfer1, D. Lieber1, N. Subramanian1, D. Stöhr1, K. Laib Sampaio1, C. Sinzger1
1Ulm
University Medical Center, Institute of Virology, Ulm, Germany
Human cytomegalovirus (HCMV) is a widely distributed herpesvirus that causes significant morbidity in immunocompromised
hosts. Inhibitors of viral DNA replication are available, but adverse effects limit their use. Alternative antiviral strategies may
include inhibition of entry. We show that soluble derivatives of the platelet-derived growth factor receptor alpha (PDGFRalpha), a putative receptor of HCMV, can inhibit HCMV infection of various cell types. A PDGFR-alpha-Fc fusion protein
binds to and neutralizes cell-free virus particles at an EC50 of 10-30 ng/ml. Treatment of particles reduced both attachment
to and fusion with cells. In line with the latter, PDGFR-alpha-Fc was also effective when applied postattachment. A peptide
scan of the extracellular domain of PDGFR-alpha identified a 40mer peptide that inhibits infection at an EC50 of 1-2 nmol/ml.
Both, peptide and fusion protein, were effective against various HCMV strains and are hence promising candidates for the
development of novel anti-HCMV therapies.
Corresponding author:
Christian Sinzger
[email protected]
Antiviral Therapy and Resistance
P205
Cationically modified dextrans inhibit HSV-1 infection at early stages
M. Pachota1, K. Pyrć1
1Jagiellonian
University, Department of Biochemistry Biophysics and Biotechnology, Kraków, Poland
Background: Human herpesvirus type 1 (HSV-1), one of the most frequent human pathogens, is known to cause orolabial,
genital and corneal lesions. It establishes a latent infection in sensory neurons and may be reactivated by various stress
stimuli. The only commercially available therapy is based on viral DNA polymerase inhibitors, acyclovir and its derivatives,
which carry some limitations, like low bioavailability and emergence of resistant strains.
Objectives: Polycationic agents are expected to inhibit HSV-1 entry, by binding to its cellular receptors or adhesion factors.
By employing a different mode of action than acyclovir, these compounds may be supplementary to conventional anti-herpes
therapy.
Methods: A set of GTMAC-substituted dextran derivatives of various molecular weights and degrees of substitution were
synthesized. The compounds were tested for cytotoxicity by XTT assay. Effect on viral replication was assessed with
standard plaque assay and real-time qPCR. Further, the mechanism of action was determined using a set of functional
assays and confocal microscopy.
Results: Long-chain dextrans (average molecular weight ~40 000 and ~100 000 Da) substituted with GTMAC in 40-60%
were found to be the most potent inhibitors of HSV-1 infection at non-cytotoxic concentrations. Inhibition of viral replication
after addition of the compound during infection carried out at 4°C indicates a blockage in viral adsorption to cell surface. The
same effect was observed for HSV-2.
Conclusion: Cationically modified dextrans inhibit HSV-1 infection at early stage, probably by interfering with binding of the
virus to the cell surface via adhesion receptors. An entry inhibitor might provide an interesting alternative or supplement for
traditional anti-HSV therapeutics.
Corresponding author:
Magdalena Pachota
[email protected]
Antiviral Therapy and Resistance
P206
Generation and characterization of newly detected combined HCMV polymerase (UL54)-and phosphotransferase
(UL97)-mutations associated with drug resistance
M. Widmann1, G. Jahn1, K. Hamprecht1, K. Göhring1
1University
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
Introduction: Drug-resistant cytomegalovirus infections cause severe problems in immunocompromised patients.
Resistance against ganciclovir (GCV), cidofovir (CDV) and foscarnet (PFA) is typically associated with mutations in either the
UL97-gene (phosphotransferase-gene) or in the UL54-gene (viral polymerase). The impact on newly emerging mutations on
drug-resistance can only be determined by Marker Transfer Analysis. In previous work we showed that the UL97
polymorphism A591V in combination with the three UL54 mutations D515E, L516M and I521T increased the level of GCV
resistance dramatically (Fischer et al., 2016). For further investigation of this phenomenon, we generated three recombinant
virus strains containing the UL97 mutation A591V with only one UL54 mutation and characterized them by phenotypic
assays.
Methods: Recombinant virus strains (A591V+D515E, A591V+L516M; A591V+I521T) were generated by en-passant
mutagenesis (Tischer et al., 2006). For the generation of recombinant HCMV all mutations were introduced into the TB40/Ederived bacterial artificial chromosome (BAC) TB40-BACKL7-UL32EGFP (kindly provided by Christian Sinzger, Ulm).
Phenotypic characterization was performed by growth assays and a modified plaque reduction assay using an HFF/Arpe coculture method. Resistance was defined as a 2-fold decrease in drug-sensitivity compared to the reference strain HCMVTB40-Bac7-UL32-EGFP.
Results: All recombinant strains were successfully generated and transfected in human foreskin fibroblasts. The
reconstituted mutants were characterized with regard to their growth-kinetics and their resistance level to antiviral agents
using co-culture plaque-reduction assay.
Conclusion: Our results implicate that polymorphisms can have an important impact on the drug resistance level in
combination with resistance-associated mutations. The findings of this work contribute to a better understanding of antiviral
drug-resistance of HCMV and also support the notion that the phenotype of a mutation needs to be regarded in the context
of its genetic background.
Corresponding author:
Manuel Widmann
[email protected]
Antiviral Therapy and Resistance
P207
Only half of the oseltamivir dose is needed to treat acute influenza viral infections
G. Montaseri1, A. Boianelli2, E. A. Hernandez-Vargas2, M. Meyer-Hermann1,3
1Helmholtz
Centre for Infection Research, Department of Systems Immunology and Braunschweig Integrated Centre of Systems Biology, Braunschweig,
Germany
2Helmholtz Centre for Infection Research, Systems Medicine of Infectious Diseases, Department of Systems Immunology and Braunschweig Integrated
Centre of Systems Biology, Braunschweig, Germany
3Technical University Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Braunschweig, Germany
Question: Influenza A virus (IAV) is a latent global threat for the human health. In view of the risk of pandemics, prophylactic
and curative treatments are essential. Oseltamivir is a neuraminidase inhibitor efficiently supporting recovery from influenza
infections. It is stockpiled worldwide in order to be prepared for the case of a pandemic outbreak. Current common clinical
practice is a constant drug dose (75 or 150 mg) administered at regular time intervals of twice a day. We aim to elaborate the
possibility of reducing the required dose of oseltamivir based on adaptive feedback control of the administered drug dose
while keeping the same virological efficacy.
Methods: We combined the data-validated mathematical model for IAV infections, which captures the viral dynamics and the
dynamics of the immune effector response, with a pharmacokinetics (PK)/pharmacodynamics (PD) model of oseltamivir.
Next, we applied an adaptive impulsive feedback control method to calculate the optimal dose of oseltamivir in dependence
on the viral load and the amount of immune effectors at the time of drug.
Results: The control-based treatment derived from adaptive feedback control clears the infection equally fast while keeping
the number of effector cells even lower than under common clinical practice (50% cell counts). Quantification of the
virological efficacy and the administered drug revealed that the adaptive control-based treatment achieves a higher
virological efficacy of 79 instead of 71% while using the same drug amount as in common clinical practice. However, the
peak doses calculated from adaptive control theory were above the safety limit of 150 mg. When limiting the adaptive dose to
75 mg, the virological efficacy is back to the level of common clinical practice but using only half of the total drug.
Conclusion: We provided evidence that with adaptive control-based drug scheduling the same virological efficacy can be
achieved with half of the total drug. Thus, adaptive adjustment of the drug dose would not only reduce the potential side
effects in the patient but also reduce the required amount of stored oseltamivir for the prevention of outbreaks.
Corresponding author:
Ghazal Montaseri
[email protected]
Antiviral Therapy and Resistance
P208
Resistance to antiviral therapies – answers form the endophyitic fungi Curvularia papendorfii
H. Jacob1, S. Philippot2, R. Spina2, M. Varbanov2
1Royal
College of Surgeons in Ireland, School of Pharmacy, Dublin, Ireland
of Lorraine, Faculty of Pharmacy, Nancy, France
2University
Viruses are ever-expanding group of microorganisms that have the capacity to cause human diseases of varying severity.
The emergence of new and evolution of pre-existing viruses are causing a significant threat to global public health. The quick
and complex evolutionary adaptation and mutation rates of viruses leading to the development of drug resistance have also
contributed hugely to the brutality of the problem. Perplexing strategies of virus evolution make antiviral therapy challenging
and selective attacks on unique biochemical features of viruses are not always possible. There is an immediate need to
develop highly effective antiviral agents as the vast majority of the current viral infections have no specific drug treatments
and prophylactic measures. In this alarming context the use of plants with antiviral properties and confirmed ethnomedical
profile could increase the chances of the successful development of novel therapeutic agents.
Our study investigated the cytotoxic and potential antiviral properties of ethyl acetate crude extract of Curvularia papendorfii
against two model viruses well known for their drug resistance: Human Coronavirus 229E (HCoV-229E) and Poliovirus strain
Sabin (PV1). Curvularia papendorfii is an endophytic fungi isolated from leaves and stems of Vernonia amygdalina, an
African medicinal plant. Various concentrations of the ethyl acetate crude extract ranging from 1 mg/mL to 256 mg/mL were
tested on L132 and CRFK cells to determine their cytotoxicity using MTT assay. The IC50 of the ethyl acetate crude extract
on L132 and CRFK cells were 37,52 mg/mL and 50,65 mg/mL respectively. Antiviral tests using ethyl acetate crude extract
were then carried out on the respiratory virus HCoV-229E and the enteric virus PV1. The results from this study provide
evidence for moderate antiviral effects of the crude extract against HCoV-229E. Even though overall very little antiviral viral
effects were witnessed against PV1, antiviral activity was observed against heat inactivated and low PV1 concentrations.
Future studies on the isolation and purification of the active constituents and the antiviral mechanism of action of the crude
extract of C. papendorfii will unravel its potential antiviral capacity and aids its progression into the development of a new,
effective antiviral agent.
Corresponding author:
Mihayl Varbanov
[email protected]
Antiviral Therapy and Resistance
P209
A high-throughput screening system for antivirals targeting the human cytomegalovirus alkaline nuclease
T. Potgieter1, A. Caceres-Nuñez1, E. Ostermann1, W. Brune1
1Heinrich
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
Human cytomegalovirus (HCMV) is a herpesvirus with a high prevalence. It is a leading cause of congenital damage in
neonates as well as serious disease in immunocompromised patients. Current treatment of HCMV largely focuses on
interfering with viral DNA replication; however side effects are common and viral strains resistant to these treatments have
been reported, indicating a need for new viral therapeutic targets. One such target is the viral alkaline nuclease, a conserved
herpesvirus enzyme. In HCMV, it is encoded by the UL98 gene. Disruption of this alkaline nuclease severely reduces
herpesviral replication, suggesting that this protein is a suitable target for antivirals. In order to screen for inhibitors of UL98
we wanted to establish a fluorescence-based high-throughput screening system. To this end the viral UL98 was expressed in
E.coli and purified. The nuclease activity of the purified protein was confirmed by digestion of plasmids and by introducing an
active site mutation with known reduced nuclease activity. The fluorescence assay makes use of a DNA oligonucleotide
substrate coupled to a fluorophore and a quencher. Upon degradation by the viral nuclease, fluorophore and quencher are
separated resulting in an increase in detectable fluorescence. As a proof of principle we show that UL98 nuclease activity is
inhibited in this system by a known inhibitor, Atanyl Blue PRL (Acid Blue 129), in a dose-dependent manner. After optimizing
the assay conditions, we will use this assay system to screen existing compound libraries for potential UL98 inhibitors. We
are also generating HCMV-UL98 mutants in order to investigate the function of UL98 in vitro as this is poorly defined.
Corresponding author:
Theodore Potgieter
[email protected]
Antiviral Therapy and Resistance
P210
Rapid acquisition of a known HSV-1 TK frameshift mutation leading to Acyclovir resistance in a patient with
HSV/EBVEncephalitis and chronic lymphocytic Leukemia
M. Karrasch1,2, H. Axer3, A. Kunze3, S. Wagner4, A. Sauerbrei2
1University
Hospital Jena, Institute of Medical Microbiology, Jena, Germany
Hospital Jena, Institute of Virology and Antivirale Therapy, Jena, Germany
3University Hospital Jena, Hans Berger Hospital of Neurology, Jena, Germany
4University Hospital Jena, Section Neuroradiology, Jena, Germany
2University
Introduction: Herpes simplex virus (HSV) infections are frequent in CLL and are usually treated with the antiviral drug
acyclovir (ACV). Resistance to ACV is mainly associated with mutations in the viral thymidine kinase (TK).
Methods: We report a CLL patient who presented with nausea, aphasia and mnestic problems. Electroencephalography
(EEG), computed tomography (CT) and magnetic resonance imaging (MRI) of the brain, and whole body positron emission
tomography (FDG-PET) was performed. Cytological/immunohistochemical examination was performed on cerebrospinal fluid
(CSF). CSF was screened for all relevant encephalitis-causing pathogens. Genotypic resistance testing with the analysis of
TK was performed at two different time points due to the severe encephalitis course.
Results: Serum hypogammaglobulinemia, CSF pleocytosis, CSF lactate elevation and intrathecal IgM production were
detected. MRI FLAIR and DWI images showed high signal edematous gray matter along the left insular cortex and the
ipsilateral medial temporal lobe. EEG showed continuous delta rhythm over frontal leads. FDG-PET revealed
hypermetabolism in vermis. Microbiological CSF analysis was negative for toxoplasma, varicella-zoster virus,
cytomegalovirus, HSV-2, human herpesvirus 6 and enterovirus, but positive for Epstein-Barr virus (EBV) and HSV-1 with
lymphocytic pleocytosis and few atypical CD-20 negative lymphocytic cells. Patient was treated with 4 cycles of rituximab for
EBV B-cell depletion. Initial genotypic HSV-1 resistance testing showed no resistance mutations and patient was started on
intravenous ACV. Follow-up genotypic testing 8 days later detected an insertion of guanine between nucleotides 430 and
436 leading to a truncated not functional TK protein due to a stop codon at amino acid site 228, resulting in ACV resistance.
Patient was switched from ACV to foscarnet. Following treatment, patient was negative for both EBV and HSV-1 in CSF.
Conclusion: CLL patients treated with inhibitors of key pathways in B-cell development have a higher probability for acquiring
HSV encephalitis. Sensitivity of those HSV strains to ACV should be monitored by regular genotypic resistance testing as
non-synonymous mutations in the TK gene can occur during ACV therapy at any time
Corresponding author:
Matthias Karrasch
[email protected]
Antiviral Therapy and Resistance
P211
Polypores as a new source for anti-influenza agents
M. Schmidtke1, M. Richter1, C. Mair2, U. Peintner3, J. M. Rollinger2, U. Grienke2
1University
Hospital Jena, Jena, Germany
of Vienna, Faculty of Life Sciences, Department of Pharmacognosy, Vienna, Austria
3University of Innsbruck, Institute of Microbiology, Innsbruck, Austria
2University
Due to the increase of viral resistance and a constant threat of pandemics, the current arsenal of anti-influenza drugs needs
extension. In this context, natural compounds have evolved as promising pool to search for novel antivirals.
Polypores, a group of fungi belonging to the class of Agaricomycetes, are a substantial part of antiviral herbal formulas used
in Traditional Chinese Medicine (TCM). However, locally grown European polypores have so far not been thoroughly
investigated in this direction. Therefore, we collected fruit bodies of several strains from ten species including Fomes
fomentarius, Fomitopsis pinicola, Ganoderma lucidum, G. applanatum, Gloeophyllum odoratum, Ischnoderma benzoinum,
Laetiporus sulphureus, Phellinus robustus, Piptoporus betulinus, and Trametes gibbosa. The morphological identification of
strains was confirmed based on rDNA ITS phylogenetic analyses.
The generated ethanol extracts where screened for their anti-influenza virus activity in a cytopathic effect (CPE) inhibition
assay using MDCK cells where they were generally well tolerated.
In particular, the extracts of three different strains of G. odoratum dose-dependently inhibited the CPE induced by influenza A
viruses with IC50 values between 8.2 and 19.5 µg/mL. First investigations regarding the constituents responsible for this
distinct anti-influenza activity led to the structural class of lanostane-type triterpenes. Fluorescence-based neuraminidase
inhibition assays revealed that the antiviral activity was not based on neuraminidase inhibition. Currently, further studies are
ongoing to elucidate the virus specificity and mechanism of antiviral activity of the identified constituents.
Corresponding author:
Michaela Schmidtke
[email protected]
Antiviral Therapy and Resistance
P212
Antiviral activity of two N-N-(bis-5-nitropyrimidyl) dispirotripiperazine (DSTP) derivatives against human
cytomegalovirus
R. Adfeldt1, M. Schmidtke2, E. Novoselova3, V. Makarov3, E. Bogner1
1Charité
Medical School, Institute of Medical Virologiy, Berlin, Germany
University Hospital Jena, IVAT, Jena, Germany
3A. N. Bakh Institute of Biochemistry RAS, Moscow, Russian Federation
2
Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in
immunocompromised adults. Transplant and AIDS patients, e.g. may develop life-threatening diseases as a consequence of
primary infection or reactivation of latent infection. The current available drugs ganciclovir, valganciclovir, cidofovir and
foscarnet target the viral DNA polymerase. Due to multiple problems caused by these drugs (limited effects, dose-dependent
toxicity, and drug resistance) development of new well-tolerated anti-HCMV compounds with novel mechanisms of action are
urgently needed. In this study we have tested two new DSTP derivates mns 13-13 and mns 19-13 in order to determine
whether both compounds are able to inhibit HCMV infection in human embryonic lung fibroblasts (HELFs). The antiviral
activity was quantified by measurement of viral plaque formation. HELF cells were treated with various concentrations of the
substances. Interestingly, cells were dose dependently protected against infection with TB40/E-pp150-GFP, whereas mns
19-13 showed the highest efficacy. These results demonstrated that both compounds are active against HCMV. The
cytotoxicity of mns 13-13 and mns 19-13 was investigated to exclude that unspecific effects could affect its potential antiHCMV activity. Both substances were well tolerated by HELF cells. Therefore, suppression of viral replication due to
cytotoxic side effects of the drugs at the concentrations used can be excluded. To determine the stage of viral replication that
is sensitive to these two compounds, the effect of time-dependent drug addition was analyzed. TB40/E-pp150-GFP infected
HELFs were treated with mns 13-13 or mns 19-13 at different times of the replication cycle. Both pretreatment with the
substances 30 minutes before and addition of them during infection resulted in suppression of viral replication by 90%.
Interestingly, an antiviral activity up to 60% was also found if the compounds were added after viral adsorption. In order to
investigate the ability to prevent virus attachment or penetration further experiments are in progress.
Corresponding author:
Rebekka Adfeldt
[email protected]
Antiviral Therapy and Resistance
P213
LNAs vs. HIV-1 RNA expression – an alternative approach to knock-down viral replication
F. Hillebrand1, H. Schaal1, S. Erkelenz2, M. Widera3, A. L. Brillen1, D. Degrandi4, K. Pfeffer4
1Heinrich
Heine University Dusseldorf, Institute of Virology, Dusseldorf, Germany
of Cologne, Institute of Genetics, Cologne, Germany
3University of Duisburg-Essen, University Hospital Essen, Institute of Virologie, Essen, Germany
4Heinrich Heine University Dusseldorf, Institute of Medical Microbiology and Hospital Hygiene, Dusseldorf, Germany
2University
Introduction: During HIV-1 replication the viral (+) RNA genome is reverse transcribed, converted into double stranded DNA
and integrated into the host genome. Transcription of the provirus generates a single pre-mRNA which is alternatively spliced
and exported into the cytoplasm either as spliced, intron-containing or unspliced mRNA. Here, the balanced and temporal
expression of the different viral mRNA species determines the successful production of new virions. Consequently, disturbing
mechanisms involved in the generation of these viral mRNAs dramatically affect HIV-1 infectivity and pathogenesis.
Objective: With the aim to develop an antisense masking strategy against HIV-1 replication we designed locked nucleic
acids (LNAs), modified antisense oligonucleotides, targeting highly conserved viral RNA sequences essential for the
generation of the different viral RNA species.
Methods: Because transfection of the LNAs is not an option for this LNA-based antiretroviral approach we just added these
mixmer LNAs within a low micromolar scale into the cell culture medium (gymnosis) of HIV-1 infected Jurkat cells. The
impact of the LNAs on the viral replication and viral RNA expression was analyzed via northern- and immunoblot as well as
RT-PCR analysis. Further, the intracellular distribution of the LNAs was analyzed by performing confocal laser scanning
microscopy.
Results: The naked delivery of the LNAs nearly completely interfered with the viral RNA expression and HIV-1 biogenesis.
Although, aiming at masking the viral RNA target sequences in the nucleus to prevent protein binding, the LNAs turned out to
exert their antiviral effect within the cytoplasm by degradation of the viral RNA species containing the LNAs target sequence.
Moreover, the LNA-mediated antiviral effect could also be observed in HIV-1 infected PBMCs from different donors.
Conclusion: The unassisted delivery of LNAs directed against sensitive viral RNA sequences has proven to dramatically
inhibit HIV-1 replication in a low micromolar scale demonstrating the potential of this LNA-based antiretroviral approach.
Corresponding author:
Frank Hillebrand
[email protected]
Antiviral Therapy and Resistance
P214
Functional analysis of Gag-p6 deletions accumulating in PI resistant HIV-1
M. M. Langer1, B. Bleekmann1, M. Dirks1, N. Pfeiffer2, E. Schülter3, S. Esser4, G. Fätkenheuer5, B. Jensen6, R. Kaiser3, J.
Verheyen1, M. Widera1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Planck Institute of Informatics, Saarbrücken, Germany
3University of Cologne, Institute of Virology, Cologne, Germany
4University Duisburg-Essen, University Hospital Essen, Clinic of Dermatology, Essen, Germany
5University of Cologne, Department of Internal Medicine, Cologne, Germany
6Heinrich Heine University Dusseldorf, Department of Gastroenterology, Hepatology and Infectious Diseases, Dusseldorf, Germany
2Max
Introduction: Mutations in the HIV-1 Gag-p6 protein can affect viral replication and have been associated with drug
resistance to protease inhibitors (PI). However, the role of insertions and deletions in Gag-p6 concerning viral replication and
drug resistance has not yet been investigated phenotypically.
Objectives: The purpose of this study was to evaluate whether deletions in Gag-p6 have an impact on Gag processing and
viral replication in presence and absence of PIs.
Patients & Methods: C-terminal Gag, PR, RT, and V3 sequences of subtype-B isolates (n=267) obtained from routine HIV-1
genotypic drug resistance tests from therapy-naïve patients (n=94), patients after treatment failure with (n=91) and without
DRM (n=82) were studied by Chi-Squared test, multivariate logistic regression, and covariation analysis. Identified deletions
were inserted into the HIV-1 molecular proviral clone pNL4-3 and analyzed in respect to viral particle production and Gag
processing. Transient transfection experiments and Western Blot analysis were performed to monitor Gag-processing in
presence and absence of PI (LPV, DRV, and ATV). Furthermore, infectivity assays were performed to evaluate the impact on
viral replication.
Results: We found that amino acid (aa) deletions Δ470-473 and Δ473 accumulate in the group of PI-resistant HIV and were
correlated with CXCR4 co-receptor usage. Data from infectivity assays indicated, that Gag Δ470-473 viruses per se have no,
and Δ473 containing viruses, respectively, had only little replicative disadvantage. As determined by Western blotting and
infectious cell culture experiments, Gag processing and viral infectivity was not affected by these deletions when tested in
presence of currently used protease inhibitors DRV, LPV, and ATV.
Conclusion: Our results provide additional phenotypical information to evaluate Gag-p6 deletions, which are frequently
observed in PI resistant HIV. In particular, deletions of Gag-p6 amino acids at position 470-473 and 473, respectively,
accumulate in PI resistant HIV and are linked to X4-tropism. However, these deletions have per se little effect on viral
infectivity and are, at least as stand-alone, unlikely to cause drug resistance towards currently used protease inhibitors DRV,
LPV, and ATV.
Corresponding author:
Mona-May Langer
[email protected]
Antiviral Therapy and Resistance
P215
Passive antibody transfer reduces canine distemper virus mortality in ferrets
M. Dörr1, S. K. Kays1, V. von Messling1
1Paul
Ehrlich Institute, Veterinary Medicine, Langen, Germany
Introduction: Canine distemper virus (CDV), a member of the Morbillivirus genus, causes severe disease with considerable
mortality in a broad range of carnivores. In addition to fever and the characteristic rash, infected animals develop respiratory
and gastro-intestinal signs and a severe immune suppression. In ferrets, this immune suppression is so severe that animals
infected with wild type strains are unable to mount an antiviral immune response and ultimately succumb to the disease. This
model is thus ideally suited to characterize the therapeutic potential of passive antibody transfer for lethal virus infections.
Objectives: To assess the efficacy of passive antibody transfer for CDV therapy in ferrets at different times after infection.
Material & Methods: First, the half-life of allogeneic hyper immune serum after intraperitoneal injection was determined by
neutralization assays. During efficacy studies, ferrets were monitored daily for fever and clinical signs, and leukocyte counts
and lymphocyte proliferation assays were performed regularly to evaluate the extent of immune suppression. To assess
treatment efficacy, the viral load in circulating lymphocytes and of the neutralizing antibodies in the serum will be quantified.
Results: We observed that serum antibody titers remained stable for at least five days after the injection of allogeneic
hyperimmune serum. A single dose treatment during the first three days after infection protected all animals from clinical
disease, whereas treatment at later time points had no effect on disease outcome. Repeated dosing for two weeks starting
as late as 8 days after infection, which coincides with the onset of clinical signs, was able to still reduce overall mortality. All
surviving animals mounted strong antibody responses, indicating that passive antibody transfer did not prevent the
development of an autologous antiviral response.
Conclusion: Passive antibody transfer has the potential to protect animals from disease and death, even if treatment is
initiated at the beginning of clinical disease, and does not inhibit a robust antiviral response. These studies constitute a
starting point for further development of passive antibody transfer as a therapeutic option in emergency situations.
Corresponding author:
Mareike Dörr
[email protected]
Antiviral Therapy and Resistance
P216
High throughput reporter screen to identify inhibitors of clinically relevant human polyomavirus infections
E. Kraus1,2,3, N. Fischer1,3, A. Grundhoff2,3
1University
Medical Center Hamburg Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
3German Center for Research on Infection (DZIF), Partner Site Hamburg-Borstel-Lübeck-Riems, Germany
2Heinrich
While human polyomavirus infections are highly prevalent in the healthy population, four of the thirteen hitherto known
human virus family members, BKV, JCV, MCPyV and TSPyV, are associated with severe diseases under
immunosuppression. While TSPyV and MCPyV induce rare disease, BK and JC virus reactivation/infection are of major
concern in transplant patients and other patients receiving immunosuppressiva. Uncontrolled BKV replication causes two
major diseases, hemorrhagic cystitis after bone-marrow transplantation (PVHC) in 5-15% of allogenic transplant patients and
polyomavirus-associated nephropathy (PVAN) after kidney transplantation in 1-10% of kidney transplant recipients. Since
there are no specific antiviral drugs available the main treatment of BK viremia is reduction of immunosuppression at the
expense of an increased risk of graft rejection.
The aim of this study is to identify and improve inhibitors active against BKV infection.
We established a reporter screen in BKV permissive human cell lines suitable for high throughput screening. Inhibitor
screens based on morphological changes induced by infection are highly challenging since BKV is slow growing with only
mild cytopathic changes in nearly all established cell lines. Our screen takes advantage of the high expression of BKV
expressed miRNA during viral replication. Based on the amount of miRNA expression during the viral life cycle we identified
immortalized human fibroblast in comparison to other cell lines most suitable for this application. In addition, we identified the
BKV Dunlop strain as a BKV strain expressing significantly higher amounts of BKV miRNA compared to other re-arranged
BKV strains tested. Sensor cell lines with a stable integrated GFP reporter cassettes sensitive to BKV miRNA expression
were generated in immortalized human fibroblasts. Optimal screening conditions (moi, single cell clone selection, time point
of infection and time point of inhibitor addition) were established using Cidofovir as the only antiviral nucleoside analog
available.
We successfully established a cell based high throughout applicable reporter screen sensitive to BKV infection, which will
allow us to test small molecule inhibitors for their specific activity in inhibiting clinically relevant human polyomavirus infection.
Corresponding author:
Nicole Fischer
[email protected]
Antiviral Therapy and Resistance
P217
Dimerization inhibitiors for ZikV, EboV, Alzheimer Aß?
H. J. Schramm1, W. Schramm2
1MPI
Biochemie, retired, Oettingen, Germany
Maxilian University Munich, Munich, Germany
2Ludwig
Oligomeric proteins and peptide aggregates can be disrupted or modified by small peptides (or mimetics) derived from the
structure stabilizing parts. This approach is valuable since viruses often form active protein structures from identical noncovalently bound subunits. For HIV protease, dimerization inhibitors (DIs) with nM activity could be identified. This widely
applicable approach also works with non-identical subunits, between sub-domains of the same protein or for disrupting toxic
complexes like amyloid Aß.
Ebola: In EboV VP30 and VP40 are possible DI targets. VP40 matrix protein exists in 3 forms: dimer (PDB: 4ldb), hexamer
(4ldd) and the RNA stabilized octamer (1h2c). In the hexamer, 2 short helices (F105 - L117) and (L163 - A188) and 2 ß-sheet
bends (Q184- A188 and F157 - P165) should allow DI. The dimer, contact D45 – I59, contains a ß segment P53 - I54 parallel to the
large ß sheet, as targets. Zika: In ZikV protein NS1 (5IY3), an anti-parallel ß sheet interface (-A182VIGT♦AVKGK191-) and the
loop (-K227WPKSHT♦LWTDGIEE238-) provide the dimer stability. Ala scanning identified binder amino acids.
DIs are also possible in the (40% homologous) West Nile Virus protein NS1 (4O6D) with the ß sheet segment
ADTGC♦AIDISR. The Cys next to the dyad may allow affinity labeling using Cys-S(S)-R peptides.
Alzheimer: In anti-parallel Aß amyloids (2lnq, D23N early onset, 5aef, ..), the ß-sheet segment -Q15(K)L(V)F(F)A(E)N(V24)- is
opposite to -G29A(I)IGL(M)V36- [() = to the "outside"]. The side-chains of the "inside a.a." form a stabilizing hydrophobic core,
while "vertical" hydrogen bonds form a ß-sheet "tube" growing by Aß addition. Blocking peptides should be able to bind to Aß
aggregates but have no Aß binding sites for further Aß docking. For reducing hydrophobic interactions in 2lnq, mutated
peptides R-Q15XLXFXAXN23-NHR'2 could be tried, e.g. with R = Palmitoyl, X = G, A or small a.a (also D-form). Some or all
peptide bonds 15-16, 17-18, 19-20, 21-22 should be replaced by -CON(CH3)- to prevent further growth by hydrogen bond
interaction. From the opposing -G29A(I)IGL(M)V36- segment, similar DI segments could be derived by N-methyl blocking and
mutations.
In aggregates with parallel sheets (2beg), the interaction of E22 and K28 stabilizes the ß-sheet "tube" which now includes turn
parts (a prion like structure switch driven by Aß aggregation?).
Corresponding author:
Hans J. Schramm
[email protected]
Antiviral Therapy and Resistance
P218
Humanized camelid heavy chain antibody that interfered RNA-binding function of HCV core protein
M. Chulanetra1, J. Thanongsaksrikul2, K. Thueng-in3, W. Chaicumpa1
1Faculty
of Medicine Siriraj Hospital, Mahidol University, Department of Parasitology, Bangkok, Thailand
of Allied Health Sciences, Thammasart University, Graduate Study in Biomedical Science, Pathumthani, Thailand
3Institute of Medicine, Suranaree University of Technology, School of Pathology, Nakhon Ratchasima, Thailand
2Faculty
HCV have been considered as a burden disease in both clinical and economic aspects. More than 50% of HCV-infected
patients have chronic hepatitis which can further progress to hepatic cirrhosis and hepatocellular carcinoma. Besides no
vaccine for HCV, standard HCV therapy; combined pegylated-interferon and nucleoside analog, has high cost and side
effects which render low patients" compliance and genotype-related effectiveness. Recently, direct-acting anti-HCV drugs
target nonstructural HCV proteins has been developed. However, there will be possibilities to generate the drug-resistance
strain due to error-prone HCV polymerase. Therefore, alternative HCV therapy might be necessary.
Core or capsid protein of HCV plays role in viral genome protection as well as has important functions in viral assembly and
viral exportation and also related to apoptosis and host immune response. Thus, core protein is another attractive target for
anti-HCV. Phage-displayed antibody library is a useful technology for selection of antigen-specific antibody in vitro. The
heavy chain antibodies (VH) produced from phage display antibody library were proven for their ability to neutralized many
toxins and treated infectious diseases. In this study, recombinant domain I of HCV core protein (D1) was produced and used
for selection the D1-specifically bound VH from humanized camelid heavy chain antibody library. The obtained VHs were
tested for D1 binding by ELISA and immunoblotting, and the cell penetrable VHs were produced. The truncated HCV
peptides were constructed for mapping VH-binding region. An immunoblotting result reveals that all selected VHs bound to
amino acid position 38-47 of HCV core. Four VHs clones were inhibited an RNA-binding function of the D1. These VHs
clones will be further tested for HCV inhibition in HCV cell culture system.
Corresponding author:
Monrat Chulanetra
[email protected]
Antiviral Therapy and Resistance
P219
An aspirin derivative impairs replication of coronavirus and other respiratory viruses
C. Müller1,2, N. Karl1, J. Ziebuhr1,2, S. Pleschka1,2
1Justus
Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Germany
2German
Coronaviruses (CoV) belong to the large family Coronaviridae within the order of Nidovirales. Among them, several human
pathogenic strains (HCoV) are known to mainly cause respiratory diseases. While most strains contribute to common coldlike illnesses others lead to severe infections. Most prominent representatives are SARS-CoV and MERS-CoV, which can
lead to fatal infections with around 10% and 39% mortality, respectively. This resulted in 8098 casualties in the 2002/2003
SARS-CoV outbreak and in 1806 documented human infections (September 2016) during the recent on-going MERS-CoV
outbreak in Saudi Arabia. Currently patients receive treatment focusing on the symptoms connected to the disease rather
than addressing the virus as the cause. Therefore, additional treatment options are urgently needed which would ideally be
widely available and show a broad affectivity against different human CoVs. Here we show that D, L-lysine acetylsalicylate +
glycine sold as "Asprin i.v. 500mg®" (LASAG), which is an approved drug inter alia in the treatment of acute pain, migraine
and fever, impairs propagation of different CoV including the highly-pathogenic MERS-CoV in vitro. We demonstrate that the
LASAG-dependent impact on virus-induced NF-κB activity coincides with (i) reduced viral titres, (ii) decreased viral protein
accumulation and viral RNA synthesis and (iii) impaired formation of viral replication transcription complexes.
Corresponding author:
Christin Müller
[email protected]
Antiviral Therapy and Resistance
P220
Antiviral activity of theaflavins from black tea against hepatitis C virus entry
P. Chowdhury1,2, M. E. Sahuc2, M. Goswami3, T. Bandyopadhyay1, J. Dubuisson2, K. Séron2
1Tocklai
Tea Research Institute, Biotechnology, Jorhat, India
Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Molecular and Cellular
virology, Lille, France
3Tocklai Tea Research Institute, Biochemistry, Jorhat, India
2Univ.
Question: Hepatitis C virus (HCV) is a significant health burden worldwide with estimated 130-170 million people affected
worldwide. In recent times, direct acting antivirals (DAA) for effectively curing HCV were developed, but the high cost limit
their use. Moreover, to avoid emergence of viral mutants, it is important to develop drugs, targeting different steps of virus life
cycle. Here, we screen theaflavins, the major black tea polyphenols with high antioxidant property, against HCV. Black tea is
the most consumed drink in the world (78%), after water,and recently it attains attention due to health benefits.
Methods: Theaflavin was extracted from prepared crush tear curl (CTC) Assam, Indian black tea using ethyl acetate. The
crude theaflavin was subjected to sephadex LH-20 column (Sigma) and fractions were collected with distinguished reddish
colour. The purity was confirmed by HPLC. The screening was done using Huh-7 hepatoma cells infected with Japanese
fulminant hepatitis-1 (JFH-1) virus containing titer-enhancing mutations. The antiviral activity of the compounds was
quantified with confocal microscopy by immunofluorescent labelling of HCV envelope protein 1 (E1).
Results:The fractions were confirmed to be theaflavin (TF1), theaflavin-monogallate (TF2) and theaflavin-3,3-digallate (TF3).
All three compounds, in dose dependent treatment, showed inhibitory effect. Time interval study showed that all compounds
inhibited the virus at entry step with TF3 showing the most prominent effect. There is no significant effect on the prophylactic
treatment of cells or in replication step.
Conclusion: Theaflavins are a new interesting inhibitors of HCV entry. Further studies are in progress to determine their
efficacy against different HCV genotypes and to understand the possible mode of action during entry.
Corresponding author:
Pritom Chowdhury
[email protected]
Antiviral Therapy and Resistance
P221
The NEDD8-activating enzyme inhibitor MLN4924 potently inhibits HBV transcription in an HBx-dependent manner
B. Qu1, M. Leuthold1, S. Urban1,2
1University
2German
Hospital Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
Centre for Infection Research (DZIF), Heidelberg, Germany
Introduction: Transcription of HBV from its authentic template requires the HBx-mediated counteraction of host-encoded
restriction factors of the SMC5/6 complex (Decorsière et al, Nature & Murphy et al, Cell Rep 2016). To fulfill that functional
HBx binds the DDB1/Cullin4 ring ligase complex, which triggers degradation of SMC5/6 complex, resulting in transcriptional
activation of the cccDNA episomes, a prerequisite for SMC5/6 ubiquitination and subsequent degradation is the neddylation
of the Cullin ring ligase. MLN4924 is a specific inhibitor of the Nedd8-activating enzyme in clinical phase I trials, which
covalently couples the protein to its substrate.
Results: Based on the hypothesis that neddylation is required to induce HBx-mediated degradation of SMC5/6, we tested
the ability of MLN4924 to interfere with HBV infection. When administered after establishment of cccDNA in HepG2 NTCP cells
stably expressing a functional HBV receptor, sodium taurocholate cotransporting polypeptide, an 80% reduction of viral
transcription and secreted antigens could be observed. Remarkably, HBV rebound was prompt after removal of the drug in
infected HepG2NTCP cells. To confirm that MLN4924 targets HBV replicative steps, we performed an infection using hepatitis
delta virus (HDV) that have HBV envelopes and a similar entry strategy as HBV. HDV replication was not affected.
To further investigate whether the inhibition is HBx-dependent, we generated two stable HepG2NTCP cells with inducible
expression of wild-type HBx or mutated HBx (R96E) that abolishes its binding activity to DDB1. We infected the cells with
HBx expressing-deficient virions and stimulated HBx expression in the presence or absence of MLN4924. Without any effect
on cccDNA formation, we observed greatly reduced pregenomic RNA level and HBeAg secretion only in the cells that
expressed wild-type HBx, compared to the cells with mutated HBx where transcription was slightly decreased, indicating that
HBx maintains HBV transcription and MLN4924 impairs the function of HBx/DDB1 complex.
Conclusion: These data indicate that inhibition of neddylation interferes with cccDNA-mediated transcription, probably by
inhibiting the HBx-mediated counteraction of transcriptional suppression. Since suppression of antigen production (e.g.
HBsAg) is highly deserved in chronically infected patients, MLN4924 might have beneficial effects in future antiviral therapies
aiming at cure chronic HBV infection.
Corresponding author:
Bingqian Qu
[email protected]
Antiviral Therapy and Resistance
P222
Cyclophilin A antagonists as Coronavirus inhibitors
A. von Brunn1, J. Carbajo-Lozoya1, B. von Brunn1, D. R. Bairad1, Y. Ma-Lauer1
1Ludwig
Maximilian University Munich and Max von Pettenkofer Institute of Virology, Munich, Germany
Although desperately needed, neither vaccines nor therapeutics are available blocking CoV infection in humans and animals.
We reasoned that knowledge of host cell proteins that take part in pivotal functions of the virus-host interplay by interaction
with viral proteins and the inhibition of these interactions should define broad-spectrum antiviral cellular targets. This
approach should also circumvent the usual fast development of drug resistances observed in the case of viral targets.
Screening the SARS-CoV orfeome against human cDNA libraries by unbiased Y2H methods we recently had identified CoV
proteins as interaction partners of human cyclophilins (Cyps). Consequently, we showed that Cyclosporin A (CsA) inhibits the
replication of human and animal CoVs. Now we demonstrate that novel nonimmunosuppressive derivatives of CsA as well as
Alisporivir, NIM811 strongly inhibit the growth of human and animal CoVs at low micromolar, non-cytotoxic concentrations in
cell culture. We show by qPCR analysis that virus replication is diminished by several orders of magnitude down to
background levels. Knockdown of the cellular CypA (CypA/PPIA) in Caco-2 cells prevents replication of HCoV-NL63
respectively, suggesting that CypA is required for virus replication. Collectively, our results uncover CypA as a host target for
controlling CoV infection and provide new strategies for urgently needed therapeutic approaches to combat newly emerging
viruses like MERS-CoV. CypA might serve as a new biomarker in anti-coronaviral research.
Corresponding author:
Albrecht von Brunn
[email protected]
Antiviral Therapy and Resistance
P223
Computer aided design of novel nucleoside analogs inhibiting dengue virus RNA polymerase
J. J. Bugert1, C. Cima1, A. Brancale1
1Intitut
für Mikrobiologie der Bundeswehr, Virologie, Munich, Germany
Dengue virus (DENV), the most widely distributed mosquito-borne human pathogen, is endemic in over 100 countries. In the
last 50 years incidence has increased 30-fold, with 50-100 million cases per year and 40% of the Worlds population at risk of
infection, accounting for 2.5 billion people. Clinical manifestations of infection with any of the existing serotypes of DENV may
vary from a self-limiting febrile illness known as dengue fever, to the life-threatening condition dengue haemorrhagic fever,
that is the cause of 500000 hospitalizations and over 20000 deaths worldwide every year. Approved antiviral agents specific
for dengue virus are not available. The crystal structure of DENV polymerase has been solved, however the polymerase
domain in complex with the template and the nascent RNA strand (ternary complex) has not been crystallised. The
polymerase needs to undergo drastic conformational changes during the synthesis of the RNA, from a closed initiationcompetent conformation to an open structure. Details of this transition are unknown. A novel reliable model of the ternary
complex of DENV polymerase was developed and employed to identify potential anti-DENV nucleoside analogues.
Nucleoside analogues designed in silico were synthesized. Compounds cima 1 and 4 were found active versus dengue virus
serotype 2 in human hepatoma cell line HUH7, with an IC50 of 8.96 +/- 0.5 and 4.58 +/- 0.2, respectively, and compared to
ribavirin, 2,5di–O-T, and ddBCNA cf2642.
Corresponding author:
Joachim Jakob Bugert
[email protected]
Antiviral Therapy and Resistance
P224
In search of sCAR-Fc resistant Coxsackie B viruses; so far no mutants in sight
S. Pinkert1, V. Brückner1, B. Dieringer1, J. Kurreck1, H. Fechner1
1Institute
of Biotechnology, TU-Berlin, Department of Applied Biotechnology, Berlin, Germany
Coxsackie B viruses (CVB) belong to the family of picornaviruses and cause a wide spectrum of diseases, ranging from mild
respiratory syndromes and hand foot mouth syndrome to life-threatening conditions such as pancreatitis, myocarditis and
encephalitis. So far, no vaccines or antivirals are available for clinical treatment mainly due to the rapid development of drug
resistance. During the last years we demonstrated in vitro and in vivo the high antiviral efficiency of the soluble receptor
protein (sCAR-Fc), containing the extracellular part of the coxsackie and adenovirus receptor (CAR) fused to the constant
domain of human IgG. Thereby we tested a wide panel of laboratory and clinical CVB strains, without finding isolates that
were not susceptible to sCAR-Fc.
Here we used three different experimental settings to analyze the existence or development of resistant mutants during
sCAR-Fc treatment, an often emerging obstacle in antiviral therapy. First, we analyzed the frequency of resistance to sCARFc in a virus population of up to 107 infectious particles using a dose-response plaque reduction assay. We found a
concentration-dependent (sCAR-Fc concentration 0.1 – 16 µg/ml) decrease in plaque formation that continued until an
apparent plateau was reached at 2 µg/ml. At this concentration plaque formation had been reduced by 5log10. The viruses
form residual plaques showed no resistance to sCAR-Fc even after five additional rounds of plaque purification under sCARFc treatment.
In a second set-up we analyzed whether resistant mutants can be established by replication inhibition assay. Therefore, virus
was pre-incubated with sCAR-Fc and replication was analyzed 24 h after infection. No alteration in inhibition efficiency by
sCAR-Fc was observed during five cycles of successive inhibition assays.
In a third approach, after pre-incubation of CVB3 with sCAR-Fc, cells were infected and further cultured for 72 h in a
continuous sCAR-Fc environment. Virus progeny displayed no altered susceptibility to sCAR-Fc compared to untreated virus.
Here we show that under selective pressure by sCAR-Fc, CVB3 does not develop resistant mutants. Thus sCAR-Fc
represents a promising candidate for use in treatment of human CVB infections.
Corresponding author:
Sandra Pinkert
[email protected]
Antiviral Therapy and Resistance
P225
Antiviral efficacy of rupintrivir and oseltamivir in human in vitro nasal epithelia
P. Alouani1, B. Boda1
1Epithelix,
Plan les Ouates, Switzerland
Objective: Respiratory viral infections cause mild to severe diseases in worldwide, such as common cold, bronchiolitis and
pneumonia and are associated with huge costs to society. To test new molecules for shortening, alleviating the diseases or
to develop new therapies, relevant in vitro human models are necessary. We propose here human reconstituted
standardized nasal epithelia, MucilAirTM for a proof of concept study of viral drug screening. The MucilAirTM respiratory
epithelium possesses defence mechanisms, comparable to in vivo situation, such as mucus production, mucociliary
clearance, and secretion of defensive molecules.
Methods: Clinically relevant Rhinovirus (A16, C15), Enterovirus (EV68) and Influenza A virus (H1N1, H3N2) strains were
added to fully differentiated MucilAirTM–Pool (mix of 14 donors) cultures at the apical side. Apically released viral genome
copy number, overall mucin secretion, cilia beating frequency, velocity of mucociliary clearance and tissue integrity were
assessed daily during 4 days. Basal media of the MucilAirTM cultures served for cytokines as well as toxicity measurements.
Different concentrations of antiviral treatments were carried out parallel and all endpoints were compared with the control
conditions.
Results: MucilAirTM cultures showed an excellent host characteristics and high rate of replication for all tested viruses,
including difficult-to-culture Rhinovirus C15. Infections in MucilAir TM cultures permitted us a detailed description of the early
viral pathomechanisms in the nasal epithelia for the different viruses. Rupintrivir efficiently inhibited the replication of HRVA16 and HRV-C15 in a dose and time dependent manner and restored the mucociliary clearance impaired by EV68.
Oseltamivir reduced the replication of H1N1 and H3N2 and restored the impaired barrier function monitored by transepithelial
electrical resistance.
Conclusion: These results demonstrate that MucilAir™ is a robust, reliable and relevant tool for antiviral drug development.
Corresponding author:
bernadett Boda
[email protected]
Antiviral Therapy and Resistance
P226
Screening and identification of inhibitors against Japanese Encephalitis Virus from an FDA-approved drug library
S. Wang1
1Chinese
Academy of Sciences, Wuhan Institute of Virology, Wuhan, China
Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. It is
estimated to be the leading cause of 67,900 Japanese encephalitis cases annually. However, there is no approved drug
available for JEV-specific treatment and the vaccines are not effective to all clinical isolates of JEV. There is an urgent need
to develop antiviral drugs against JEV infection. In this study, we performed a high-throughput screening against JEV from
an FDA-approved drug library. Through the RVP (replicon recombinant viral particles containing luciferase)-based screening
system, 28 hit compounds with inhibition ratio > 90% were identified in the first round screening. Then 9 hit compounds were
further confirmed by dose-dependent and cell viability assays. The antiviral effects of 5 drug candidates with SI >10 against
JEV AT3 strain infection were confirmed by plaque assay, quantitive PCR and indirect immunofluorescence assay. In the the
time-of-addition assay, the 5 drug candidate were all proved to impair the replication stage of infection. In addition, the target
of 3 drug candidates was calcium channels and three other calcium-associated inhibitors were also proved to affect JEV
infection in a dose-dependent manner. Therefore, the 3 identified calcium channel inhibitors might inhibit the activities of
calcium channel and decrease the calcium concentrations, thus affect JEV replication. Through selection of manidipine (the
most effective drug candidate)-resistant virus, we found substitution of glutamine at amino acid 130 (Q130) of the
nonstructural protein 4B (NS4B) with arginine might confer JEV resistance to manidipine. Moreover, the antiviral activities of
5 candidates on some other flavivirus including Zika virus were also validated. Our study provided 5 antiviral candidates and
identified a novel antiviral target for treatment of JEV infection.
Corresponding author:
Shaobo Wang
[email protected]
Antiviral Therapy and Resistance
P227
Nafamostat is a new potent inhibitor of trypsin-independent influenza A viruses spread
P. Zmora1,2, A. S. Moldenhauer2, S. Erdelt2, F. Dahlmann3, K. Sewald3, Y. Zhao4, C. Bruns4, S. Pöhlmann2
1Max
Planck Institute of Dynamics of Complex Technical Systems, Magdeburg, Germany
Primate Center GmbH, Infection Biology Unit, Göttingen, Germany
3Fraunhofer Institute of Toxicology and Experimental Medicine, Preclinical Pharmacology and Immunology, Hanover, Germany
4Otto von Guericke University, Department of Surgery, Magdeburg, Germany
2German
The influenza A viruses (FLUAV) depend on cleavage-activation of their hemagglutinin (HA) protein by type II
transmembrane serine proteases (TTSP), i.e. TMPRSS2, to acquire infectivity and the responsible enzymes are potential
targets for antiviral intervention. Therefore, the aim of this study was to identify novel serine proteases inhibitors which
effectively block HA proteolytic processing by TTSPs.
Here, we showed that human oesophagus derived cells (OE19, OE21 and OE33) support trypsin independent spread of
influenza A viruses. Additionally, by siRNA knock-down we demonstrated that TMPRSS2 facilitate the HA proteolytic
processing in OE19 and OE33 cells, while TMPRSS4 is responsible for the trypsin-independent FLUAV spread in OE21
cells. Moreover, we tested 3 novel serine protease inhibitors: nafamostat, gabexate and alvelestat, and found that while
gabexate and alvelestat did not affect HA cleavage and activation at all, nafamostat blocks HA proteolytic cleavage by all
known TTSPs which results in the inhibition of virus spread in the cell culture. Additionally we demonstrated that nafamostat
blocks the FLUAV HA activation and therefore virus spread in the non-human primate (Cynomolgus and Rhesus macaque)
precision cut lung slices (PCLS) without any cytotoxic effect.
Collectively, our results identify novel serine protease inhibitor – nafamostat, which effectively blocks the HA proteolytic
cleavage and inhibit influenza A virus spread in cells supporting trypsin independent spread of the virus as well as in the nonhuman primate PCLS.
Corresponding author:
Pawel Zmora
[email protected]
Evolution of Viruses – Viruses in Evolution
P228
Comparative study of in vivo evolution of hepaciviral glycoproteins in their natural hosts
D. Todt1, B. Richard J.P.1, S. Walter1, J. Doerrbecker1, S. Pfaender1,2, H. Wedemeyer3, T. Pietschmann1, E. Steinmann1
1Twincore
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
of Bern, Institute of Virology and Immunology, Vetsuisse Faculty, Bern, Switzerland
3Hanover Medical School, Department of Gastroenterology, Hepatology und Endocrinology, Hanover, Germany
2University
Introduction: Although recent developments in hepatitis C virus (HCV) therapy have led to more powerful and better
tolerated drugs, the virus is still a major health burden. The restricted host species tropism of HCV hampered the
development of a robust immunocompetent animal model in the past, impeding mechanistic analysis of virus pathogenesis,
immune control and prophylactic vaccine development. A new approach in the development of HCV animal models could be
the application of another phylogenetically closely related member of the genus hepacivirus.
Objectives: The non-primate hepacivirus (NPHV), which is the closest homolog of HCV described to date, was initially
identified in horses and could drive the understanding of hepacivirus biology in vivo. To expedite the use of NPHV as
surrogate model for HCV infections, we compare in vivo evolution of the viral glycoproteins in their natural hosts.
Material & Methods: In this study, we made use of next generation sequencing methods to extensively study NPHV
glycoprotein mutant spectra in periodically drawn serum samples of several chronically as well as acutely infected horses. In
addition, we compared the intra-host viral population identified in serum and liver samples.
Results: Pronounced differences in the number of sites exhibiting single nucleotide variations (SNV) as well as the
frequencies of their occurrences between viral populations in acutely and chronically infected horses was observed, whereas
no differences between serum and liver samples were noted. In addition, the pattern of SNVs was stable over the monitored
period of 1.5 years and their frequencies did not change significantly. Interestingly, we could not identify significant
mutational cold or hot spots within E1E2 that correlate with HCV hypervariable regions (HVR).
Conclusion: A unique collection of sera from acutely and chronically HCV infected patients receiving different antiviral
treatment regimens is currently under investigation employing the same methods. Understanding the differences and
similarities between this newly discovered virus and HCV will advance our knowledge of hepacivirus evolution and
pathogenesis in vivo.
Corresponding author:
Daniel Todt
[email protected]
Evolution of Viruses – Viruses in Evolution
P229
Identification of novel polyomaviruses in mammals from multiple orders and experimental analysis of large T
antigen mRNA splicing
B. Ehlers1, C. Walter1, S. Liebmann1, N. Ben Salem1, D. Fischer1, H. Preugschas1, N. Buchwald1, S. Broll1, T. Teichmann1,
M. Pauly1, C. A. Szentiks2, M. Martin3, B. Pérez de Val3, D. Richter4, A. Gedvilaite5, R. G. Ulrich6, F. H. Leendertz1, S.
Calvignac-Spencer1
1Robert
Koch Institute, Berlin, Germany
Institute of Zoo and Wildlife Research (IZW), Berlin, Germany
3Centre de Recerca en Sanitat Animal (CReSA), Barcelona, Spain
4Technical University Braunschweig, Braunschweig, Germany
5Vilnius University, Vilnius, Lithuania
6Friedrich Loeffler Institute, Greifswald - Isle of Riems, Germany
2Leibniz
Question: To get further insight into polyomavirus (PyV) evolution, we aimed at (i) identifying yet unknown PyVs in mammals
that are close relatives of or display environmental proximity to humans, (ii) determining full genomes and (iii) annotating
coding regions. As the large T antigen (LTAg) is translated from spliced mRNA, experimental confirmation of predicted splice
sites is important. Unfortunately, experimental data are available for only few of the >100 known PyVs. Thus, an element of
uncertainty is inherent in splice site annotations of most PyVs. Here, we predicted LTAg mRNA splice sites in newly
discovered PyVs and aimed at experimentally confirming them in cell culture.
Methods: PyVs were identified with generic PCR. Full genomes were amplified with long-range PCR and sequenced. PyV
assignment to genera was done by LTAg-based phylogeny. Host association was analysed with PyV-specific PCR. LTAg
mRNA splice sites were predicted in silico by using the Human Splicing Finder database
(http://www.umd.be/HSF3/index.html). Splice sites were experimentally determined by transfection of synthetic PyV early
regions into several mammalian cell lines, mRNA was isolated, cDNA synthesized and PCR with primers flanking predicted
splice sites performed. PCR products were sequenced and compared with the genomic sequences.
Results: Fourteen PyVs were discovered in hosts of 5 mammalian orders, and assigned to the taxonomic genera AlphaBeta-, Deltapolyomavirus or a proposed novel genus. Full genome sequence and the natural host of the novel PyVs were
determined. Coding sequences were annotated. For nearly all early regions the predicted LTAg splice sites could be
confirmed experimentally. As these splice sites are highly conserved in closely related PyV genomes, the experimental data
were used to critically review LTAg splice site annotations of PyV genome sequences deposited in GenBank. For 10/104
genomes, the results suggested a revision of the previously annotated LTAg splice sites.
Conclusion: PyVs are more ubiquitous than previously thought. At large, their evolution seems to be mostly driven by
codivergence with their hosts. Experimental identification of splice sites is important for all kinds of LTAg analyses, including
LTAg-based taxonomical classification of novel PyVs.
Corresponding author:
Bernhard Ehlers
[email protected]
Evolution of Viruses – Viruses in Evolution
P230
Puumala virus prevalence and S-segment sequence evolution in the bank vole (Myodes glareolus)
F. Binder1, S. Jadgmann1, U. M. Rosenfeld1, R. Ryll1, D. Reil2, C. Imholt2, G. Heckel3,4, J. Jacob2, R. G. Ulrich1
1Friedrich
Loeffler Institute, INNT, Greifswald - Isle of Riems, Germany
Kühn Institute, Münster, Germany
3University of Bern, Institute of Ecology and Evolution, Bern, Switzerland
4Swiss Insitute of Bioinformatics, Lausanne, Switzerland
2Julius
The S-segment of vole-borne hantaviruses contains two major overlapping open reading frames (ORF) coding for the
nucleocapsid (N) protein and a non-structural (NSs) protein, an interferon-antagonist. In Germany, Puumala virus (PUUV),
which is transmitted by the bank vole (Myodes glareolus) is the main causative agent of human hantavirus infections. The
aim of this study was to identify the influence of bank vole population dynamics on PUUV prevalence and sequenceevolution regarding the N- and N/NSs coding region of the PUUV S-segment. For this purpose, blood and lung tissue
samples of 851 bank voles trapped during 2010-2014 in Baden-Wuerttemberg and North Rhine-Westphalia were analyzed.
Our investigations showed that 242 animals (28.4%) were positive for PUUV-specific antibodies, whereas in 194 animals
(22.8%) PUUV-specific RNA could be detected. Simultaneous presence of PUUV specific antibodies and RNA could be
detected in 141 (16.7%) voles. In the hantavirus outbreak years 2010 and 2012 a higher PUUV prevalence could be
detected compared to 2011/2013/2014. From spring to autumn a decrease in the prevalence of persistent infections was
observed in bank voles. Sequence types of the N-ORF and NSs-ORF showed temporal and/or local distribution variation.
During the whole observation period, one sequence-type each could be detected in Baden-Wuerttemberg as well as in North
Rhine-Westphalia. The NSs-ORF showed a higher number of non-synonymous mutations than the overlapping and nonoverlapping N-ORF regions. Further analysis of sequence variations revealed positive selection for the NSs-ORF and a
negative (purifying) selection for the non-overlapping N-ORF. These results suggest an influence of bank vole population
dynamics on molecular PUUV evolution.
Corresponding author:
Florian Binder
[email protected]
Evolution of Viruses – Viruses in Evolution
P231
Molecular characterization of avian influenza H9N2 isolates from recent outbreaks in poultry of Bangladesh
R. Parvin1, J. A. Begum1,2, K. Heenemann2, T. Vahlenkamp2, E. H. Chowdhury1, M. R. Islam1
1Department
2University
of Pathology, Faculty of Veterinary Science, Mymensingh, Bangladesh
of Leipzig, Institute of Virology, Leipzig, Germany
Avian influenza virus (AIV) H9N2 is wide spread in nature, generally caused mild illness and became panzootic in the mid1980s among chickens, ducks, turkeys, pheasants, quails, ostrich and migratory birds. In Bangladesh both, highly
pathogenic AIV H5N1 and AIV H9N2 viruses are co-circulating among commercial poultry and village chickens since their
first reported cases from a retrospective analysis of a sample from a poultry farm collected in September, 2006. Since then
H9N2 viruses have been frequently isolated from different live bird markets, environments (wild/migratory birds) as well as
from commercial poultry flocks in Bangladesh. Due to a limitation in sequence data from Bangladeshi field isolates there is a
gap on knowledge in virus characteristics and their evolutions. The aim of the study was to enlarge sequence information to
follow the genetic evolution and pandemic potential of H9N2 viruses currently circulating in Bangladesh. Fourteen (14)
different samples from commercial chickens and ducks with clinical signs of respiratory infections were collected. Real time
RT-PCR targeting M gene confirmed 11 out of 14 samples were positive for influenza type A virus. Subtyping was performed
by RT-PCR using 383bp and 244 bp fragments of the H9 and N2 gene, respectively. Full length amplification of HA and NA
gene of three selected viruses were completed. Nucleotide blast search and sequence homology analysis of HA and NA
genes revealed 94-99 % nucleotide homology of the isolated H9N2 viruses to other contemporary Bangladeshi H9N2
isolates along with other South Asian and Middle Eastern isolates. The HA gene of the Bangladeshi AIV H9N2 exhibited
particular genetic features and mostly belonged to the G1 lineage which has the ability to donate gene to other HPAI viruses.
Keywords: Avian Influenza H9N2, Phylogeny, Genetic Evolution, Bangladesh Poultry
Corresponding author:
Rokshana Parvin
[email protected]
Evolution of Viruses – Viruses in Evolution
P232
PoSeiDon: a web server for the detection of evolutionary recombination events and positive selection
M. Hölzer1, M. Marz1
1Friedrich
Schiller University Jena, Faculty of Mathematics and Computer Science, Jena, Germany
Question: So-called 'arms races' between host and virus result in high selection pressure on the host to evolve a defense
against the pathogens, while the virus itself establishes countermeasures to evade the host immune system. One approach
to study selection pressure involves the comparison of orthologous genes to detect sites (codons) that underwent positive
(diversifying) selection. For instance, certain amino acid changes are favored if they increase the hosts fitness against a viral
infection. In that case, the ratio between the non-synonymous (dN) and synonymous (dS) substitution rate may reach values
greater than 1 and we call such a site positive selected. Since recombination can have a profound impact on evolutionary
processes and can adversely affect the accurate detection of positive selection, screening for breakpoints to define
recombinant parts should be a default step in each comparative evolutionary study.
Material & Methods: We developed a pipeline called PoSeiDon, that comprises an assembly of different scripts and tools
(Fig. 1) that allow for the detection of recombination and positive selection in protein-coding sequences. The input is a
multiple FASTA file of homologous coding sequences that is automatically transferred into a codon-based alignment. Based
on the alignment, PoSeiDon calculates putative recombination breakpoints, maximum-likelihood-based phylogenetic trees,
dN/dS ratios at each site and their impact on the positive selection.
Results: With this, PoSeiDon is an easy-to-use, web-based pipeline for the accurate detection of site-specific positive
selection and recombination events in protein-coding sequences. PoSeiDon does not only detect positively selected sites in
an alignment of homologous sequences, but also possible recombination events, that could otherwise adversely affect the
positive selection detection. The output of PoSeiDon is summarized in a user-friendly and clear manner, allowing researches
to study positive selection in various genes.
Conclusion: Viral infections are thought to be one of the major driving forces regarding selective pressure acting on living
organisms. With PoSeiDon, an easy-to-use and web-based pipeline, researchers can detect positively selected sites to gain
insights into the evolution of a gene to develop counter measurements against viruses that are in an arms-race with their
host.
Figure 1: Workflow of the PoSeiDon pipeline and example of the HTML output.
PoSeiDon is freely available at http://www.rna.uni-jena.de/poseidon.
Corresponding author:
Martin Hölzer
[email protected]
Figure 1
Evolution of Viruses – Viruses in Evolution
P233
Whole genome sequencing of eleven herpes simplex virus type 2 isolates
K. Döring1, M. Groth2, R. Zell1, A. Sauerbrei1
1University
2Leibniz
Hospital Jena, Institute of Virology and Antiviral Therapy, Jena, Germany
Institute on Aging, Fritz Lipmann Institute, Genome Research, Jena, Germany
Introduction: Herpes simplex virus type 2 (HSV-2) commonly causes genital herpes infections which belong to the most
frequent sexually transmitted diseases worldwide. Until 2014, the genome of only one HSV-2 strain (HG52) was sequenced
and, hence, little was known about the genomic DNA variation of HSV-2 strains.
Objectives: In this study, we sequenced the genomes of ten clinical HSV-2 isolates and of the laboratory reference strain
MS. Aim of this work was to investigate possible sequence variation due to their geographic origin.
Patients & Methods: Between 1993 and 2011, ten clinical HSV-2 isolates were collected in Germany from patients
originating from various countries of Europe (e.g. Germany, Bulgaria, Italy), South America (Brazil, Ecuador), and Southeast
Asia (Thailand). For sequence determination, the Illumina sequencing technology was used. The nearly complete genomes
were compared to 41 recently published genomes and the laboratory reference strain HG52. Thus, a non-redundant genome
alignment consisting of UL, IRL, a", IRS and US of 53 genomes was examined.
Results: The overall mean p-distance of all 53 HSV-2 sequences was d=0.266%, which means that HSV-2 has a lower
diversity than HSV-1 (d=0.756%). Phylogenetic analyses of the non-redundant genomes revealed no evidence of genotypes,
geographic clusters or groups characterized by uniform single nucleotide polymorphisms. Likewise, phylogenetic trees based
on subgenomic sequences (US4, US7, US8) failed to demonstrate any consistent typing.
Conclusion: Phylogenetic analysis confirmed that HSV-2, unlike HSV-1, has high sequence conservation without
remarkable differences. Inconsistent clustering of non-redundant genomes and several open reading frames argues against
genogroups as suggested by Norberg et al. (2007).
Corresponding author:
Kristin Döring
[email protected]
Evolution of Viruses – Viruses in Evolution
P234
Evolutionary dynamics and endemic co-circulation of avian influenza H5N1 and H9N2 in Egypt provoke diagnostic
challenges
M. Naguib1, M. Beer1, T. C. Harder1
1Friedrich
Loeffler Institute, Greifswald - Isle of Riems, Germany
Highly pathogenic avian influenza virus (HP AIV) H5N1 of clade 2.2 in Egypt continues to evolve since its introduction into
Egypt in 2006. The endemic status of these zoonotic viruses not only caused sharp losses in poultry production but also
seriously threatened public health in Egypt. Since 2011, potentially zoonotic H9N2 viruses likewise have established an
endemic status in poultry in Egypt. The evolutionary dynamics and demographic viral history was inferred by Bayesian
skyline plot and BEAST analysis for each clade and lineage of these viruses revealing fluctuations over time likely related to
host population densities and vaccine-driven effects.
The endemic co-circulation of the two subtypes increases risks for mixed infections of poultry with HP AIV H5N1 and H9N2
which raises concerns of the generation and emergence of reassortants between these viruses. Hence, the timely detection
of such reassortants which might express unforeseeable phenotypic properties is of public health concern in Egypt. As a
rapid tool for detection of reassortants discriminative SYBR-green reverse transcription real-time PCR assays, targeting the
internal genes of the Egyptian H5N1 and H9N2 virus lineages, were developed to screen viral RNAs extracted from virus
isolates and clinical samples.
In addition, experimental infection studies in chickens carried out at FLI"s biosecurity animal facilities showed that prior
LPAIV H9N2 infection but not vaccination influenced the course of consecutive infection with lethal Egyptian HPAIV H5N1.
Lower virus shedding titers and marginally prolonged survival were recorded in chickens with AIV H9N2-based heterologous
post-infection but not post-vaccination immunity upon challenge with a lethal dose of HPAIV H5N1. Thus, a high incidence of
H9N2 infections in poultry populations in Egypt may interfere with syndromic surveillance of HPAIV infection.
Corresponding author:
Mahmoud Naguib
[email protected]
Evolution of Viruses – Viruses in Evolution
P235
V-table©
The virosphere structured in one comprehensive table
V. Novitska1
1Taras
Shevchenko National University of Kyiv, Virology, Kiev, Ukraine
V-table© is an interactive spherical table of viruses arranged according to their taxonomy, structure and phylogeny
simultaneously. The structure of V-table© provides an overview on morphology, chemical characteristics and host type of 111
viral families and 26 unassigned genera in one comprehensive table. It allows organizing and comparing viruses,
understanding the relationships among diverse viruses, facilitates learning virology and is designed to be expanded.
The structure of V-table© is based on arrangement of multiple parameters, which define for each viral species and family
unique place in a table on a surface of a sphere. The place in V-table© is defined by specific characteristics of listed
parameters.
Each parameter covers the entire surface of the sphere, dividing it into a certain number of parts according to the
characteristics of a specific parameter. Each parameter consists of distinct number of characteristics e.g. the parameter II Type of genetic information - has 7 characteristics representing 7 types of genetic information of viruses according to
Baltimore classification. The parameters I – VI have multiple characteristics. The parameters are type of host, type of genetic
information, type of capsid symmetry, T-number (if any), capsid size and genome size.These parameters divide the spherical
table into layers (rows), vertical slices (columns), sectors and cards and define the unique place in a table for every viral
family according to their set of characteristics. Viral species and genera within a particular family usually have identical or
similar characteristics of parameters. Thus, card of viral family is a unit of V-table©. Along with parameters, which define the
place of the specific viral family in V-table©, there are supporting parameters VII–X, which usually have only two
characteristics (variants).
The functions of V-table© are providing structured and easy to overview information about every virus, organizing knowledge
about viruses, analyzing and comparing viral species, genera, families and clades according to one or several specific
parameters simultaneously. Using V-table© helps perceiving and understanding viral diversity. It is the solution for gathering
information about viruses in a structured and informative manner.
Copyright © 2016 [COPYRIGHT VICTORIA NOVITSKA]. All Rights Reserved.
Corresponding author:
Victoria Novitska
[email protected]
Evolution of Viruses – Viruses in Evolution
P236
Ancient recombination events between human herpes simplex viruses
S. Burrel1, D. Boutolleau1, D. Ryu2, H. Agut1, K. Merkel2, F. H. Leendertz2, S. Calvignac-Spencer2
1Hopital
2Robert
La Pitie - Salpetriere, Paris, France
Koch Institute, Berlin, Germany
Introduction: Human herpes simplex virus 1 and 2 (HSV-1 and 2; species Human alphaherpesvirus 1 and 2, genus
Simplexvirus, subfamily Alphaherpesvirus, family Herpesviridae, order Herpesvirales) usually cause recurrent mucosal
lesions but are also, more rarely, responsible of severe diseases like neonatal morbidity and meningo-encephalitis. Despite
their (relatively) close relatedness, these two pathogens have unambiguously been considered as evolutionary independent
units.
Objectives: In an effort to further elucidate the evolutionary history of HSV-2, we aimed at sequencing and analyzing the
genomes of multiple isolates representing a variant characterized by very divergent UL30 gene sequences and an apparent
association with the African continent.
Material & Methods: We derived isolates from 18 clinical samples from which we sequenced nearly full genomes using insolution hybridization capture coupled to next-generation sequencing. We compared these genomes to those publicly
available, performing a number of phylogenomic analyses.
Results: Surprisingly, genome-wide recombination analyses showed that all HSV-2 genomes sequenced to date comprise
HSV-1 fragments. Phylogenomic trees also showed that two main HSV-2 lineages exist. One lineage is mostly restricted to
sub-Saharan Africa while the other has reached a global distribution. Interestingly, only the worldwide lineage is
characterized by ancient recombination events with HSV-1.
Conclusion: Our findings highlight the complexity of HSV-2 evolution, a virus of likely zoonotic origin which later recombined
with its human-adapted relative. They also suggest that co-infections with HSV-1 and 2 may have genomic and potentially
functional consequences and should therefore be monitored more closely.
Corresponding author:
Sébastien Calvignac-Spencer
[email protected]
Evolution of Viruses – Viruses in Evolution
P237
In silico analyses of evolutionary dynamics of N-linked glycosylation sites within the influenza A virus
hemagglutinin (HA) protein
D. Anhlan1, W. Hafezi1, J. Kühn1, P. Nymadawa2, S. Ludwig1, E. R. Hrincius1
1University
2Mongolian
of Münster, Institute of Virology, Münster, Germany
Academy of Medical Sciences and National Influenza Center, National Center of Communicable Diseases, Ulaanbaatar, Mongolia
Influenza A viruses (IAV) are very dynamic pathogens with a wide host range. The rapidly mutating Hemagglutinin (HA)
surface protein is one of most critical IAV pathogenicity determinant. The HA glycoprotein is posttranslationally modified by
the addition of N-linked glycans, which can contribute to the escape from existing host immunity. In addition, differences in
glycosylation patterns of the IAV HA have been linked to IAV induced immune responses and pathogenicity as well.
Therefore, knowledge about HA glycosylation sites evolution (time/spatial scale) has to be gained and their biological
consequences have to be studied.
To track N-glycosylation sites in recent history, we analyzed HA amino acid sequences of human H1N1 IAV from 2009
(introduction of the 2009 pandemic H1N1) until 2016 and human H3N2 IAV from 2010 until 2016. According to our results for
the existence of potential N-linked glycosylation sites (NXS/T motif, with X for any aa except proline) within the HA protein,
evolutionary dynamics of N-glycosylation sites for human H1N1 and H3N2 HA were found. Interestingly we observed an
unique emergence of a potential glycosylation site on position 179 of most recent seasonal H1N1 IAV. Since the introduction
of the 2009 pandemic H1N1 into humans, the percentage of HA proteins carrying this motif raised from as less as 1.0% to
over 90% of investigated sequences in 2016. Similarly, the executed analyses revealed evolutionary dynamics in H3N2 HA
proteins regarding the existence of N-linked glycosylation sites as well. Deeper, more directed HA sequence analyses will
shed further light into the involvement of socio-economic and cultural living circumstances (e.g. densely populated versus
rural areas in different parts of the world (e.g. Europe vs. Mongolia)) as potential driving forces of IAV adaptation and coevolution. Studying the biological consequences of the highlighted glycosylation site 179 in the H1N1 HA by analysing recent
clinical isolates with differences in HA N-linked glycosylation patterns will clarify the impact on IAV infection biology as well.
Taken together, our results suggest that IAV have a potential requirement for specific HA glycosylation sites during
adaptation to and co-evolution with the human host and highlight that understanding the biological impact of these sites is
demanded.
Corresponding author:
Darisuren Anhlan
[email protected]
Evolution of Viruses – Viruses in Evolution
P238
Virulence determinants of a recent H7N7 avian influenza virus in chickens
D. Scheibner1, J. Veits1, T. C. Mettenleiter1, E. M. Abdelwhab1
1Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
Avian influenza viruses (AIVs) have a multipartite single-stranded RNA genome with eight gene segments coding for at least
10 viral proteins. According to the variation in the antigenicity of the surface glycoproteins hemagglutinin (HA) and
neuraminidase (NA), there are 16 and 9 HA and NA distinct subtypes, respectively. Some H5 and H7 viruses exhibit high
virulence in poultry inducing up to 100% mortality. These highly pathogenic (HP) strains evolve from low pathogenic (LP)
progenitors by reassortment and/or point mutations where changing the monobasic HA cleavage site (CS) in the LP viruses
to a polybasic motif is essential for the activation of the HP virus in multiple organs by furin-like proteases. In 2015, both LP
and HP H7N7 AIVs were isolated from farmed poultry in the same farm in Emsland, Germany providing a unique opportunity
to analyze in detail requirements for transition from LP progenitor to HP progeny. In the current study, genetic relatedness of
both viruses and virulence markers of the HP virus in chickens were studied using reverse genetics and animal experiments.
Four viruses were generated: LP, HP, LP carrying polybasic CS (LP_pCS) and LP carrying the whole HA from HP
(LP_HAhp). Except for the recombinant LP, after intravenous infection of 6-week old chickens all other tested viruses
exhibited an Intravenous Pathogenicity Index (IVPI) exceeding 1.2 specifying HPAIV. However, whereas after oculonasal
inoculation of the recombinant HPAIV, all inoculated animals and their contacts animals died, insertion of the polybasic CS or
the HA from HPAIV into the progenitor LPAIV increased virulence but not to the level of the isogenic HPAIV. Thus, mutations
in other gene segments appear to be required as well for the exhibition of full pathogenicity and efficient transmission of the
recent German H7N7.
Corresponding author:
David Scheibner
[email protected]
Evolution of Viruses – Viruses in Evolution
P239
Functional characterization of adaptive mutations during the West African Ebola virus outbreak
E. Dietzel1,2, G. Schudt1, V. Krähling1,2, M. Matrosovich1,2, S. Becker1,2
1Philipps
2German
University Marburg, Institute of Virology, Marburg, Germany
Centre for Infection Research (DZIF), Partner Site Gießen-Marburg-Langen, Marburg, Germany
Introduction: Ebola virus (EBOV) is a filovirus and causes Ebola virus disease which is characterized by severe fever
accompanied by systemic inflammation and damage to the endothelial cell barrier leading to shock and multi-organ failure
with high case/fatality rates.
The Ebola virus outbreak in West Africa claimed more than 11,000 lives, threatened to destabilize a whole region, and
showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African
outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and
disease remained obscure.
Objectives: We investigated the functional significance of three nonsynonymous mutations that emerged early during the
West African EBOV outbreak.
Material & Methods: A newly developed virus-like particle was used to analyze the impact of the mutations on viral
transcription and replication and reverse genetics were used to obtain recombinant EBOV for virus growth and growth
competition analyses.
Results: Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three
mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding
domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating
nucleoprotein NP. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral
transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of
GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth
advantage over the prototype isolate Makona C7 lacking the mutations.
Conclusion: Virus variants with improved fitness emerged early during the West African EBOV outbreak. The adaptive
changes in the Ebola virus genome during virus circulation in humans prompt further studies on the potential role of these
changes in virus transmissibility and pathogenicity.
Corresponding author:
Erik Dietzel
[email protected]
Evolution of Viruses – Viruses in Evolution
P241
Electrostatics plays important role in transcription regulation in viral genomes
A. Osypov1,2, G. Krutinin1, E. Krutinina1, S. Kamzolova1
1ICB
RAS, Pushchino MR, Russian Federation
RAS, Moscow, Russian Federation
2IHNA&NPh
DNA is highly charged and electrostatics influences its interactions with proteins.
E. potential (EP) is distributed non-uniformly along DNA molecule and correlates (not exactly) with GC content, strongly
depending on the sequence arrangement and its context. Binding sites of transcription factors of different protein families in
different taxa are located in long areas of high EP. EP distribution on transcription factors protein molecule surface reflects
that of their binding sites. Promoters in average have high value and heterogeneity of EP profile. The transcription starting
sites of prokaryotic genomes are characterized by extensive (hundreds of bp) zone of high EP and some peculiarities directly
around TSS. This is associated with protein binding and formation of physical properties due to transcription machinery.
Specific details of the TSS EP architecture are similar in related taxa. A direct correlation was found between the potential
value and binding frequency of RNA polymerase to phage lambda DNA, supporting the idea of the role of electrostatics in
these interactions. There is a correspondence between the scale of the electrostatic profile patterns of promoter regions for
bacterial and viral RNA polymerases and these proteins size. Direct correlation between the promoters biological functions
and their fine electrostatic structure was shown in T7 and T3 phage for the virus RNAP promoters. Promoters from mutated
T7 bacteriophage demonstrate adaptation to RNA polymerase from T3 bacteriophage by changing their electrostatic
properties. Specific electrostatic up-elements, which are responsible for modulating promoter activities through ADPribosylation of the RNA polymerase alpha-subunit were identified in the far upstream regions of T4 phage early promoters, E.
coli ribosomal, and some other promoters. Strong lambda and lambda-like phage promoters have pronounced electrostatic
up-elements, the sequence texts of which are quite different, compared to the absence of it in weak promoters. Other
genome elements, such as terminators, attachment sites etc., also show electrostatic peculiarities.
The work was supported by RFBR grants 14-44-03683 & 16-04-01865
References:
A. A. Osypov et al. (2012) DEPPDB – DNA Electrostatic Potential Properties Database. Electrostatic Properties of Genome
DNA elements, J Bioinform Comput Biol, 10(2) 1241004
Corresponding author:
Alexander Osypov
[email protected]
Evolution of Viruses – Viruses in Evolution
P242
Genesis, evolution and prevalence of H5N6 avian influenza viruses causing sporadic human infections
Y. Bi1, Q. Chen1, Q. Wang1, J. Chen1, T. Jin1, G. Wong1, J. Liu1, W. Liu1, Y. Liu1, W. Shi1, D. Liu1, G. F. Gao1
1Chinese
Academy of Sciences, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Beijing, China
A history of exposure to live poultry and/or live poultry markets (LPMs) was documented for the majority of human AIV
infections. Furthermore, many studies have described the important role of LPMs in the evolution of avian influenza viruses
(AIVs). In 2014, a novel H5N6 reassortant was first reported causing human infection. To date, a total of 17 human infections
with H5N6 have been reported, which all emerged in China in addition to more than 16 outbreaks in poultry. However, the
genesis, evolution, and situation of the novel H5N6 AIV has not been thoroughly illuminated. Constant surveillance of LPMs
is currently the best way to predict and identify emerging AIVs that pose a potential threat to public health. Through
surveillance of LPMs from 16 provinces and municipalities in China during 2014–2016, we identified 3,174 AIV positive
samples and isolated and sequenced 1,135 AIVs covering 31 subtypes. Our analysis shows that H5N6 has replaced H5N1
as one of the dominant AIV subtypes in southern China, especially in ducks. Phylogenetic analysis reveals that H5N6 arose
from reassortments of H5 and H6N6 viruses, with the hemagglutinin and neuraminidase combinations being strongly lineage
specific H5N6 viruses constitute at least 34 distinct genotypes derived from various evolutionary pathways. Notably,
genotype G1.2 virus, with internal genes from the chicken H9N2/H7N9 gene pool, was responsible for at least five human
H5N6 infections. Our findings highlight H5N6 AIVs as potential threats to public health and agriculture.
Corresponding author:
Yuhai Bi
[email protected]
Vaccines
P243
Cell-permeable capsids as universal antigen carrier platform for the induction of an antigen-specific CD8+ T-cell
response
S. Akhras1, M. Toda2, K. Boller3, K. Himmelsbach1, F. Elgner1, S. Scheurer2, M. Gratz1, S. Vieths2, E. Hildt1,4
1Paul
Ehrlich Institute, Virology, Langen, Germany
Ehrlich Institute, Allergology, Langen, Germany
3Paul Ehrlich Institute, Immunology, Langen, Germany
4German Center for Infection Research (DZIF), Braunschweig, Germany
2Paul
Introduction: Vaccine platforms that can be flexibly loaded with various antigens can contribute to decrease the response
time to emerging infections. For many pathogens and chronic infections, the induction of a robust cytotoxic T lymphocytes
(CTLs) response is desirable to control or eliminate the infection. Such a response can be induced by delivering the antigen
into the cytoplasm of antigen-presenting cells (APCs).
Objectives: Our aim is to generate a universal vaccine platform that can be flexibly loaded with variety of antigens for the
induction of a robust immune response.
Results & Methods: Based on the structurally well characterized hepatitis B virus (HBV) capsid, cell-permeable carrier
capsids were generated to serve as a universal vaccine platform. The cell permeability was achieved by fusion of the cellpermeable translocation motif (TLM) peptide to the capsid-forming core protein. In addition, the carrier capsids were further
modified by inserting strep-tag into the spike tip forming domain of the core protein. This insertion enabled the flexible
loading of the assembled capsids, via strep-tag/streptavidin binding, with various antigens. The successful antigen loading
on the surface of the capsids was proved by electron microscopy, density gradient centrifugation and surface plasmon
resonance spectroscopy. Confocal immunofluorescence microscopy demonstrated the capacity of these cell-permeable
carrier capsids to transport their antigen cargo across the plasma membrane into the cytoplasm. In order to study the
suitability of the generated cell-permeable carrier capsid to serve as a universal vaccine platform, ovalbumin was used as a
model antigen by loading it onto the surface of the carrier capsid. Compared to free ovalbumin, ovalbumin-loaded carrier
capsid enabled significantly enhanced activation of the APCs and ovalbumin-specific CD8+ T-cells. This activation was
correlated with an enhanced specific killing capacity of the activated ovalbumin-specific CTLs.
Conclusion: These data demonstrate the capacity of the TLM-capsid to serve as a universal carrier to deliver foreign
antigens into the cytoplasm of APCs, leading to enhanced MHC class I-mediated presentation and the induction of an
antigen-specific CTLs response.
Corresponding author:
Sami Akhras
[email protected]
Vaccines
P244
Development of a new approach for therapeutic vaccination against chronic HBV infection
T. Zahn1, S. Akhras1, E. Hildt2
1Paul
Ehrlich Institute, Virology, Langen, Germany
Center for Infection Research (DZIF), Braunschweig, Germany
2German
There are 300 million people worldwide suffering from chronic HBV infection. In many cases, an insufficient CTL response is
causative for the establishment of a chronic infection leading to an incomplete elimination of infected hepatocytes. This
project aims to develop a new strategy for combatting chronic infection based on a preventive and therapeutic vaccine.
Therefore, a novel vaccine platform was developed which is based on cell-permeable virus like particles (VLPs). This
universal antigen carrier can be flexibly loaded with various antigens. Due to the highly ordered structure of the antigens
presented on the surface of the carrier, a robust B-cell response is triggered – the membrane permeability enables the
transfer of the antigen into the cytoplasm of antigen presenting cells (APCs) and thereby the induction of a CTL response.
Cell permeability of the carrier capsids is achieved by fusion of the translocation motif (TLM), a 12 aa encompassing peptide,
to the N-terminus of HBV core protein via a flexible linker. By insertion of a strep-tag into the spike tip region of the core
protein, various HBV-specific fusion proteins conjugated with streptavidin can be loaded onto the surface of the carrier
capsid. Due to the membrane permeability of the carrier capsid, the antigen-cargo is transferred into the cytoplasm of APCs,
enabling the immunoproteasomal processing and subsequent MHC class I-dependent presentation that finally leads to an
efficient induction of a CTL-response. In this way, HBV-positive cells can be eliminated by induction of a robust T cell
response and reinfection of hepatocytes prevented by induction of protective antibodies.
For this purpose, cell-permeable VLPs that are loaded with various HBV-specific antigens are produced, purified and
characterized via biophysical methods. In addition, membrane permeability, antigen transfer and induction of specific CTLs
by the antigen loaded carrier capsids are examined.
Corresponding author:
Tobias Zahn
[email protected]
Vaccines
P245
Immunization with DNA plasmids coding for Crimean Congo hemorrhagic fever viral capsid and envelope proteins
and/or virus like particles induce protection and survival in challenged mice
J. Hinkula1,2, S. Devignot3,4, S. Åkerström5, H. Karlberg6, E. Wattrang5, S. Bereczky6, M. Mousavi-Jazi6, C. Risinger7, G.
Lindegren6, C. Vernersson5, J. Paweska8, P. Jansen van Vuren8, O. Blixt7, A. Brun9, F. Weber3,4, A. Mirazimi6,5,10
1University
of Linköping, Department of Clinical and Experimental Medicine, Linköping, Sweden
iInstitute, Microbiology and Tumorbiology Center, Solna, Sweden
3Justus Liebig University Giessen, Institute of Virology, Giessen, Germany
4Philips University Marburg, Institute of Virology, Marburg, Germany
5National Veterinary Institute, Uppsala, Sweden
6Folkhälsomyndigheten, Solna, Sweden
7University of Copenhagen, Department of Chemistry, Frederiksberg, Denmark
8National Institute of Communicable Diseases, Centre for Emerging and Zoonotic Diseases, Sandringham, South Africa
9Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CISA), Madrid, Spain
10Karolinska Institute, Department of Medicine, Solna, Sweden
2Karolinska
Crimean Congo Hemorrhagic fever virus (CCHFV) is a Bunyavirus causing severe hemorrhagic fever disease in humans,
with high mortality rates. Although CCHFV represents a threat for public health in many countries, there is still no FDAapproved vaccine. The requirement of high-containment laboratory and the lack of animal model severely hampered the
study of the immune response and protection of vaccine candidates.
Using the recently developed Interferon-alpha-receptor knock-out (IFNAR -/-) mouse model, which mimics the human CCHF
disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine
encoding an ubiquitin-linked version of CCHFV Gc, Gn, and N, and transcriptionally-competent virus-like particles (tc-VLPs).
Contrary to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses.
We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine.
Interestingly, there was no correlation with the neutralizing antibody titers, which were higher in the tc-VLP vaccinated mice.
In this study, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response, resisted the CCHFV
challenge. Moreover, we were also able to identify new linear B-cell epitopes regions that are very conserved between
CCHFV strains, and localized in the glycoproteins. Altogether, our results suggest that a predominantly Th1-type immune
response, combined with mainly neutralizing antibodies against the glycoproteins, provide the most efficient protective
immunity against CCHFV challenge.
Corresponding author:
Stephanie Devignot
[email protected]
Vaccines
P246
Induction of complex immune responses and strong protection against retrovirus challenge by adenovirus-based
immunization depends on the order of vaccine delivery
M. Kaulfuß1, I. Wensing1, S. Windmann1, C. Hrycak1, W. Bayer1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
In the Friend retrovirus mouse model we developed potent adenovirus-based vaccines in the past that were designed to
induce either strong Friend virus GagL85-93-specific CD8+ T cell or antibody responses, respectively. To optimize the
immunization outcome we evaluated vaccination strategies using combinations of these vaccines.
While the vaccines on their own confer strong protection from a subsequent Friend virus challenge, the simple combination
of the vaccines for the establishment of an optimized immunization protocol did not result in a further improvement of vaccine
effectivity. We demonstrate that the co-immunization with GagL85-93/leader-gag encoding vectors and envelope-encoding
vectors abrogates the induction of GagL85-93-specific CD8+ T cells, and that immunization with an envelope encoding vector
also abolishes induction of GagL85-93-specific CD8+ T cells by a subsequent immunization. The immunosuppressive effect
was long-lasting, but did not seem to be mediated by regulatory T cells. Interestingly, the antibody response to adenovirusdelivered immunogens was in fact enhanced when the mice were adenovirus-experienced from a prior immunization.
Therefore, a two-immunizations-based vaccination protocol was established that employs successive immunizations with first
the GagL85-93/leader-gag encoding vector followed by the envelope-encoding vector, which mediated robust protection in a
highly stringent mouse challenge model and was comparable in its efficacy to a live-attenuated retrovirus vaccine.
Interestingly, the Ad-based vaccine induced more prominent cellular than humoral immune responses, while the attenuated
retrovirus induced no detectable cellular but high neutralizing antibody responses, indicating that different immune
mechanisms can indeed confer a high level of protection from retrovirus infection.
Our results highlight that the order of vaccine delivery is important for the immunization outcome as retroviral surface
envelope protein can exert suppressive effects on simultaneously or subsequently administered immunogens, and preexisting immunity in Ad-experienced recipients can actually lead to improved transgene-specific antibody responses.
Corresponding author:
Wibke Bayer
[email protected]
Vaccines
P247
An mRNA-based vaccine technology for next generation prophylactic vaccines
B. Petsch1, J. Lutz1, S. Lazzaro1, K. Schmidt1, P. Baumhof1, R. Heidenreich1, M. Fotin-Mleczek1
1CureVac
AG, Vaccines, Tübingen, Germany
In recent years messenger RNA (mRNA) is increasingly used for therapeutic as well as for prophylactic vaccination. First
approaches utilized mRNA for therapeutic cancer vaccination, followed by the use of messenger RNAs (mRNA) for
prophylactic vaccination that draws much attention from industrial and academic groups.
RNActive®, an mRNA based vaccination technology was applied successfully to a variety of viral pathogens such Influenza,
Rabies, RSV, Ebola and others in animal models. We already published data on protection against lethal influenza or rabies
virus challenge infection in mice for our mRNA vaccines administered intradermal (i.d.). We also demonstrated the induction
of virus neutralizing antibody responses (VNT) in pigs for rabies virus (Petsch et al., 2012; Schnee et al., 2016). However,
immunological responses after intramuscular (i.m.) injection of this mRNA vaccine formulation were less efficient compared
to i.d.
Here we describe the next generation mRNA-based vaccines that induce potent immune responses in mice for low µg
mRNA doses when applied intramuscular. Immune responses strongly increased in terms of humoral immune response for
Influenza HA as analyzed by hemagglutinin inhibition assay (HAI) and cellular immune response as demonstrated by
intracellular cytokine staining (ICS) of T cells. Comparable immunological responses were observed for the rabies virus
glycoprotein G, as analyzed by virus neutralizing antibody assay and ICS as well.
Moreover, we could demonstrate the induction of potent and long lasting immune responses for Influenza HA in non-human
primates (NHPs) as well as strong induction of anti-rabies VNT in NHPs.
References:
Petsch B, Schnee M, Vogel AB, Lange E, Hoffmann B, Voss D, Schlake T, Thess A, Kallen KJ, Stitz L, Kramps T. Protective
efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection. Nat Biotechnol. 2012
Dec;30(12):1210-6.
Schnee M, Vogel AB, Voss D, Petsch B, Baumhof P, Kramps T, Stitz L. An mRNA Vaccine Encoding Rabies Virus
Glycoprotein Induces Protection against Lethal Infection in Mice and Correlates of Protection in Adult and Newborn Pigs.
PLoS Negl Trop Dis. 2016 Jun 23;10(6):e0004746.
Corresponding author:
Benjamin Petsch
[email protected]
Vaccines
P248
Heterologous prime-boost therapeutic vaccination stimulates strong cellular immune and breaks immune tolerance
in HBV transgenic mice
A. Kosinska1, K. Schulze1, C. Jäger2, N. Röder2, J. Su2, T. Bauer2,3, C. Guzman3,1, U. Protzer2,3
1Helmholtz
Centre Braunschweig, Department of Vaccinology and Applied Microbiology, Braunschweig, Germany
University Munich and Helmholtz Centre Munich, Munich, Germany
3German Center for Infection Research (DZIF), Munich, Germany
2Technical
Although hepatitis B cure is highly desired, it is rarely achieved. HBV persistence was found to correlate with a failure of an
efficient virus-specific B- and T-cell response. Therefore, induction of HBV-specific immune responses by therapeutic
vaccination is a promising strategy to treat chronic hepatitis B. We have recently reported a therapeutic vaccination approach
based on heterologous protein-prime/modified vaccinia virus Ankara (MVA) vector-boost vaccination scheme. To prime Tcells and induce neutralizing antibodies, we use particulate HBV core and surface antigens (HBcAg and HBsAg). However,
lack of a safe and effective adjuvant inducing a balanced Th1/Th2 CD4+ T cell response may be an obstacle for initiating
clinical trials, and triggering the cytoplasmic pattern recognition receptor STING is an interesting alternative. Our objective
was to investigate the efficacy of bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) as a potential new adjuvant
for a therapeutic hepatitis B vaccine.
C57BL/6 mice and syngeneic HBV transgenic (HBVtg) mice received two protein primes and a MVA boost immunization.
Protein antigens were combined and adjuvanted with c-di-AMP or a previously established combination of CpG with
polyphosphazenes (PCEP). The efficacy of the different vaccine formulations and application routes were compared with
respect to inducing humoral and cellular immune responses. Prime-boost vaccination of C57BL/6 mice induced not only high
antibody titers against both antigens but also multispecific, polyfunctional HBV-specific CD4+ and CD8+ T-cell responses.
Hereby, c-di-AMP as an adjuvant seemed to be superior in priming multispecific cytotoxic T-cell responses than CpG or/and
PCEP. Even in high antigenemic HBVtg mice, formulation of the protein vaccine with either c-di-AMP or a combination of
CpG and PCEP led to anti-HBs seroconversion in all animals. Moreover, both vaccine formulations induced significant HBsand HBc-specific CD8+ T-cell responses detectable and in liver associated lymphocytes in the HBVtg mice, accompanied by
mild T-cell-induced liver damage.
Conclusion: Results of this study demonstrate that ci-di-AMP is a potent adjuvant for protein prime-MVA boost vaccination
in a mouse model of chronic HBV infection reflecting vertical transmission.
Corresponding author:
Anna Kosinska
[email protected]
Vaccines
P249
Increasing the antigenic spectrum of EBV virus-like particles
D. van Zyl1, J. Mautner 2, H. J. Delecluse1
1German
Cancer Research Center (DKFZ), Pathogenesis of virus-associated tumors F100, Heidelberg, Germany
Centre Munich, Clinical Cooperation Group Pediatric Tumor Immunology, Munich, Germany
2Helmholtz
Introduction: The Epstein-Barr virus (EBV) is a ubiquitous DNA virus that can infect epithelial cells and B-lymphocytes. It is
currently estimated that more than 90% of adults worldwide are infected with EBV1. EBV infection is usually asymptomatic;
however, in a minority of cases infectious mononucleosis (IM) can occur. Furthermore, EBV is also aetiologically linked to
many different types of cancer2. The global significance of these EBV-associated cancers is exemplified by the fact that they
account for ~200,000 new cases each year3. This underscores the need to develop a prophylactic EBV vaccine.
Objectives: Our group recently developed an EBV vaccine candidate in the form of DNA-free virus-like particles (VLPs)4.
This approach is dramatically different from other EBV vaccine candidates in that it is comprised of multiple EBV antigens.
Despite this unique advantage, our VLPs completely lack latent antigens. To address this shortcoming, we have developed a
novel method for increasing the antigenic spectrum of EBV VLPs.
Material & Methods: Through the use of recombination we have constructed several VLPs that contain different tegumentlatent antigen fusions.
Results & Conclusion: We have found that the enhanced VLPs can successfully stimulate various T cell clones in vitro. We
are currently extending our interrogation of the modified VLPs to a humanized mouse model.
References:
[1] Balfour, H.J., 2014, Progress, prospects, and problems in Epstein-Barr virus vaccine development, Curr Opin Virol., 6: 1-5
(2014).
[2] Cohen, J.I., Mocarski, E.S., Raab-Traub, N., Corey, L., Nabel, G.J., Cohen, J.I., Mocarski, E.S., Raab-Traub, N., Corey,
L., and Nabel, G.J., 2013, The need and challenges for development of an Epstein-Barr virus vaccine, Vaccine, 31 Suppl 2:
B194-6.
[3] Hui, E.P., Taylor, G.S., Jia, H., Ma, B.B., Chan, S.L., Ho, R., Wong, W.L., Wilson, S., Johnson, B.F., Edwards, C.,
Stocken, D.D., Rickinson, A.B., Steven, N.M., and Chan, A.T., Phase I trial of recombinant modified vaccinia Ankara
encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients, Cancer Res., 73(6): 1676-88.
[4] Pavlova, S., Feederle, R., Gärtner, K., Fuchs, W., Granzow, H., and Delecluse, H.J., 2013, An Epstein-Barr Virus Mutant
Produces Immunogenic Defective Particles Devoid of Viral DNA, J Virol., 87(4): 2011-22.
Corresponding author:
Dwain van Zyl
[email protected]
Vaccines
P250
Development of vaccination strategies for vector-based prophylactic immunization in the Friend retrovirus model
N. Bongard1, U. Dittmer1, M. Trilling1, V. T. K. Le-Trilling1, M. Rückborn1, W. Bayer1
1University
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Nearly 37 million people worldwide are living with HIV. Until today no vaccine against HIV is available. The most promising
candidate at the moment is a vaccine based on human cytomegalovirus, because the prophylactic vaccination of rhesus
macaques with RhCMV/SIV vectors resulted in 50% protection against simian immunodeficiency virus. Here we use a
vaccine based on mouse cytomegalovirus, which expresses env of FV, to investigate the prophylactic potential in the Friend
virus model. FV is an immuno-suppressive retroviral complex, consisting of the apathogenic Friend murine leukemia virus (FMuLV) and the replication-defective but pathogenic spleen focus-forming virus. FV causes an acute and lethal infection in
susceptible mice. In our prophylactic vaccination studies we compared one immunization with a prime/boost immunization
with regard to env-specific CD4+ T cell responses, antibody titers, and the protection from FV challenge infection. We
discovered a CD4+ T cell response also after a single immunization, however little protection. On the other hand we saw a
very strong protection after two immunizations with mCMV.env. Surprisingly all strategies induce a high frequency of the envspecific CD4+ T cells, which does not seem to correlate with this protection. As there was no obvious correlation between
CD4+ responses and protection, and we observed low antibody responses, the exact mechanism of protection remains
elusive and shall be investigated in following experiments.
Corresponding author:
Nadine Bongard
[email protected]
Vaccines
P251
Development of an HIV-Vaccine based on the VSV-GP vector
C. A. Bresk1, R. Tober1, M. Krismer1, T. Hofer1, S. Wilmschen1, G. Effantin2, H. X. Liao3, B. F. Haynes3, G. Schoehn2, W.
Weissenhorn2, Z. Banki1, L. Egerer1, D. von Laer1, J. Kimpel1
1Medical
University Innsbruck, Division of Virology, Innsbruck, Austria
of Grenoble Alpes, Institut de Biologie Structurale (IBS), Grenoble, France
3Duke University School of Medicine, Department of Medicine, Durham, United States
2University
Background: Our group has recently shown that VSV pseudotyped with the glycoprotein (GP) of the lymphocytic
choriomeningitis virus, VSV-GP, is a potent vaccine vector, overcoming limitations of wild type VSV.
Objective: Here, we evaluated the potential of VSV-GP as a vaccine vector for HIV infection.
Methods: We incorporated antigens from HIV or marker genes into the genome of VSV-GP and generated infectious viruses
via reverse genetics. These viruses were analyzed in vitro for expression, infectivity, localization and conformation of the
antigen. In mice distribution and kinetics of infected cells, antigen-specific and vector-specific immune responses were
analyzed.
Results: Infectious viruses containing antigens from HIV were generated. The addition of the additional antigen did not
attenuate VSV-GP replication. HIV envelope variants were expressed in VSV-GP infected cells and incorporated into VSVGP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV such as MPER (membrane-proximal
external region), CD4 binding side, V1V2 loop and V3 loop were present on the surface of VSV-GP-env particles. After
intramuscular immunization of mice, viral replication was limited to injection side and the draining lymph nodes. No
neutralizing antibodies against VSV-GP were induced even after seven boost immunizations. However, high HIV antibody
titers were elicited in mice.
Conclusion: Taken together, VSV-GP is non-neurotoxic, induces potent immune responses, enables boosting and thus is a
promising novel vaccine vector platform.
Corresponding author:
Christiane Anika Bresk
[email protected]
Vaccines
P252
Novel influenza vaccine candidates based on Modified Vaccinia virus Ankara expressing different neuraminidase
consensus sequences
L. Marr1, K. Roose2, A. T. Lülf1, A. Freudenstein1, X. Saelens2, G. Sutter1, A. Volz1
1Ludwig
Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
of Ghent, Center for Medical Biotechnology, Ghent, Belgium
2University
Each year about 5 million severe Influenza infections are registered worldwide leading to 0.5 million deaths. Current
influenza vaccines require annual updating and are only partially effective due to a possible mismatch with the circulating
virus strains. Therefore it is important to develop a universal influenza vaccine that leads to a broad protection against as
many influenza viruses as possible, especially also against new pandemic subtypes.
In this study we use Modified Vaccinia virus Ankara (MVA), which was developed at the University of Munich, as a vaccine
production platform. MVA is a replication deficient and safety tested Vaccinia virus (VACV) that is already licensed as a
replacement smallpox vaccine in Europe. We generated and tested novel candidate influenza vaccines based on MVA
encoding different neuraminidase (NA) sequences. NA in vaccines is known to strongly induce the production of Influenzaspecific antibodies and to reduce the amount of released virus from infected cells.
In order to contribute to the development of a broad protective vaccine, we used different consensus sequences of the
neuraminidase subtypes N1 and N2 for the generation of recombinant MVAs. Both of these subtypes commonly occur in
human and swine. The MVA-NA candidate vaccines were successfully generated, quality controlled and tested according to
standard procedures.
Primary in vivo data clearly support the use of neuraminidase consensus sequences for the development of universal
influenza vaccines.
Corresponding author:
Lisa Marr
[email protected]
Vaccines
P253
Combination adjuvants as a source of vaccines effectivity increase
A. Bogoyavlenskiy1, A. Turmagambetova1, P. Alexyuk1, M. Alexyuk1, I. Zaitceva1, E. Omirtaeva1, V. Berezin1
1Institute
of Microbiology and Virology, Virology, Almaty, Kazakhstan
Many current vaccines induce a suboptimal immune response and require either large antigen doses or boosting
immunizations. Improving vaccine are possibly using by nonspecific immunostimulators – adjuvants. Unfortunately, most
modern immunostimulators can stimulate various parts of the immune response. The activation of numerous arms of the
immune response offers a compelling explanation for the strong and durable protection against infection that is induced by
this vaccine. Combination adjuvants represent an attempt to recapitulate this beneficial immune stimulation in a controlled
and defined way.
Our studies have shown that combination adjuvants should consider the mechanism action of each of the auxiliary
substance. Combining different ways immunostimulation by adjuvants the immune response can enhance is not less than 48 times. When combined with the same mechanism of adjuvant action of enhancement of the immune response does not
exceed about 2-4 times.
The combination of immunostimulants with different mechanisms of action may be the new development direction of
vaccinology.
Corresponding author:
Andrey Bogoyavlenskiy
[email protected]
Vaccines
P254
Selection of nanobodies with broad neutralizing activity against primary HIV-1 strains of different subtypes using
soluble subtype C gp140 immunogens
U. Dietrich1, K. Koch1, S. Kalusche1, J. Torres2, R. Stanfield2, W. Danquah3, H. von Briesen4, I. Wilson2, U. Wernery5, F.
Koch-Nolte3, A. Ward2
1Georg
Speyer Haus, Experimental Therapy, Frankfurt a.M., Germany
Research Institute, Integrative structural and computaional biology, La Jolla, United States
3University Medical Center Hamburg Eppendorf, Immunology, Hamburg, Germany
4Fraunhofer IBMT, Sulzbach, Germany
5Central Vetreinary Research Laboratory, Dubai, United Arab Emirates
2Scripps
Nanobodies (VHH), which correspond to the variable domains of heavy chain only antibodies from Camelidae, were selected
by phage display from dromedaries immunized with different soluble trimeric envelope proteins derived from the most
prevalent HIV-1 subtype C. We identified 25 distinct VHH families that bound to subtype C trimeric Env, of which 6
neutralized heterologous primary tier 2 isolates of various HIV-1 subtypes in a standardized in vitro neutralization assay. The
neutralization breadth of two selected VHHs in combination covers 19 out of 21 HIV-1 strains from a standardized panel of
epidemiologically relevant HIV-1 subtypes including all 12 strains from a global reference panel recently defined for the
standardized assessment of vaccine-elicited neutralizing antibodies. The CD4 binding site was preferentially targeted by the
broadly neutralizing VHHs as determined by competition ELISAs with known monoclonal antibodies and 3D models of VHHEnv complexes derived from negative stain electron microscopy. Due to their potency and their complementary neutralization
patterns covering the majority of epidemiologically relevant HIV-1 subtypes as well as to the intrinsic properties of
nanobodies including small size, high solubility and stability, long HCDR3 regions and high homology to the human VH3
genes, the nanobodies identified here are excellent candidates for further clinical development for prophylactic and
therapeutic applications.
Corresponding author:
Ursula Dietrich
[email protected]
Vaccines
P255
Inactivation of Influenza A Virus and RSV via low-energy electron irradiation provides a versatile method for
efficacious vaccine production
L. Bayer1, J. Fertey1, E. Hiller2, S. Bailer2, S. Rupp2, A. Pohl3, C. Wetzel3, S. Ulbert1, T. Grunwald1
1Fraunhofer
IZI, Immunology, Leipzig, Germany
IGB, Stuttgart, Germany
3Fraunhofer FEP, Dresden, Germany
2Fraunhofer
Question: Inactivation of pathogens for vaccine production is commonly achieved by incubation with toxic chemicals such as
formaldehyde or beta-propiolactone. This time consuming inactivation procedure requires extensive downstream processing
in order to remove or inactivate the chemicals. Moreover, the inactivation efficiency varies and the antigenic properties of the
virus are often altered, leading to weaker immune responses and reduced antibody specificity upon immunization. In specific
cases, such as human Respiratory Syncytial Virus (RSV), vaccines based on formaldehyde-inactivation can even lead to an
enhanced onset of the disease and are therefore not used. Currently, there is no licensed vaccine for RSV available. We
tested low-energy electron irradiation (LEEI) as an alternative inactivation method, as ionizing radiation acts rapidly and
predominantly damages nucleic acids, leaving antigenic structures largely intact.
Methods: As RSV and Influenza A virus belong to the most important respiratory viruses, we analyzed the potential of LEEI
to produce effective vaccines with these two viruses. Infectivity of both viruses was tested in cell culture-based assays before
and after LEEI treatment. We conducted ELISAs to monitor surface protein integrity, and analyzed viral RNA in an
electrophoresis based assays. To investigate the immunogenic potential of the LEEI inactivated virus solution the LEEI
material was injected into BALB/c mice, followed by a boost 4 weeks later. ELISAs and virus neutralization tests were used
to analyze the antibody response. Finally, mice were challenged with Influenza A and RSV, respectively, and sacrificed three
days later. The viral load in the animals lungs was determined by quantitative PCR.
Results: Protein integrity was reproducibly measured around 80% after inactivation, compared to non-irradiated virus.
Fragmentation of the viral RNA occurred in a dose-dependent manner, correlating with loss of infectivity. Vaccinated mice
showed strong immune responses, high titers of virus neutralizing antibodies, and a significant reduction of viral load in the
lungs compared to non-vaccinated mice.
Conclusion: LEEI has the potential to provide a fast and effective method for the production of safe and efficacious human
and veterinarian vaccines. (Fertey et al, 2016)
Corresponding author:
Lea Bayer
[email protected]
Vaccines
P256
Immunogenicity and protective capacity of recombinant vaccinia virus MVA delivering influenza A virus M2 protein
ectodomain (M2e) antigens
A. Volz1,2, K. Roose1,2, A. Freudenstein1, G. Rimmelzwaan3, X. Saelens2, G. Sutter1
1Ludwig
Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
Ghent, Medical Biotechnology Center, Department of Biomedical Molecular Biology, Ghent, Belgium
3Erasmus Medical Centre, Department of Viroscience, Rotterdam, Netherlands
2VIB
The development of a universal influenza vaccine has gained high priority due to the permanent threat of a new pandemic
caused by novel influenza viruses that may emerge from birds or pigs. Current seasonal influenza vaccines fail to afford
broad-protective and long-lasting immunity, and they provide no or very little efficacy against avian or pandemic influenza
viruses. To develop a universal influenza vaccine that provides longer-lasting and broader protection against multiple strains
of influenza A virus both broad-reactive antibody (B-cell) and T-cell responses need to be induced. Modified Vaccinia virus
Ankara (MVA), a highly attenuated strain of vaccinia virus originating from growth selection on chicken embryo fibroblasts
serves as an advanced recombinant poxvirus vector in preclinical and clinical developmentof new vaccines against infectious
disease and cancer.
Here, we generated and evaluated recombinant MVA candidate vaccines that deliver synthetic chimeric antigens as fusion
proteins and carriers for the ectodomain (M2e) of the influenza A virus membrane protein M2. M2e is considered as a very
promising target antigen for the induction of a broad influenza A virus neutralizing antibody response. We developed an
expression construct encoding three tandem copies of M2e (representing both avian and human type M2e sequences)
genetically fused to sequences of the MVA membrane protein A56 and tested the antigen production of MVA-A56-M2e
viruses.. In vitro characterizations confirmed that the A56-M2e fusion proteins are glycosylated and expressed on the surface
of MVA-A56-M2e infected cells. Intramuscular immunizations with MVA-A56-M2e induced influenza A virus neutralizing
antibodies in C57BL/6 mice and protected the animals against lethal challenge infections with influenza A virus.and Thus,
further studies are warranted to evaluate recombinant MVA-A56-M2e vaccines in other preclinical models and eventually as
candidate vaccines in humans.
Corresponding author:
Asisa Volz
[email protected]
Vaccines
P257
Potent humoral and cellular immunity induced by a measles virus-derived Zika virus vaccine
C. Nürnberger1, A. Fiedler1, B. Bodmer1, M. Muehlebach1
1Paul
Ehrlich Institute, Veterinary Medicine, Langen, Germany
Question: Zika virus (ZIKV) is a mosquito-borne flavivirus, which causes considerable problems in South America. Although
ZIKV infections are usually asymptomatic or mild, the present epidemic causes severe birth defects such as microcephaly in
newborns, when ZIKV has been transmitted transplacentally to the fetus. Unfortunately, no ZIKV-vaccines are available, yet.
Recombinant measles virus (MV) constitutes a promising vaccine vector platform to induce immunity against foreign
pathogens by additional expression of respective antigens. Thereby, a recombinant MV encoding ZIKV antigens has been
generated. We aimed to analyze the efficacy of this vaccine to induce humoral and cellular immune responses.
Methods: Recombinant Schwarz-strain MV that additionally encodes the ZIKV prM and the soluble domain of the E protein
(MV-ZIKV-sE) was made available to us. To determine its immunogenicity, IFNAR -/--CD46Ge mice were immunized twice
with this vaccine or respective controls. Serum was taken before vaccination, after prime, and boost vaccination, while
splenocytes were prepared 4 days post boost. ZIKV-specific humoral responses were detected by ELISA, and neutralizing
antibodies were quantified by plaque reduction neutralizing titers (PRNT) or virus neutralizing titers (VNT). Cellular immunity
was assayed by IFN-γ ELISpot and T cell proliferation after re-stimulation of splenocytes with overlapping peptides or MV
bulk antigen.
Results: MV-ZIKV-sE expressed the respective ZIKV proteins and replicated with vaccine-strain characteristics. This
vaccine revealed robust induction of humoral and cellular immune responses directed against ZIKV and MV. While no prMspecific antibodies became detectable, MV-ZIKV-sE induced anti-E antibodies in the range of 1,000 to 5,000 U/ml 21 days
after boosting, with a PRNT50 in some animals exceeding 1,280 and a mean VNT of 30. Our data also indicated effective
boosting of humoral anti-ZIKV immunity. ELISpot revealed about 200 ZIKV-reactive IFN-g secreting cells per 1x106
splenocytes, with about 40% of proliferating T cells after stimulation with ZIKV E-peptides.
Conclusion: Based on the safe and immunogenic replicating vaccine platform MV-Schwarz, a prototypic ZIKV vaccine has
been generated that triggers significant humoral and cellular anti-ZIKV immune responses.
Corresponding author:
Michael Muehlebach
[email protected]
Vaccines
P258
Neuraminidase-expressing vesicular stomatitis virus replicons induce subtype-specific protection against influenza
A virus
L. Walz1, S. K. Kays1, G. Zimmer2, V. von Messling1
1Paul
Ehrlich Institute, Veterinary Medicine, Langen, Germany
of Virology and Immunology, Mittelhäusern, Switzerland
2Institute
Introduction: Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths
worldwide. Currently licensed inactivated vaccines primarily induce antibody responses against the hemagglutinin (HA)
glycoprotein. The resulting antigenic drift of HA requires a frequent update of the vaccine composition. Hence, there is a
need for vaccines that not only protect against the homologous strain but also against heterologous viruses of the same or
even heterologous subtypes.
Objectives: To investigate the potential of the more conserved neuraminidase (NA) protein as a target antigen for subtypespecific vaccines.
Material & methods: Vesicular stomatitis virus (VSV)-based replicons expressing a panel of N1 proteins with different
phylogenetic distances were generated and characterized in vitro. C57BL/6 mice were immunized intra-muscularly with the
respective replicons using a prime-boost regimen. Total and functional antibody responses were measured at different times
after immunization, and interferon gamma ELISpot assays were performed to gain insight in the extent of cellular immune
responses. All animals were challenged with a lethal dose of mouse-adapted H1N1 PR/8/34 to assess the level of protection
conferred by the different candidate vaccines.
Results: All N1 proteins were efficiently incorporated in the replicon particles and were expressed at similar levels in infected
cells. Immunization with the replicon carrying the matched N1 resulted in robust humoral and cellular immune responses and
protected against the homologous influenza virus with similar efficacy as the matched HA protein, illustrating the potential of
the NA protein as target antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the
level of cross-reactive antibody titers, whereas no protection was observed after immunization with heterologous NA
subtypes.
Conclusion: This study reveals that NA-expressing VSV replicons constitute a promising platform for the development of
more broadly protective influenza vaccines.
Corresponding author:
Lisa Walz
[email protected]
Vaccines
P259
Biodistribution of the recombinant vaccine MVA-MERS-S following single dose intramuscular inoculation in mice
M. Langenmayer1, S. Adam-Neumair1, G. Sutter1, A. Volz1
1Ludwig
Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient virus serving as well-established vector
virus in vaccine development against infectious or neoplastic diseases. In previous clinical evaluations various recombinant
MVA vaccines have shown an excellent safety profile suggesting the possibility of safe MVA-based immunizations even in
immunocompromised individuals and in persons with severe comorbidities. Still it is highly desirable to collect any available
information about the non-clinical safety of a new candidate MVA vector vaccine.
In this study we used a recombinant MVA vector vaccine expressing the full length spike protein of Middle Eastern
Respiratory Syndrome coronavirus (MVA-MERS-S; Song et al. 2013 J Virol 87:11950, Volz et al. 2015 J Virol 89:8651) to
assess biodistribution in mice after a single dose intramuscular inoculation. Control mice were inoculated in the same
manner with a well-characterized control recombinant virus MVA-GFP-mCherry (Lülf et al. 2016 Virology 499:322).
Mice were necropsied at different time points and a full set of tissues was examined histologically. Immunohistochemistry
was applied to identify viral and recombinant antigens in situ.
Lesions were similar in animals inoculated with both recombinant MVA viruses. Gross lesions were restricted to the site of
inoculation (mild edema and swelling) and draining lymph nodes (hyperplasia). Histologically the parenteral site was
expanded by edema and inflammation while muscles displayed focal necrosis. Paracortical cellularity of the draining lymph
nodes was increased. Lesions attributable to MVA were not detected in tissues other than the parenteral site. Both MVA and
recombinant antigen were detected only at the site of inoculation in mesenchymal cells.
In conclusion, a single intramuscular inoculation of recombinant MVA-MERS-S does not lead to histologically or
immunohistochemically detectable lesions in tissues peripheral to the parenteral site and draining lymph nodes. The lesions
and antigen distribution pattern are indistinguishable from those found following the use of the control recombinant MVAGFP-mCherry. Levels of inflammation and the hyperplasia of draining lymph nodes are considered in line with the expected
immunological response to the vaccine inoculation.
Corresponding author:
Martin Langenmayer
[email protected]
Vaccines
P260
Novel recombinant DNA vaccine candidates for human respiratory syncytial virus – Preclinical evaluation of
immunogenicity and protection efficiency
F. Almajhdi1, H. Amer1, M. Farrag1, M. Hamad1, P. Öhlschläger2
1King
Saud University, Botany and Microbiology, College of Science, Riyadh, Saudi Arabia
University of Applied Sciences, Juelich, Germany
2Aachen
Development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for the researchers
worldwide. DNA-based immunization is currently promising approach for generation of many human vaccines for different
age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice.
Three different versions of codon-optimized HRSV fusion (F) gene were individually cloned into pPOE vector. The
recombinant clones either express full-length (pPOE-F), secretory (pPOE-TF), or M282-90 linked (pPOE-FM2) forms of F
protein. Distinctive expression of F protein was identified in HEp-2 cells transfected with the different recombinant vectors
using ELISA and immunofluorescence. Mice immunization verified the potential of recombinant vectors to elicit significant
levels of neutralizing antibodies and CD8+ T-cell lymphocytes. In particular, pPOE-TF showed higher level of gene
expression in cell culture and better induction of humoral and cellular immune responses. Following mice challenge, all
recombinant vectors protected mice from disease enhancement as compared to mice immunized with formalin-inactivated
HRSV and empty vector. They also showed lower lung virus titer, restrictive pulmonary histopathology and lower level of
inflammatory cytokines in lungs. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms
of safety, immunogenicity and protection efficiency. These data encourage further valuation in other animal models and in
clinical trials.
Corresponding author:
Fahad Almajhdi
[email protected]
Figure 1
Vaccines
P261
Low genetic stability of recombinant influenza A viruses carrying a foreign gene in the neuraminidase segment
V. Czudai-Matwich1,2, C. Paul1, S. Thoma1, L. Enns1, S. Becker1,2, M. Matrosovich1,2
1Philipps
2German
University Marburg, Institute of Virology, Marburg, Germany
Centre for Infection Research (DZIF), Giessen-Marburg-Langen, Germany
Introduction: Emerging viral diseases pose a continuous threat to human health. As part of DZIF, we aim to develop a novel
inactivated vaccine platform based on influenza A viruses (IAV) expressing protective antigenic epitopes of other emerging
viruses against which no vaccine or therapeutic treatment is available, such as MERS-CoV and CCHFV.
Objectives:
1. Establishment of recombinant IAVs, in which the globular enzymatic domain of the viral neuraminidase (NA) is
substituted by protective antigenic domains of other viruses.
2. Optimization of antigen yield and immunogenicity of recombinant viruses by defining optimal replication parameters
and/or modification of NA fusion proteins. Analysis of viral genetic stability.
Material & Methods:
pHW2000-NA-GFP reverse genetics plasmids were generated by fusing GFP to the N-terminal domain of the NA required for
the fusion protein incorporation into viral particles. Recombinant IAVs expressing NA-GFP proteins (rPR8-NA-GFP) were
created by reverse genetics and propagated in the presence of V. cholerae NA to compensate for the lack of NA activity.
Protein yield was determined by BCA-assay, SDS-PAGE and ELISA. Genetic stability of the viruses during serial passaging
was analyzed by plaque-screen for GFP expression and sequencing.
Results: rPR8-NA-GFP virus was rescued and replication conditions in cell culture and chicken eggs were optimized by
titration of exogenous NA. Purified rPR8-NA-GFP viruses contained up to 10% NA-GFP protein with respect to total viral
protein. However, a rapid loss of GFP signal was observed during virus passaging owing to deletions that occurred at
random inside and adjacent to the GFP ORF. Similar observations were made using generated NA-MERS-containing
recombinant virus, suggesting that rapid loss of foreign antigens by IAV represents a general phenomenon.
Conclusion: Although chimeric influenza virus carrying foreign antigen represents an attractive vaccine platform, our data
suggest that there is a strong selection of variants with deletions of genes that are not required for replication. This critical
problem should be considered and addressed in the future studies.
Corresponding author:
Volker Czudai-Matwich
[email protected]
Vaccines
P262
Naked DNA immunization with full-length attachment gene of human respiratory syncytial virus induces safe and
protective immune response
H. Amer1, F. Almajhdi1, M. Hamad1, M. Farrag1
1King
Saud University, Botany and Microbiology, College of Science, Riyadh, Saudi Arabia
Development of potent vaccine for human respiratory syncytial virus (HRSV) that confers better protection than natural
infection remains a global challenge. Vaccination with naked DNA is currently considered successful approach for the control
of many viral diseases. In this study, the potential of DNA vaccination using full-length attachment gene of HRSV type A
Saudi strain cloned in the mammalian expression vector pcDNA3.1+ (pcDNA/GA) was evaluated in BALB/C mouse model.
The expression efficiency of pcDNA/GA was first confirmed in HEp-2 cells on both RNA and protein levels. BALB/C mice
immunized with pcDNA/GA exhibited high antibody titers in ELISA with superior neutralization activity. ELISOPT assay also
revealed the ability of pcDNA/GA to induce HRSV-specific CD8+ T cells in immunized mice. Following challenge of
immunized mice with the wild-type virus strain, no clinical disease outcomes, no lung viral load, and a significant diminish of
pulmonary immunopathology were recorded compared to control mice. The pulmonary cytokine profile in pcDNA/GA
immunized mice after challenge displayed notable upregulation of Th1-associated cytokines while that of FI-RSV immunized
mice exhibited high levels of Th2-associated cytokines. In conclusion, the DNA vaccine candidate pcDNA/GA has proven
distinct efficacy and safety in mouse model. Further evaluation in other animal models such as cotton rats and non-human
primates is necessary before its use in clinical trials.
Corresponding author:
Haitham Amer
[email protected]
Vaccines
P263
A new RNA-based adjuvant enhances virus-specific vaccine responses by locally activating DC and inducing type I
IFN responses
A. Ziegler1, C. Soldner1, S. Lienenklaus2, J. Spanier1, S. Trittel3, P. Riese3, T. Kramps4, S. Weiss5, R. Heidenreich6, C. A.
Guzmán3,6, K. J. Kallen7, M. Fotin-Mleczek6, U. Kalinke1
1Twincore
GmbH, Center for Experimental and Clinical Infection Research, Hanover, Germany
Medical School, Institute of Laboratory Animal Science and Central Animal Facility, Hanover, Germany
3Helmholtz Centre for Infection Research, Department of Vaccinology and Applied Microbiology, Braunschweig, Germany
4Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim, Germany
5Helmholtz Centre for Infection Research, Department of Molecular Immunology, Braunschweig, Germany
6CureVac AG, Tübingen, Germany
7eTheRNA immunotherapies NV, Niel, Belgium
2Hanover
Question: Among new adjuvants conferring a TH1-shift, RNAdjuvant® consisting of non-coding RNA complexed with a
cationic carrier peptide is a promising candidate. We studied the immunoenhancing effects of RNAdjuvant ® on dendritic cells
(DC), cytokine release, antiviral protection and antibody responses. Additionally, we elucidated the signaling pathways
involved in the sensing of RNAdjuvant®.
Methods: To study the direct effects of RNAdjuvant® on DC, bone marrow-derived DC were stimulated with RNAdjuvant® in
vitro and analyzed by flow cytometry. In preclinical mouse studies with two different vaccination models, the
immunoenhancing effects of RNAdjuvant® were investigated in vivo. To elucidate the sensing mechanism of RNAdjuvant®,
experiments with mice deficient in various pattern recognition receptors were conducted. Additionally, an IFN-β reporter
mouse was used to study spatiotemporal conditions of the induced cytokine response.
Results: Upon RNAdjuvant® treatment, bone marrow-derived myeloid and plasmacytoid DC showed Toll-like receptor
(TLR)7-dependent IFN-I induction and upregulation of CD69 and CD86. This result was verified by the ex vivo analysis of
splenic DC. Immunization of mice revealed that RNAdjuvant® enhanced vaccine-induced antiviral protection and significantly
enhanced neutralizing antibody responses. In particular, IgG2b/c antibody subclasses were elevated, indicating a TH1-biased
immune response. RNAdjuvant®-mediated enhancement of antibody responses was conferred by concomitant MyD88- and
Cardif-dependent signaling. Thus, upon administration of RNAdjuvant® two different signaling pathways were induced.
Notably, transient lymphopenia as a sensitive readout of serum cytokine responses was only detected upon intravenous but
not upon intramuscular (i.m.) injection of RNAdjuvant®. Furthermore, upon i.m. injection of RNAdjuvant® IFN-β induction and
CD69 upregulation on DC were only detectable within draining lymphoid organs, indicating that RNAdjuvant ® induced
primarily local effects.
Conclusion: RNAdjuvant® is a novel efficacious adjuvants that augments TH1 responses in a MyD88- and Cardif-dependent
manner by triggering primarily local effects. Thus, RNAdjuvant® holds promise to be a particularly well tolerated new adjuvant
also in humans.
Corresponding author:
Annett Ziegler
[email protected]
Vaccines
P264
Cross-protection induced by Japanese encephalitis vaccines against different serotypes of Dengue viruses in mice
J. Li1, N. Gao1, D. Fan1, H. Chen1, Z. Sheng1, S. Fu2, G. Liang2, J. An1
1School
of Basic Medical Sciences,Chinese Capital Medical University, Microbiology, Beijing, China
Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
2National
Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) are closely related mosquito-borne flaviviruses that cause
very high global disease burdens. Although cross-reactivity and cross-protection within flaviviruses have been demonstrated,
the effect of JEV vaccination on susceptibility to DENV infection has not been well elucidated. In this study, we found that
vaccination with the JEV inactivated vaccine (INV) and live attenuated vaccine (LAV) could induce cross-immune responses
and cross-protection against DENV1-4 in mice. Despite the theoretical risk of immune enhancement, no increased mortality
was observed in our mouse model. Additionally, low but consistently detectable cross-neutralizing antibodies against DENV2
and DENV3 were also observed in the sera of JEV vaccine-immunized human donors. Moreover, we further demonstrated
that innovation ofa DNA vaccine(expressing JEV prM-E protein) also induced cross-immunity and cross-protection against
DENVs in mice.The results suggested that both JEV-LAV and JEV-INV can elicit strong cross-immunity and protection
against DENVs andprM-E antigens is the important for the cross-protection. Taken together, this study demonstrated that
inoculation with JEV vaccines may influence the distribution of DENVs in co-circulated areas and that the cross-protection
induced by JEV vaccines against DENVs might provide important information in terms of DENV prevention.
Corresponding author:
Jieqiong Li
[email protected]
Vaccines
P265
Immunization with electroporation enhances the protective effect of a DNA vaccine candidate expressing prME
antigen against dengue virus serotype 2 infection
H. Chen1, X. Zheng1, R. Wang1, N. Gao1, Z. Sheng1, D. Fan1, K. Feng1, X. Liao1, J. An1
1School
of Basic Medical Sciences,Chinese Capital Medical University, Microbiology, Beijing, China
Question: We aimed to use the dengue virus (DV) serotype 2 as a proof of principal for testing the efficacy of a DNA vaccine
candicate via in vivo electroporation in mice and rabbits prior to the development of a tetravalent vaccine.
Methods: Different dosages of DNA pVAX1-D2ME encoding DV2 prME genes were vaccinated in mice via intramuscular
injection or in vivo electroporation, immune responses and protection were determined. In DNA-vaccinated rabbits via
electroporation, antibody titer and protein expression were tested.
Results: In mice, 50μg of pVAX1-D2ME via electroporation elicited effective anti-DV2 responses and conferred significant
protection against DV2 challenge. Moreover, anti-DV2 IgG and neutralizing antibodies were successfully induced in rabbits
immunized with pVAX1-D2ME via electroporation and the expression of the interest protein was observed at local sites.
Conclusion: Enhanced immunogenicity and protective effect conferred by pVAX1-D2ME via electroporation show great
promise for the development of a dengue tetravalent DNA vaccine.
Corresponding author:
Hui Chen
[email protected]
Vaccines
P266
Recombinant modified vaccinia virus Ankara generating Ebola virus-like particles
M. Schweneker1, A. S. Laimbacher2, G. Zimmer3, S. Wagner1, E. M. Schraner4, M. Wolferstätter1, M. Klingenberg1, U.
Dirmeier1, R. Steigerwald1, H. Lauterbach1, H. Hochrein1, P. Chaplin1, M. Suter5, J. Hausmann1
1Bavarian
Nordic GmbH, Martinsried, Germany
of Zurich, Institute of Virology, Zurich, Switzerland
3Institute of Virology und Immunology, Mittelhäusern, Switzerland
4Veterinar-Anatomic and Institute of Virology, Zurich, Switzerland
5Vetsuisse, Dekanat, Zurich, Switzerland
2University
There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death
caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV-VLPs) consisting of the matrix protein VP40, the
glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in non-human primates against Ebola
virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic® (MVA-BN®) recombinant coexpressing VP40 and glycoprotein (GP) of EBOV Mayinga and the nucleoprotein (NP) of Taï Forest virus (TAFV) (MVA-BNEBOV-VLP) to launch non-infectious EBOV-VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated
organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression
levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP
produced large amounts of EBOV-VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5,
thus resembling authentic EBOV particles. The heterologous TAFV-NP enhanced EBOV-VP40-driven VLP formation with
comparable efficiency as the homologous EBOV-NP in a transient expression assay, and both NPs were incorporated into
EBOV-VLPs. The levels of EBOV-GP-specific neutralizing and binding antibodies induced by MVA-BN-EBOV-VLP and MVABN-EBOV-GP in mice were similar, raising the question whether the quality rather than the quantity of the GP-specific
antibody response might be altered by an EBOV-VLP-generating MVA recombinant.
Corresponding author:
Jürgen Hausmann
[email protected]
Vaccines
P267
Validation of MST to determine binding affinities of HIV-1 Env to bNAbs in solution – Benchmarking against ELISA,
SPR-Spectroscopy and Flow Cytometry
B. Zimmer1, K. Kuttenberger2, T. Fischer1, S. Schmidt2, A. Gabele1, T. H. Bruun1, T. Schubert2, R. Wagner1
1University
of Regensburg, Institute of Medical Microbiology and Hygiene, Molecular Microbiology (Virology), AG Prof. Dr. Ralf Wagner, Regensburg,
Germany
22bind GmbH Molecular interaction services, Regensburg, Germany
Anti-retroviral therapy of HIV infections has made significant progress over the last decades. Despite these efforts, only
about 36% of infected patients worldwide have access to the expensive medication, indicating the need for an efficient
vaccine. However, available HIV-1 Envelope (Env) immunogens have so far not been able to induce broadly neutralizing
antibody (bnAb) responses, which are considered crucial for the development of efficient vaccines.
In vaccine development the precise characterization of the binding of HIV-1 envelope immunogens towards bnAbs is
important. Different methods have been evaluated to quantify this interaction, depending on the production strategy of the
envelope protein. While the characterization of antibody-Env interactions of cell surface exposed Env molecules is mostly
done by flow cytometry, virus like particle (VLP) exposed or truncated soluble envelope variants are characterized by other
methods like ELISA or SPR-spectroscopy (SPR).
Microscale Thermophoresis (MST) is a novel powerful method which allows the quantification of biomolecular interactions in
solution. To explore the options of the MST to quantify antibody-Env interactions we generated a set of genetically
engineered HIV-1 Env variants with distinct binding properties to bnAbs. These Env variants were produced both, as soluble
proteins in different oligomerization states (monomers, trimers) and membrane-bound trimers on VLPs. Finally the affinity
parameters were determined by MST and compared with the results from ELISA and SPR measurements. Additionally, the
results were compared with affinities obtained from cytometer based titration assays with cells, expressing membrane-bound
Env on the cell surface.
With this, we were able to show that i) the relative affinities of the Env proteins, initially obtained from FACS titration
experiments with membrane-bound Env variants, are transferable to the corresponding soluble and VLP exposed variants
and ii) MST is capable to quantify the antibody-Env interaction from basic (Ab vs monomers) to complex measurements (Ab
vs VLPs). However, the obtained KD values were only comparable within the respective method, indicating that the
measurements are influenced by the different measurement conditions of the different methods used.
Corresponding author:
Benjamin Zimmer
[email protected]
Vaccines
P268
A novel recombinant Orf virus (ORFV) vector system as a therapeutic strategy against CRPV-induced tumors
T. Iftner1,2, M. Schneider1, R. Amann2, T. Feger2, H. G. Rammensee2, F. Stubenrauch1
1University
2University
Hospital Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
Hospital Tübingen, Department of Immunology, Tübingen, Germany
Objectives: The prophylactic vaccination against different human tumorvirus (HPV) types as primary prevention is well
established. For treatment of HPV-associated cancers, however, no therapeutic vaccine is currently available. The ORFV
vector platform provides a suitable system for the development of a novel intervention strategy because the ORFV vector is
replication deficient, shows an excellent safety profile and is well tolerated. Furthermore, repeated boost immunizations or
immunizations against different target antigens are possible. Finally, the intrinsic adjuvant capability triggers a humoral and
cellular response and the viral vector system induces a strong immune response against the inserted antigen.
Methods: The ORFV vector was used to develop four different recombinants each expressing one of the codon-optimized
cottontail rabbit tumorvirus (CRPV) proteins E1, E2, LE6 or E7. Nine outbred New Zealand White rabbits were challenged
with CRPV-DNA via gene gun. After tumor growth was detected, six rabbits were vaccinated with 5*107 pfu using a
combination of the four recombinant ORFV vaccines. The other three rabbits were vaccinated with an empty ORFV virus
vector. Tumor development was measured. An Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect E1-, E2-,
LE6- and E7-specific antibodies after vaccination. Furthermore, a delayed-type hypersensitivity (DTH) skin test was
performed on four rabbits to verify a cell-mediated immune response.
Results: The measurement of the tumor size showed a significant (p< 0,001) reduction of tumor growth already two weeks
after vaccination. The overall response rate was 83% (5/6 animals). CRPV-specific antibodies were detectable in the serum
after vaccination. After the intracutaneous injection of the antigens into the ear skin the DTH test showed a strong reaction
against E6 and E2 protein.
Conclusion: The recombinant ORFV vector system could be a useful vaccine for papillomavirus-induced tumors
Corresponding author:
Thomas Iftner
[email protected]
Vaccines
P269
Development of an Enzyme-Linked-Lectin-Assay to measure influenza A virus neuraminidase specific antibodies
H. Sediri1, S. Ho2, J. Waldock3, K. Hoschler2, O. G. Engelhardt3, R. Wagner1
1Paul
Ehrlich Institute, Virology, Langen, Germany
Health England, London, United Kingdom
3NIBSC, London, United Kingdom
2Public
The influenza virus major surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are the principal targets of the
protective immune response. HA mediates both binding to cell surface receptors and fusion of viral endosomal membranes
following endocytosis of the virus, releasing the viral nucleocapsid into the cytoplasm of the target cell. NA removes sialic
acid from the HA of progeny virus particles, and thus facilitates the release of the progeny virus from the infected cells.
HA antibodies, induced after vaccination, have been extensively characterized. They generally confer protection by blocking
the attachment and fusion of the virus onto its target cells. Although a permissive infection can be initiated in the presence of
immunity against NA, NA antibodies have been demonstrated to have various important roles during infection such as
decrease of disease severity, duration of illness and virus shedding.
Several methods are available for HA antibody titration such as Hemagglutinin Inhibition tests (HAI) or Microneutralization
test (MNT). However there is no standardized method for NA antibody titration. The main goal of our project is to develop,
optimize and standardize a serological test measuring NA antibodies titer.
It has been suggested that HA-specific antibodies in human sera interfere with the access of NA to its substrate, leading to
an overestimation of the NA-antibody titer. We conducted a comparative analysis of NA antibody titer following an EnzymeLinked-Lectin-Assay (ELLA) serological titration with viruses of different HA-NA combinations. These viruses were either
matched or mismatched for the anti-HA antibodies present in the tested serum samples. This analysis highlighted the impact
of HA antibodies for NA antibody titration.
These observations suggest that serological test aiming at the specific titration of NA antibodies require a new source of NA
antigen to prevent any interference by anti-HA antibodies.
This work has received support from the EU/EFPIA Innovative Medicines Initiative Joint Undertaking FLUCOP grant n°
115672.
Corresponding author:
Hanna Sediri
[email protected]
Vaccines
P270
Impact of HCMV pentameric complex-specific depletion on neutralizing capacity of immunoglobulin preparations in
vitro
M. S. Schampera1, J. Arellano-Galindo1, G. Jahn1, K. Hamprecht1
1University
of Tübingen, Institute of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
Introduction: In the absence of a highly efficient HCMV vaccine and in context of the recent RCT-study of Revello et al.,
2014, which could not confirm the preventive effects of HCMV-specific hyperimmunoglobulins (HIG) on maternofetal HCMV
transmission as reported originally by Nigro et al., 2005, we analyzed the neutralization capacity (NC) of HIGs and standard
IVIGs in vitro by depletion of pentameric complex (PC) related antibodies.
Objectives: We investigated the NC-changes using a recombinant HCMV PC and defined UL130 peptides in order to
analyze their contribution to NC.
Material & methods: We analyzed the NC in vitro using the HIG preparation Cytotect® and a standard intravenous
immunoglobulin (IVIG) Kiovig®. The depletion of antibodies was performed using a recombinant PC and two UL130
peptides. We used a six-fold histidine-tail on the PC, which was linked to magnetic beads containing His-Tag mouse mAbs
on the surface. We used a protocol for a long-term cell-free HCMV neutralization plaque reduction assay (PRA) using human
foreskin fibroblasts (HFF) and human retinal epithelial cells (ARPE-19) cells as target cells. Each primary HFF- and ARPE19-adapted clinical isolate from amniotic fluid was propagated until cell-free HCMV shedding, respectively. For calibration we
previously generated serum pools (N=100) from two cohorts of mothers: seronegative and latently HCMV infected mothers at
birth. For control, untreated immunoglobulin preparations were investigated.
Results: HIG and standard IVIG preparations lead to a significant decrease in NC after incubation with the recombinant PC
compared to untreated samples. However, the impact of depletion was not significant against infected HFF monolayers.
Conclusion: We demonstrated that polyclonal HCMV specific antibodies present in HIG and standard IVIGs bind to the
recombinant PC and therefore may play an important role for the neutralization activity of HIG and IVIG preparations. The
antibodies targeting the recombinant PC are more effective for viral neutralization in ARPE-19- than in HFF-microcultures.
Further epitope analyses of the PC-binding NT-antibodies could lead to a better understanding of specificity and efficiency of
these antibodies to prevent potentially maternofetal HCMV-transmission.
Corresponding author:
Matthias Stefan Schampera
[email protected]
Vaccines
P271
Generation and characterization of recombinant Vesicular stomatitis viruses coding for several filovirus proteins
J. Westmeier1, S. Becker1, O. Dolnik1
1Philipps
University Marburg, Institute of Virology, Marburg, Germany
The members of the family Filoviridae, Ebola virus (EBOV) and Marburg virus (MARV), cause severe fever disease with high
mortality rates in humans. The recent EBOV outbreak in West Africa (2014-2016) have shown the need for the development
of vaccines or post-exposure treatment against all filoviruses as they present a public health concern. While there are many
vaccines under development, one promising system is based on recombinant Vesicular stomatitis virus (rVSV). VSV,
belonging to the family Rhabdoviridae, is one of the best studied RNA viruses and successfully used as a vaccine vector for
other viral diseases.
In this study, we aim to produce rVSV expressing not only the filoviral glycoproteins (GPs), but also the nucleoprotein in
order to enhance the immunogenicity. rVSV containing the EBOV or MARV GPs are created using a unique XhoI-NheI linker
site between the VSV M and L genes in a full-length plasmid containing the whole VSV anti-genome except the gene for the
VSV glycoprotein. Furthermore, a second filoviral gene is then inserted with help of a unique MluI restriction site between the
GP and VSV L genes. It has been shown that also an insertion of a third foreign open reading frame between the VSV N and
P genes is feasible (Mire et al., 2015 [1]). The main focus of the study is to create and to characterize bivalent rVSV vaccine
candidates. In addition, as a tool for easier quantification and standardization of the infection, an additional open reading
frame for a reporter gene like eGFP or luciferase is introduced into the rVSV genome using unique restriction sites. The
obtained viruses will be useful tools to analyze the immune response of vaccinees during clinical trials and for testing of
antivirals.
References:
[1] Mire CE, Geisbert JB, Versteeg KM, et al. A single-vector, single-injection trivalent filovirus vaccine: proof of concept
study in outbred guinea pigs. J Infect Dis 2015; 212:S384–8
Corresponding author:
Jaana Westmeier
[email protected]
Vaccines
P272
Generation and biochemical characterization of HIV-1 envelope immunogens with increased stability and affinity to
broadly neutralizing antibodies
C. Schmalzl1, V. Graßmann1, H. Hierl1, M. Glögl1, A. Kliche1, D. Peterhoff1, R. Wagner1
1University
of Regensburg, Institute of Medical Microbiology & Hygiene, Molecular Microbiology (Virology), AG Prof. Dr. Ralf Wagner, Regensburg, Germany
One of the main goals of Human Immunodeficiency Virus (HIV) vaccine design is the generation of HIV-1 envelope (Env)
immunogens able to induce broadly neutralizing antibodies (bNAbs). Improved conformational stability and antigenicity are
key criteria recently shown to be essential to achieve autologous HIV-1 tier 2 neutralization.
For the rational design of such next generation Env immunogens, an Env-Alanine-Scan using mammalian cell display in a
high throughput FACS assay was performed, identifying mutations in a clade C Env candidate, which resulted in a gain of
binding (GOB) for bNAbs PG9 (conformational epitope) and VRC01 (CD4 binding site) as well as improved Env trimer
structure. Further analysis demonstrated that these phenotypes could also be transferred to HIV-1 Envs derived from
different clades including those trimers that were stabilized by incorporation of additional cysteins (SOSIPs). Notably, these
mutations also proved to be combinable.
Single and double mutants showing the highest affinity to the tested bNAbs were further purified as soluble gp140 trimers.
The PG9 GOB mutation led to an increase of the trimer fraction (vs. monomers/dimers) in most isolates, indicating a
transferable positive effect on trimer structure. Furthermore, the biochemical characterization via ELISA showed an
increased affinity of the mutants to both PG9 and VRC01.
In order to further increase trimer stability and decrease the presentation of non-neutralizing antibody (nNAb) epitopes
usually hidden in the inner part of the Env trimer, chemical cross-linking was performed for the selected Env mutants.
Analysis via ELISA revealed a decreased binding of nNAbs 17b and F105, primarily binding to more flexible or CD4 induced
Env conformations, while maintaining the affinity for selected bNAbs targeting different epitopes. This, as well as nanoDSF
measurements verifying the stabilizing effect of the cross-linking by increased melting temperatures, indicates a positive
effect of the cross-linking on the stability of the Env trimers.
In summary, we were able to generate more stable trimeric Env variants with improved antigenicity, which are promising
candidates for testing in preclinical animal models to elucidate their immunogenic potential.
Corresponding author:
Christina Schmalzl
[email protected]
Vaccines
P273
Cytomegalovirus mutants lacking interferon antagonists as candidates for a live attenuated vaccine
A. Jagnjic1, K. Wohlgemuth1, W. Bayer1, K. Hoffmann2, H. Hengel2, V. T. K. Le-Trilling1, M. Trilling1
1University
2Albert
Duisburg-Essen, University Hospital Essen, Institute of Virology, Essen, Germany
Ludwigs University, University Hospital Freiburg, Institute of Virology, Freiburg, Germany
Cytomegalovirus (CMV) infections frequently cause morbidity and mortality in immune-compromised and immune-immature
individuals like congenitally infected newborns. A protective vaccine does not exist. Live attenuated viruses have been used
very successfully as vaccines - including vaccines against veterinarian and human herpesviruses (e.g. the varicella vaccine
strain VZV-Oka).
We have characterized the mouse cytomegalovirus (MCMV) encoded interferon antagonist pM27 for which an analogous
STAT2 degrading protein also exists in human CMV. pM27 acts by recruiting STAT2 to DDB1 containing ubiquitin ligase
complexes. Site-directed mutagenesis of a conserved CxCxxC motif in pM27 impaired the interaction with DDB1 and the
ability to degrade STAT2. pM27-deficient MCMV mutants are extremely interferon susceptible and highly attenuated in vivo.
Here we explore the potential of CMV mutants lacking functional interferon antagonists as live attenuated vaccine viruses.
Despite its transient and low level replication, ΔM27-MCMV raised considerable humoral immune responses (e.g. IgG
against viral proteins and purified virions as well as neutralizing antibodies) in different mouse strains. The functional
impaired CxCxxC mutant similarly mounted IgG responses.
Experiments addressing Fc receptor activation and antibody-dependent cellular cytotoxicity of IgG, T cell responses as well
as protective capacities are currently ongoing.
Corresponding author:
Andreja Jagnjic
[email protected]
Vaccines
P274
Vaccinia virus MVA vector expressing a novel synthetic HCMV pp65-IE1 fusion antigen efficiently induces IE1- and
pp65-specific CD8 T cells in HLA-A0201 transgenic mice
E. Link1,2, C. Brandmüller1, A. Volz1,2, G. Sutter1,2, M. H. Lehmann1,2
1Ludwig
Maximilian University Munich, Institute of Infectious Diseases and Zoonoses, Munich, Germany
Centre for Infection Research (DZIF), Munich, Germany
2German
Infection with human cytomegalovirus (HCMV) affects a majority of humans worldwide. HCMV is efficiently controlled by the
immune system of a healthy individual but it can cause severe disease in immunocompromised patients for example after
organ transplantation.
Cytotoxic T cells control HCMV infection, and the HCMV proteins pp65 and IE1 contain large numbers of CD8 T cell epitopes
and serve as targets of immunodominant T cell responses in healthy carriers.
Modified Vaccinia virus Ankara (MVA), a highly attenuated and replication-deficient vaccinia virus strain, has been shown to
induce cytotoxic T cell responses when used as vector vaccine delivering various viral or tumor-associated antigens.
Additionally, its well-established clinical safety suggests MVA as suitable viral vector for experimental vaccination in
immunocompromised persons. Therefore, MVA has been chosen as a means for delivering HCMV pp65 and IE1 antigens to
induce immunity.
Here, we generated a recombinant MVA expressing a synthetic pp65-IE-1 fusion gene (MVA-syn65_IE1) for evaluation as a
candidate HCMV vaccine. HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/ class II-knockout mice (HHD mice) were
intramuscularly vaccinated twice with MVA-syn65_IE1 as well as with MVA-pp65 and MVA-IE1. Mice were euthanized eight
days after boost vaccination and spleen cells were analysed using murine IFN-γ ELISPOT. Importantly, pp65-specific (NLV)
and IE1-specific (VLE, ILE) T cells were detected in MVA-syn65_IE1 vaccinated mice. The responses to the respective
HCMV antigen were comparable to those induced in mice vaccinated with MVA-pp65 or MVA-IE1 alone. MVA-syn65_IE1
was generated under serum-free conditions and proved stable during low-MOI-passages in chicken embryo fibroblasts
strongly supporting its suitability to serve as HCMV-specific candidate vaccine for clinical evaluation.
Corresponding author:
Michael H. Lehmann
[email protected]
Viral Pathogenesis and Persistance
P275
Characterization of Feline Coronavirus 7b Accessory Protein
D. Florek1, J. Ziebuhr2, H. J. Thiel1, G. Tekes1
1Justus
2Justus
Liebig University Giessen, Institute of Virology, Giessen, Germany
Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
Introduction: Coronaviruses are important pathogens of animals and humans. Their approximately 30 kb large positivestranded RNA genome comprises a variable number of accessory genes at different positions in the 3"-terminal part of the
genome. Homologs of specific accessory genes are conserved in closely related coronaviruses. There is evidence that
accessory gene products are important for virulence in the natural host, but the precise functions of the vast majority of
accessory proteins remain to be investigated.
Objectives: The feline coronavirus (FCoV) genome encodes five accessory proteins termed 3a, 3b, 3c, 7a and 7b with so far
unknown functions. In the present work, we analyzed the expression, localization and posttranslational modification of the
FCoV 7b protein in infected cells.
Material & Methods: Our previously established reverse genetic system was used to generate a panel of recombinant
FCoVs (flag tag at N- or C-terminus of 7b, deletion of N-linked glycosylation site, changes of C-terminal KDEL-like sequence)
for the characterization of accessory protein 7b.
Results & Conclusion: Using a vaccinia virus-based, T7 RNA polymerase-driven expression system it has been reported
by others that 7b is a ~26-kDa secreted glycoprotein, which contains a C-terminal KDEL-like endoplasmic reticulum (ER)
retention signal. In several respects, our study contradicts these previous publications. Using a set of recombinant FCoV
mutants, we were able to show that (i) 7b is not secreted from FCoV-infected cells and (ii) the C-terminal KDEL-like
sequence acts as a Golgi- rather than ER-retention signal. In additional experiments, we determined the signalase cleavage
site for 7b and investigated the significance of N-linked glycosylation for intracellular transport of 7b. The combined data
provide insight into the expression, localization and posttranslational modification of the FCoV 7b protein. Further studies are
underway to identify possible functions of this protein in viral replication and pathogenesis.
Corresponding author:
Dominik Florek
[email protected]
Viral Pathogenesis and Persistance
P276
Transmission of low-pathogenic avian influenza viruses in chickens – Role of the hemagglutinin in organ tropism
and cloacal shedding
G. Zimmer1, T. Ludersdorfer1, M. Tippenhauer1, S. Locher1
1Institute
of Virology and Immunology (IVI), Mittelhäusern, Switzerland
In wild waterfowl, avian influenza viruses (AIV) primarily replicate in the gastrointestinal tract and are shed into the
environment at large quantities facilitating transmission to domestic poultry. Continuous circulation of H5 and H7 AIVs in
domestic poultry is believed to favour the evolution of highly pathogenic AIVs (HPAIV). To understand the adaptation
process of AIV in poultry, we experimentally infected chickens via the intratracheal route with a number of low-pathogenic
AIV (LPAIV) H5 and H7 isolates from wild waterfowl. While some H7 viruses were shed from the gastrointestinal tract of
infected chickens and transmitted to contact animals without prior adaptation, none of the H5 viruses was passed to the
sentinels. An HPAIV (H5N1) isolate from chickens was efficiently transmitted to contact birds but failed to do so when the
proteolytic cleavage site of HA was changed from a polybasic to a monobasic sequence motif, indicating that this virus was
not fully adapted to chickens. Genetic reassortment between a non-transmittable H5N1 and a transmittable H7N7 revealed
that the hemagglutinin (HA) was critical for cloacal shedding and transmission of LPAIV. Likewise, a 7:1 reassortant
harbouring the HA from a low-pathogenic H5N2 virus that has been isolated from domestic chickens was efficiently
transmitted to sentinel birds. Potential molecular determinants for transmission such as receptor specificity, glycosylation and
pH stability of HA are currently analyzed using reassortant H5 viruses with chimeric and mutant HAs. In summary, our
findings suggest that the HA of LPAIV is a key factor for the enterotropism in domestic chickens, which favours cloacal
shedding, transmission, and further adaptation. Our results also suggest that at least some HPAIV (H5Nx) strains have
already evolved in wild birds before they are transmitted to domestic poultry.
Corresponding author:
Gert Zimmer
[email protected]
Viral Pathogenesis and Persistance
P277
Immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1
M. Mühle1, M. Lehmann2, K. Hoffmann3, D. Stern1, T. Kroniger1, W. Luttmann2, J. Denner1
1Robert
Koch Institute, Berlin, Germany
GmbH, Friesoythe, Germany
3Berlin Center of Regenerative Therapies, Berlin, Germany
2ImmunoTools
Introduction: There is increasing evidence that the transmembrane envelope (TM) protein gp41 of the human
immunodeficiency virus - 1 (HIV-1) plays a role in the immunopathogenesis leading to the acquired immunodeficiency
syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as
well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly
conserved domain of all retroviral TM proteins, the immunosuppressive domain. Single mutations in the immunosuppressive
domain abrogated the effect.
Objectives: To study the immunosuppressive properties in more detail, a newly designed gp41 produced in human cells was
used.
Material & Methods: A secreted, soluble recombinant gp41 was produced in 293 cells and characterised. The conformation
was analysed by ELISA and surface plasmon resonance using antibodies against specific conformational states. Binding to
immune cells was studied by FACS analysis using specific antibodies against immune cell subpopulations.
Immunosuppressive properties were studied using cytokine microarrays and a murine model of cytotoxic T cell immunity.
Results: The recombinant gp41 was glycosylated, it assembled into trimers and had a six-helix bundle conformation. The
recombinant gp41 bound to monocytes and to a lesser extend to lymphocytes and triggered the production of specific
cytokines when added to human peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the
antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production.
Conclusion: This is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive
properties. Our data provide additional evidence that gp41 might be directly involved in HIV-1 immunopathogenesis through
modulation of the cytokine release and active inhibition of immune responses.
Corresponding author:
Joachim Denner
[email protected]
Viral Pathogenesis and Persistance
P278
Characterization of cowpox virus proteins based on protein interaction studies
I. Schlenther1, L. Schrick1, J. Döllinger2, A. Nitsche1
1Robert
2Robert
Koch Institute, Centre for Biological Threats and Special Pathogens (ZBS) 1 - Highly Pathogenic Viruses, Berlin, Germany
Koch Institute, Centre for Biological Threats and Special Pathogens (ZBS) 6 - Proteomics and Spectroscopy, Berlin, Germany
The various species of the genus Orthopoxvirus (OPV) show high genetic similarity; however they can vary significantly in
their in vivo host range and virulence. Among the OPV, Variola virus (VARV) was restricted to humans in nature, whereas
Cowpox viruses (CPXV) display the widest host range with variable virulence in their hosts.
CPXV code for the largest set of proteins containing a C-terminal F-Box and multiple Ankyrin-domains (ANK) as proteinprotein interaction-mediating motifs. A subset of 4 of these 14 ANK-proteins is only partially characterized, highly conserved
within the CPXV and absent or severely truncated in OPV-species with a narrow host range, like VARV. As this may indicate
a potential role of these viral proteins for host range extension, interaction studies are performed to characterize their
molecular function.
For the identification of cellular interaction partner pull-down assays with immobilized recombinant viral proteins and native
HeLa cell lysate were performed, followed by quantitative mass spectrometry (nLC MS/MS). Protocols were optimized
thoroughly.
Using the optimized pull-down protocol first potential cellular interaction partners were identified, related to processes like
nucleocytoplasmic transport, translation, apoptosis or cytoskeletal re-arrangement.
To investigate their biological relevance, replication kinetics of both CPXV wildtype and respective deletion mutants will be
compared in cell lines either expressing or lacking the cellular interaction partners. The latter will be generated using
CRISPR/Cas9-mediated gene editing.
Corresponding author:
Ilka Schlenther
[email protected]
Viral Pathogenesis and Persistance
P279
Newcastle disease virus W protein – investigation of its role during infection
J. Karsunke1, A. Karger1, K. Franzke2, A. Groseth3, T. C. Mettenleiter1, A. Römer-Oberdörfer1
1Friedrich
Loeffler Institute, Institute of Molecular Virology and Cell Biology, Greifswald - Isle of Riems, Germany
Loeffler Institute, Institute of Infectology, Greifswald - Isle of Riems, Germany
3Friedrich Loeffler Institute, Working Group Arenavirus Biology, Greifswald - Insel Riems, Germany
2Friedrich
Co-transcriptional mRNA editing of the phosphoprotein (P) gene is a general principle for viruses of the family
Paramyxoviridae to enlarge their coding capacity. By the insertion of non-templated nucleotides, proteins with interferon
(IFN) antagonistic activities are expressed having beneficial effects on replication or infection like the V protein of Newcastle
disease virus (NDV), an avian paramyxovirus causing high economic losses in poultry worldwide. While V protein is
expressed after insertion of one non-templated G nucleotide, insertion of two nucleotides leads to the synthesis of mRNA
encoding the so far hypothetic NDV W protein.
We demonstrate the existence of the NDV W protein and analyze its localization in infected cells, its function, and its
essentiality for viral replication.
To investigate subcellular localization, expression plasmids for NDV P, V or W protein, were transfected into quail muscle
(QM9) cells. Using confocal microscopy and a W protein-specific antiserum NDV W protein could be detected in the nuclei of
transfected and infected cells. A potential nuclear localization sequence (NLS) within the W specific carboxy-terminal part of
the protein was identified and validated by mutation of crucial basic residues. Furthermore, recombinant NDV with either
deletions of the editing sites of the P gene and an additionally introduced V gene open reading frame (ORF), or a stop
mutation in the W gene reading frame within the P gene were constructed and characterized. Prevention of W protein
expression did not alter NDV replication kinetics on chicken embryo fibroblasts (CEF) in the presence of the V protein. An
interferon assay was used to characterize the interferon antagonistic activity of W protein.
In conclusion, we demonstrated expression of NDV W protein during infection and show that it localizes to the nuclei
mediated by a potential NLS.
Corresponding author:
Julia Karsunke
[email protected]
Viral Pathogenesis and Persistance
P280
Seminal plasma inhibits human Cytomegalovirus infection
S. Lippold1, J. Müller2, J. Münch2, J. von Einem1
1Ulm
2Ulm
University Medical Center, Institute of Virology, Ulm, Germany
University Medical Center, Institute of Molecular Virology, Ulm, Germany
Introduction: Human Cytomegalovirus (HCMV) is a ubiquitous and widespread human pathogen with a high
seroprevalence. One possible way of HCMV transmission is through sexual contact. In agreement with this, HCMV is
frequently detected in semen, suggesting that this body fluid may serve as vector for virus transmission. It has been shown
recently that semen-derived amyloid fibrils enhance HCMV infection in cell culture (Tang et al. 2013), as it was first reported
for human immunodeficiency virus (HIV) (Münch et al. 2007). These fibrils form by self-assembling peptides derived from
semen proteins PAP and SEM1&2. The best characterized seminal amyloid is termed semen-derived enhancers of viral
infection (SEVI) and consists of PAP248-286 peptides.
Objectives: The aim of this work was to investigate the effect of SEVI and whole semen on HCMV infection.
Material & Methods: Two different endotheliotropic HCMV strains were incubated for 1 h at 37°C with different
concentrations of SEVI, seminal plasma (SP; semen devoid of spermatocytes) and heat-inactivated SP. These mixtures
were then added to either fibroblasts or endothelial cells for 1 h at 37°C. Subsequently, cells were washed and incubated
with medium overnight. HCMV infection rates were quantified with indirect immunofluorescence experiments and flow
cytometry.
Results: Synthetic SEVI fibrils enhanced HCMV infection up to 50% confirming previous data (Tang et al. 2013). In contrast,
infection was severely reduced in both cell lines when performed in presence of as little as 5% of SP, while HIV infection was
clearly enhanced. Consistently, heat inactivation of semen-derived amyloid fibrils in SP had no effect on the inhibiting effect
of SP on HCMV infection. Furthermore, the inhibitory effect of SP could not be overcome by addition of SEVI to SP. Finally,
incubation of cells with SP prior infection had no effect on the susceptibility of these cells to HCMV infection.
Conclusion: Although an enhancing effect of SEVI on HCMV infection was confirmed, it is counteracted by an inhibitory
effect in the context of semen. This inhibition of HCMV infection by SP appears to be a direct effect on the virus and not due
to an impaired susceptibility of cells. Therefore, we conclude, that SP contains components that suppress HCMV infectivity.
Corresponding author:
Sina Lippold
[email protected]
Viral Pathogenesis and Persistance
P281
Construction of Chimeric Dengue Virus Type 2 to explore the determinant of pathogenicity in mice
P. Dhole1, A. Saito1, E. Nakayama1, T. Shioda1, T. Kawagishi2, T. Kobayashi2, K. Limkittikul3, T. Kurosu4
1RIMD,
Osaka University, Viral Infections, Osaka, Japan
Osaka University, Osaka, Japan
3Mahidol University, Faculty of Tropical Medicine, Bangkok, Thailand
4National Institute of Infectious Diseases, Department of Virology I, Tokyo, Japan
2RIMD,
In past few years, vector-borne flaviviral infections have been challenging human population inhabiting tropical and subtropical areas of the world. Among these, dengue virus (DENV) annually causes approximately 390 million infections and
500,000 deaths worldwide. To develop anti-DENV vaccines and drugs, we need a suitable small animal model in which we
can reproduce symptoms of humans. We recently reported that infection of immunodeficient mice lacking IFNα/β/γ receptor
gene with a DENV type 2 (DENV-2), P04/08 strain resulted in 100% mortality. This phenotype is in sharp contrast with that of
a lab-adapted DENV-2 16681 strain that does not kill mice. We compared the viral genome sequence of 16681 strain with
those of two DENV strains P04/08 and PL046 which kill mice. We found several amino acid differences among those three
strains but we have focused on NS5 gene, since NS5 protein has bi-functional activity (RNA-dependent RNA polymerase
and methyl-transferase). For instance, at 2738th amino acid position, 16681 has tyrosine while P04/08 and PL046 have
histidine. Moreover, at 3359th position 16681 has alanine while on the other hand, both P04/08 and PL046 have valine and
threonine respectively. These results suggest that polymorphism in NS5 gene may be responsible for the pathogenicity in
mice. To test our hypothesis, we aimed to construct a chimeric 16681 virus which incorporates NS5 gene of P04/08 strain.
Initially, we generated a linearized vector containing pCMV-DV2-16681 and an insert by amplifying NS5 gene of P04/08 with
gene specific primers to make a 15 bp overlap with the vector end. We are now performing In-Fusion cloning reaction to
ligate fragments of vector and insert. The resultant plasmid DNA will be used for transfection on hamster cells to produce
chimeric virus. We will test the replication capability of chimeric viruses in vitro prior to experiments in mice.
Corresponding author:
Priya Dhole
[email protected]
Viral Pathogenesis and Persistance
P282
Role of the protein m139 in murine cytomegalovirus replication in macrophages
O. Puhach1, E. Ostermann1, W. Brune1
1Heinrich
Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
Blood monocytes and differentiated macrophages are important players in the pathogenesis of cytomegalovirus infection.
They contribute to viral dissemination within the host and establishment of latency. In addition, macrophages play an
important role in early inflammatory and innate immune responses. Murine cytomegalovirus (MCMV) genes m139, m140 and
m141 have been described as important for efficient virus replication in differentiated macrophages in vitro and viral
dissemination in vivo. Proteins encoded by these genes form a stable complex and colocalize in transfected cells, where
they are thought to optimize MCMV replication in a cooperative manner. However, the underlying mechanisms have
remained unknown.
In this study we explored the role of m139 during infection of fibroblasts and differentiated macrophages. We show that
deletion of m139 and its putative binding partners m140 and m141 leads to a replication defect and reduced expression of
the viral immediate-early protein 3 and the early proteins e1 (M112–113) and M142 in macrophages, but not in fibroblasts.
Immunofluorescence analysis showed accumulation of m139 in viral replication compartments within the nucleus at early
times post infection and relocalization to perinuclear viral assembly compartments at later time points. This subcellular
distribution was independent of m140 and m141. The recruitment of m139 to nuclear replication compartments was mediated
by the viral e1 (M112–113) proteins. By using an interferon-beta reporter assay we found that an MCMV mutant lacking
m139 caused a stronger IFN-beta induction in macrophages than wildtype MCMV. However, the levels of phosphorylated
Interferon Regulatory Factor 3 (IRF3) during infection were not affected by m139, suggesting that the m139 protein might
affect IFN-beta production independent or downstream of IRF3.
These results indicate an involvement of m139 in MCMV gene expression and replication in macrophages. The requirement
of m139 for limiting MCMV-induced activation of the IFN-beta promoter suggests that the effects of m139 on viral replication
in macrophages might be IFN-dependent.
Corresponding author:
Olha Puhach
[email protected]
Viral Pathogenesis and Persistance
P283
Merkel cell polyomavirus encoded small viral tumor antigen contributes to metastasis formation in a xenograft
mouse model
M. Czech-Sioli1, J. Knips2,3, M. Spohn4, I. Moll5, M. Heiland3, A. Grundhoff4, U. Schumacher2, N. Fischer1
1University
Medical Center Hamburg Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany
Medical Center Hamburg Eppendorf, Institute of Anatomy and Experimental Morphology, Hamburg, Germany
3University Medical Center Hamburg Eppendorf, Department of Oral and Maxillofacial Surgery, Hamburg, Germany
4Heinrich Pette Institute, Leibniz Institute of Experimental Virology, Hamburg, Germany
5University Medical Center Hamburg Eppendorf, Department of Dermatology, Hamburg, Germany
2University
Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high
propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is
monoclonally integrated in up to 95 % of tumors. While there are presently no animal models to study the role of authentic
MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted
MCC derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two
MCPyV positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC
cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing
a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice.
Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all
tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of
explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in
inflammatory response, growth factor activity and Wnt signaling pathway are significantly upregulated, suggesting that sTAntigen is the driver of the observed differences in metastasis formation.
Corresponding author:
Nicole Fischer
[email protected]
Viral Pathogenesis and Persistance
P284
Impact of virus-induced ERK signaling on the expression of Na,K-ATPase and ENaC during influenza A virus
infection
I. Kuznetsova1, C. Peteranderl2, J. Schulze3, S. Herold2, T. Wolff3, S. Pleschka1
1Justus
Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
Giessen & Marburg Lung Center, Department of Internal Medicine II, Giessen, Germany
3Robert Koch Institute, Division of Influenza and Other Respiratory Viruses, Berlin, Germany
2Universities
Introduction: Influenza A virus (IAV) infection may cause life-threatening conditions such as Acute Lung Injury (ALI) and
Acute Respiratory Distress Syndrome (ARDS), which are characterized by lung edema formation. Clearance of alveolar fluid
is strongly dependent on active transport of sodium across alveolar epithelial cells. The main ion channels that contribute to
the establishment of an osmolytic gradient are Epithelial Sodium Channel (ENaC) and Sodium-Potassium Adenosine
Triphosphatase (NKA). Previously it was shown that IAV infection has a negative effect on the membrane expression of
ENaC and NKA and that Raf/MEK/ERK signaling is involved in the regulation of the expression and activity of both channels.
Moreover, IAV-induced activation of the Raf/MEK/ERK cascade in infected cells is needed for productive IAV replication.
Objectives: Our aim was to illuminate the role of virus-induced ERK activity for ENaC and NKA expression in IAV-infected
cells.
Material & Methods: We analyzed influence of the MEK inhibitors U0126, CL1040 and Trametinib on influenza virus
A/Puerto Rico/8/34(H1N1) (PR8) growth in Calu-3 cells and virus-induced ERK phosphorylation by titration and "in cellwestern-blot" analysis, respectively. Also, the effect of the viral infection (+/-) MEK-inhibitors on the total amount of ENaCß or
NKAα was determined by immunoblotting.
Results: MEK-inhibition decreased the viral titer, prevented ERK phosphorylation and reduced the decrease of the ENaCß
protein during PR8 infection. Furthermore, total amount of NKAα was analyzed and data will be presented.
Conclusion: Reduction of the total amount of ENaCß but not NKAα during IAV infection seems to be connected to virusinduced ERK-activation and can be restored by using MEK-inhibitors, which also impair IAV replication.
Corresponding author:
Irina Kuznetsova
[email protected]
Viral Pathogenesis and Persistance
P285
importance of the ciliary activity of the airway epithelium in preventing influenza virus infection
Y. Fu1, F. Meng1, G. Herrler1
1University
of Veterinary Medicine Hanover, Institute of Virology, Hanover, Germany
The airway epithelium is equipped with the mucociliary clearance system to prevent the detrimental effect of foreign
substances including infection by microorganisms. This defense mechanism is based on the mucins produced by mucusproducing cells and the ciliary activity of specialized epithelial cells that transport the mucus out of the respiratory tract. We
applied precision-cut lung slices as a culture system of differentiated airway epithelial cells to analyze the importance of the
ciliary activity for the infection by influenza viruses.
Conditions were established that result in reversible ciliastasis. By applying this technique, we are able to compare infection
in the presence and absence of ciliary activity. This approach will help to determine how efficient is the ciliary activity in
preventing virus infection.
Recently, we have shown that the ciliary activity of PCLS can serve as a virulence marker for the respective virus. This
finding was obtained by analyzing swine influenza viruses of the subtypes H1N1, H1N2, and H3N2. Depending on their
virulence, they caused partial or complete ciliostasis.
Currently, we are applying this approach to determine whether isolates of the pandemic H1N1 virus recovered from humans
and swine show differences in their ciliostatic effect and thus in their virulence properties.
Corresponding author:
Yuguang Fu
[email protected]
Viral Pathogenesis and Persistance
P286
Prepackaged nonstructural protein 1 of Influenza A virus supports ribonucleoprotein import into the nucleus
T. W. Sha1, M. Gerlach2
1Kyoto
University, Integrated Life Science, Kyoto, Japan
Liebig University Giessen, Institute of Virology, Giessen, Germany
2Justus
The nonstructural protein 1 (NS1) of Influenza A virus (FLUAV) is a key player in inhibiting type I interferon (IFN) production
in host cells. Other roles besides IFN antagonism have not been well studied yet. Recently, it was also shown that NS1 is
packaged in the virion but the significance of this prepackaged NS1 is unclear. Thus, we investigated the role of
prepackaged FLUAV NS1 on viral replication.
We observed a delayed translocation of incoming FLUAV ribonucleoprotein complexes (RNP) into the nucleus with
recombinant FLUAV virus lacking NS1 compared to its wild type virus. This delay of nuclear RNP import could be also
observed in IFN incompetent cells. This suggests that prepackaged NS1 exerts a supportive effect on RNP nuclear import
independent of NS1 transmitted IFN antagonism.
It was shown that FLUAV virus lacking NS1 is attenuated compared to its wild type virus. This might due to delayed RNP
import into the nucleus and hence delayed onset of virus replication. Moreover, identification of the novel function of NS1 as
a nuclear import factor increases our knowledge about the broad spectra activities of FLUAV NS1.
Corresponding author:
Tim Wai Sha
[email protected]
Viral Pathogenesis and Persistance
P287
Pathogenesis and transmission of the novel highly pathogenic avian influenza H5N8 2016 virus in ferrets and mice
D. Hoffmann1, M. Naguib1, T. C. Harder1, C. Grund1, M. Beer1
1Friedrich
Loeffler Institute, IVD, Greifswald - Isle of Riems, Germany
Introduction: The recent incursion of a new reassortant of HPAI H5N8 virus clade 2.3.4.4b in 2016 resulted in an epidemic
outbreak among wild birds and frequent infection of poultry throughout Europe. As some HPAIV bear zoonotic propensities
the potential of this new reassortant virus to infect mammalian species including humans is of special interest.
In this study, the potential of cross-species infection was evaluated by determining the pathogenicity and transmissibility of
HPAI H5N8 2016 virus in different mammalian animal models.
Material & Methods: The virulence and pathotype of A/tufted duck/Germany/AR8444/2016 H5N8 was assessed by
experimental inoculation of ferrets and balb/c mice. Viral titers in nasal washes or organ samples (3 and 6 DPI) were
determined by virus titration and segment-specific qPCR. Sero-responses were measured by ELISA detecting antibodies
against NP protein and H5 protein, as well as by hemagglutination inhibition assays.
Results: The novel reassortant H5N8 virus replicated in mouse lungs without prior adaptation. An infectious dose of 102
TCID50/animal resulted in a survival rate of 40%, while animals receiving higher dosages had to be euthanized until 9 DPI.
Substantial virus titers in lungs were found at 3 DPI and systemic spread of infectious virus to the brain was verified from
mice euthanized 6 DPI. Taken together HPAIV AR8444/2016 H5N8 replicate in mice and generally demonstrated a highpathogenicity phenotype.
Eight ferrets were i.n. inoculated with 106 TCID50/animal. Four ferrets served as transmission sentinels by direct contact.
Four ferrets were humanely euthanized on 3 DPI to assess viral spread in organs. No respiratory symptoms and only minor
changes in body weight and body temperature were detected. From a single nasal wash sample virus was re-isolated, and
moderate viral titers were determined from various organ samples. The naive ferrets contact did not seroconvert, confirming
that AR8444/2016 H5N8 was not transmitted between ferrets.
Conclusion: To assess virulence and transmissibility of HPAIV in humans, it is imperative to examine their pathobiological
properties using mammalian models. Clearly, the new H5N8 virus evaluated here exhibited a mild virulent phenotype in
ferrets and was not transmissible, while it demonstrated high-pathogenicity in mice.
Corresponding author:
Donata Hoffmann
[email protected]
Viral Pathogenesis and Persistance
P288
Characterization of the ORF4-encoded accessory protein of human coronavirus 229E
A. Friedrich1, G. Tekes2, J. Ziebuhr1
1Justus
2Justus
Liebig University Giessen, Institute of Medical Virology, Giessen, Germany
Liebig University Giessen, Institute of Virology, Giessen, Germany
Introduction: Coronaviruses (CoV) are enveloped, positive-sensed, single-stranded RNA viruses with genomes of ~30 kb.
Along with the viral structural proteins and replicase-gene encoded nonstructural proteins, CoVs encode a varying number of
"accessory" proteins with no detectable homology to other viral and cellular proteins. Recently, a putative accessory protein
encoded in a genome region between the spike (S) and envelope (E) protein genes was identified in most viruses belonging
to the genera Alpha- and Betacoronavirus. The proposed phylogenetic relationship is mainly based on similar
transmembrane topologies and, to date, there is limited information regarding the functional roles of these distantly related
proteins.
Objective: To characterize the functional role(s) of the ORF4-encoded accessory protein in coronavirus replication.
Material & Methods: A reverse genetic approach to generate a set of recombinant HCoV-229E viruses and confocal
microscopy were used to characterize the HCoV-22E ORF4-encoded protein.
Results & Conclusion: HCoV-229E mutants have been generated using a vaccinia virus-based reverse genetic approach
suitable to produce and genetically manipulate full-length HCoV-229E genome RNA. The HCoV-229E ORF4a/b sequence
was (i) reconstituted to yield a continuous ORF4, (ii) replaced with the ORF4 of a field isolate, (iii) replaced with the
equivalent accessory protein gene from other alphacoronaviruses, (iv) deleted from the HCoV-229E genome. The analysis of
the first mutants (i+ii) revealed only minimal differences in the in vitro growth kinetics compared to wild-type HCoV-229E (Inf1) in Huh7 and MRC5 cells. ORF4 protein-specific polyclonal antisera were used to analyze the subcellular localization of the
protein. Further characterization of the remaining chimeric viruses is underway and will include studies of viral replication and
antiviral host responses in primary human cells (macrophages, dendritic cells and human airway epithelial cells) to gain more
insight into possible functions of this accessory protein in viral tropism and pathogenesis.
Corresponding author:
Alexandra Friedrich
[email protected]
Viral Pathogenesis and Persistance
P289
Barrier function of the differentiated airway epithelium retains after infection by influenza viruses
N. H. Wu1, D. L. Shin1, W. Yang1, F. Meng1, R. Dijkman2,3, M. Matrosovich4, V. Thiel2,3, P. Valentin-Weigand5, G. Herrler1
1University
of Veterinary Medicine Hanover, Institute of Virology, Hanover, Germany
of Virology and Immunology, Federal Department of Home Affairs, Bern, Switzerland
3University of Bern, Institute of Virology and Immunology, Vetsuisse Faculty, Bern, Switzerland
4Philipps University Marburg, Institute of Virology, Marburg, Germany
5University of Veterinary Medicine Hanover, Institute of Microbiology, Hanover, Germany
2Institute
Mucociliary clearance is the first line of defence of the airway epithelium against infections by respiratory pathogens. In an ex
vivo culture model, precision-cut lung slices, we have previously shown that impairment of the ciliary clearance after swine
influenza virus infection enhanced the susceptibility of the epithelial cells to secondary bacterial infection. To further study
morphological and functional changes in the airway epithelium after virus infection, we applied an air-liquid interface culture
system for well-differentiated porcine bronchial epithelial cells (PBEC) to analyze the respiratory epithelium for virus-induced
cellular damages. PBEC were infected via the apical membrane domain by swine influenza viruses of different subtypes.
Virus was continuously released from the apical surface up to eight-day post-infection. In the course of infection, the
differentiated PBEC underwent apoptosis and a dramatic loss of cilia, accompanied by a reduction of the epithelial thickness.
Despite these detrimental effects, the barrier function of the PBEC was retained, as indicated by the transepithelial
resistance and the presence of tight junctions. The basal cells had started to differentiate into specialized cells to replace the
lost cells. Our results suggest that basal cells help to maintain the barrier function of the airway epithelium and thus prevent
the spread of infection to deeper tissue layers. Our culture model allows analyzing the regeneration of the airway epithelium
after virus infection. In the future, we will use this culture system to analyze the susceptibility of the regenerating epithelium to
secondary viral/bacterial infections.
Corresponding author:
Dai-Lun Shin
[email protected]
Viral Pathogenesis and Persistance
P290
Human Endogenous RetroViruses (HERVs), the missing link between exogenous pathogens and mis-understood
auto-immune diseases
B. Charvet1,2, J. Medina1,2, S. Levet1,2, H. Perron1,2
1GeNeuro,
2GeNeuro
Plan les Ouates, Switzerland
Innovation, Lyon, France
Herpesviruses and retroviruses viruses have long been associated with numerous mis-understood auto-immune diseases,
but failed to provide any clear direct link with their pathogenesis. Recently, Human Endogenous Retroviruses (HERVs) were
considered as a new type of pathogens of endogenous origin. A large majority of HERV sequences are inactivated by
cellular mechanisms or by accumulation of mutations. Nevertheless, few HERVs sequences are still encoding functional
proteins but their transcription is strongly regulated (Seifarth et al., 2005).
Among HERV sequences coding for proteins, certain were domesticated like ERVWE1, encoding a retroviral envelope
protein (Env), Syncytin, which became a crucial partner during placenta formation. However, the majority of other HERVs
sequences encoding proteins, most often Env proteins, were associated with various diseases, e.g., HERV-K and
Amyotrophic Lateral Sclerosis, HERV-W and multiple sclerosis (MS), neuropsychiatric disorders like schizophrenia or bipolar
disorder (Balada et al., 2010 ; Frank et al., 2005 ; Perron et al., 2008 ; Nath et al., 2015). Indeed, Env Proteins encoded by
HERV-W family genes possess superantigen properties, inducing a strong pro-inflammatory response, via TLR-4 activation
(Rolland et al., 2006; Duperray et al., 2015). The most studied HERV-W sequences belong to the MS RetroViral element
(MSRV) isolated from purified viral particles isolated from MS patients cell cultures (Perron et al., 1989, 1991 and 1997;
Serra et al., 2001; Dolei et al., 2002).
Pathologic HERV copies expression still remain "dormant" in physiological conditions but they can be "awaken" by
environmental factors such as Herpesviruses or Retroviruses. Exogenous viruses can disrupt repressive mechanisms of
HERV copies by DNA methylation inhibition (Sutkowski et al., 2004; Hsiao et al., 2009), can directly or indirectl