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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