Download Bat ID Program final - Rocky Mountain Virology Club

Infectious Diseases of
Bats Symposium
June 26-27, 2014
Colorado State University
Fort Collins, CO, USA
Infectious Diseases of Bats Symposium!
Fort Collins, CO, USA
Table of Contents
Presentations
3
Posters
8
Presentation Abstracts
10
Poster Abstracts
27
Acknowledgments and Financial Support
35
Campus Map
36
MAX Bus Schedule
37
List of Attendees
38
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Infectious Diseases of Bats Symposium!
Fort Collins, CO, USA
Program
Venue: Lory Student Center and Allison Hall, Colorado State University
Wednesday, June 25
5:30 p.m. Registration, PowerPoint file transfer, Allison Hall
6:00 p.m. Reception - Wine, beer and snacks, Allison Hall
Thursday, June 26
7:00 a.m. Registration, North Ballroom, Lory Student Center
8:00 a.m. Tony Schountz. Colorado State University. Welcoming remarks
8:10 a.m. Charles H. Calisher. Are bats the source of (almost) all vertebrate viruses (and God
knows what else)? Colorado State University
8:30 a.m. Session I - Coronaviruses (Katherine Holmes, Moderator)
8:30 a.m. Susanna Lau. Bat coronaviruses: diversity, evolution and interspecies
transmission. University of Hong Kong, Hong Kong, China
9:00 a.m. Christian Drosten. Origins, hosts and sources of MERS coronavirus. University of
Bonn Medical Centre, Bonn, Germany
9:30 a.m. Vincent Munster. The ecology of Middle East respiratory syndrome coronavirus
(MERS-CoV) in a reservoir host. Rocky Mountain Laboratories, NIAID, Hamilton, MT
10:00 a.m. Break
10:30 a.m. Joseph Prescott, E. de Wit, D. Falzarano, D. Scott, H. Feldmann, V. Munster.
Immunosuppression in the rhesus macaque model of Middle East respiratory
syndrome. Rocky Mountain Laboratories, NIAID, Hamilton, MT
10:45 a.m. Supaporn Wacharapluesadee1, P. Duengkae2, A. Rodparn1, T. Kaewpom1, P.
Maneeorn3, S. Yinsakmongkon1, N. Sittidetboripat1, C. Chareesaen3, N. Khlangsap3, A.
Pidthong3, K. J. Olival4, J. H. Epstein4, P. Daszak4, P. Blair5, T. Hemachudha1.
Surveillance for and diversity of coronaviruses in bats from eastern Thailand.
World Health Organization Collaborating Centre for Research and Training on Viral
Zoonoses, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand1, Faculty of
Forestry, Kasetsart University, Bangkok, Thailand2, Department of National Parks,
Wildlife and Plant Conservation, Bangkok, Thailand3, EcoHealth Alliance, New York,
USA4, Naval Medical Research Center-Asia, US Embassy Singapore, PSA Sembawang,
Singapore5
11:00 a.m. Luis Góes1, A. C. Campos1, G. Ambar2, A. Neto2, E. L. Durigon1. Comparison of 3
nested PCR pancoronavirus detection assays in bats from Brazil. Universidade de
São Paulo - USP, São Paulo, SP, Brazil1, Universidade Estadual Paulista Júlio de
Mesquita Filho - UNESP, Rio Claro, SP, Brazil2
11:15 a.m. Session II - White-Nose Syndrome (Paul Cryan, Moderator)
11:15 a.m. Paul Cryan. Ecology of white-nose syndrome. USGS Fort Collins Science Center,
Fort Collins, CO
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Infectious Diseases of Bats Symposium!
Fort Collins, CO, USA
11:30 a.m. David Blehert. White-nose syndrome: a deadly mycosis of hibernating bats. United
States Geological Survey, Madison, WI
12:00 p.m. Lunch and Poster Session
2:00 p.m. DeeAnn Reeder. The immunological response to the fungal pathogen
(Pseudogymnoascus destructans) that causes white-nose syndrome in bats.
Bucknell University, Lewisburg, PA
2:30 p.m. Kate Langwig1, W. Frick1, T. Kunz2, J. Foster3, A. M. Kilpatrick1. Infection loads, not
exposure, drive impacts of white-nose syndrome among species. University of
California Santa Cruz1, Boston University2. Northern Arizona University, Flagstaff, AZ3
3:00 p.m. Break
3:30 p.m. Session III - Rabies (Amy Gilbert, Moderator)
3:30 p.m. R. Sachidanandam1, O. Attie1, A. Jayaprakash1, R. S. Shabman2,3, A. Davis4 and
Christopher F. Basler2. Transcriptome profiling reveals robust interferon responses
in the brain of rabies infected Myotis lucifugus bats and identifies a novel gamma
herpesvirus in a bat cell line.. Dept. Genetics, Icahn School of Medicine at Mount
Sinai, New York1, Dept. Microbiology, Icahn School of Medicine at Mount Sinai, New
York2, Virology group, J. Craig Venter Institute3, Rabies Laboratory, Wadsworth Center,
New York State Department of Health, NY4
4:00 p.m. Amy T. Gilbert. Lyssaviruses in bats; current knowledge and future directions.
National Wildlife Research Center, USDA, Fort Collins, CO
4:15 p.m. Monica Borucki1, H. Chen-Harris1, S. Messenger2, D. Wadford2, J. Allen1. Analysis of
viral populations for improved biosurveillance and prediction of viral emergence.
Lawrence Livermore National Laboratory1, California Department of Public Health, CA2
4:30 p.m. Lisa Worledge, H. Miller. Bat disease surveillance in the UK: the role of
conservation volunteers. Bat Conservation Trust, London, UK.
4:45 p.m. Dana Mitzel, D. Merrill, K. Dearen, R. Russell, P. Kuehl, Robert Baker. Aerosol
infection of mice with rabies virus, and comparison to intramuscular and
intranasal routes of infection. Lovelace Respiratory Research Institute, Albuquerque,
NM
5:00 p.m. Open Discussion
6:00 p.m. Recess
Friday, June 27
7:30 a.m. Registration, North Ballroom, Lory Student Center
8:00 a.m. Lin-Fa Wang. From cell lines to bat genomes: are we ready to study virus-bat
interactions in a serious manner? Duke-National University of Singapore Graduate
Medical School, Singapore
8:30 a.m. Peter Daszak. Linking environmental conservation with human and wildlife health.
EcoHealth Alliance, New York City, NY
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9:00 a.m. Session IV - Filoviruses and Paramyxoviruses (Charles Calisher, Moderator)
9:00 a.m. Jonathan Towner. Replication of marburgviruses in their reservoir host, Egyptian
fruit bats (Rousettus aegyptiacus). U.S. Centers for Disease Control and Prevention,
Atlanta, GA
9:30 a.m. J. Paweska1, Petrus Jansen van Vuren1, S. McCulloch2, A. Kemp1, N. Storm2, A.
Grobbelaar1, T. Scott2, M. Geldenhuys2, M. Mortlock2, N. Moolla1, A. Coetzer2, L. Nel2,
W. Markotter2. Marburg virus infection in Egyptian fruit bats (Rousettus
aegyptiacus), South Africa. Center for Emerging and Zoonotic Diseases, National
Institute for Communicable Diseases of the National Health Laboratory Service,
Sandringham, Gauteng, South Africa1, Viral Zoonosis Group, Department of
Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences,
University of Pretoria, Pretoria, Gauteng, South Africa2
9:45 a.m. Amy Schuh, B. Amman, M. Jones, T. Sealy, L. Uebelhoer, B. Martin, S. Nichol, J.
Towner. Investigation of bat-to-bat transmission of Marburg virus in the Egyptian
fruit bat, Rousettus aegyptiacus. U.S. Centers for Disease Control and Prevention,
Atlanta, GA
10:00 a.m. Break
10:30 a.m. Melinda Ng1, E. Ndungo1, M. Kaczmarek2, A. Herbert3, R. Biswas1, A. Demogines2, M.
Muller5, J. H. Kuhn4, J. M. Dye3, S. Sawyer2, K. Chandran1. Molecular evolution of the
filovirus receptor NPC1 in bats. Albert Einstein College of Medicine, Bronx, NY1,
University of Texas at Austin, Austin2, US Army Medical Research Institute of
Infectious Diseases, Fort Detrick, MD3, Tunnell Consulting and NIH/NIAID
Integrated Research Facility, Ford Detrick, MD4, Universitats Klinikumbonn,
Bonn, Germany5
10:45 a.m. Jon Epstein. The ecology of Nipah virus in Bangladesh. EcoHealth Alliance, New
York City, NY
11:15 a.m. Emmie de Wit, J. Prescott, D. Falzarano, T. Bushmaker, D. Scott, H. Feldmann, V.
Munster. Modeling the zoonotic transmission cycle of Nipah virus in Bangladesh.
Rocky Mountain Laboratories, NIAID, Hamilton, MT
11:30 a.m. Brian Amman1, C. Albariño1, T. Sealy1, B. Bird1, A. Schuh1, S. Campbell1, U. Stroeher1,
M. Jones1, M. Vodzak2, D. Reeder2, S. Nichol1, J. Towner1. Sosuga virus
(Paramyxoviridae) detected in Egyptian fruit bats (Rousettus aegyptiacus) from
Uganda. Centers for Disease Control and Prevention, Atlanta, GA1, Bucknell University,
Lewisburg, PA2
12:00 p.m. Lunch
1:00 p.m. Session V - Immunology of Bats (Tony Schountz, Moderator)
1:00 p.m. Michelle Baker. Innate antiviral immunity in the Australian black flying fox,
Pteropus alecto. Australian Animal Health Laboratory, Geelong, Australia
1:30 p.m. Aaron Irving1, M. Ahn1, L. Wang1,2. Interrogating inflammasome signaling pathways
in the black flying fox, Pteropus alecto. Duke-NUS Graduate Medical School,
Singapore, Singapore1, Australian Animal Health Laboratory, CSIRO Livestock
Industries, East Geelong, Victoria, Australia2
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1:45 p.m. Nicole B. Glennon1, O. Jabado2, M. K. Lo3, M. L Shaw1. Characterizing the antiviral
response of the large flying fox (Pteropus vampyrus), an important ecological
reservoir of henipaviruses. Icahn School of Medicine at Mount Sinai Department of
Microbiology, New York, NY1, Icahn School of Medicine at Mount Sinai Department of
Genetics and Genomic Sciences, New York, NY2, Center for Disease Control &
Prevention Viral Special Pathogens Branch, Atlanta, GA3
2:00 p.m. Neeltje van Doremalen1, K. Cameron2, S. Milne-Price1, S. Olson2, A. Ondzie2, T.
Bushmaker1, J-V. Mombouli2, P. Reed2, V. Munster1. Development of a serological
assay for the investigation of emerging viruses in central African fruit bats.
Laboratory of Virology, Virus Ecology Unit, Division of Intramural Research,
NIAID, Rocky Mountain Laboratories, Hamilton, MT1, Wildlife Conservation
Society, Bronx NY2, Laboratoire National de Santé Publique, Brazzaville, Congo3
2:15 p.m. Session VI - Other Infectious Agents of Bats (Rebekah Kading, Joel Rovnak,
Moderators)
2:15 p.m. Elizabeth Cook1, G. Michuki2, C. Rumberia2, A. Kiyong'a2, J. Akoko2, A. Ogendo3, E.
Dobson2, M. Bronsvoort1, S. Kemp2, B. Agwanda4, E. Fevre5. A metagenomic
approach identifies plasmodium species in bats in Kenya. University of
Edinburgh, Edinburgh, UK1, International Livestock Research Institute, Nairobi,
Kenya2, Kenya Field Epidemiology and Laboratory Training Program, Nairobi,
Kenya3, National Museums of Kenya, Nairobi, Kenya4, University of Liverpool,
Liverpool, UK5
2:30 p.m. David Morán1, D. Alvarez1, M. F. Rizzo3, Y. Bai3, L. F. Peruski2, M. Kosoy3. Geographic
distribution and diversity of bartonella in bats from Guatemala. Centro de Estudios
en Salud, Universidad del Valle de Guatemala, Guatemala, Guatemala1, Centers for
Disease Control and Prevention, Central American Region, Guatemala, Guatemala2,
Centers for Disease Control and Prevention, Division of Vector Borne Diseases, Fort
Collins, CO3
2:45 p.m. Ying Bai. Bartonella infections in bats. U.S. Centers for Disease Control and
Prevention, Fort Collins, CO
3:00 p.m. Break
3:30 p.m. Kevin Olival1, C. Weekley2. Viral discovery in bats: a quantitative review of ~100
studies from the last seven years. EcoHealth Alliance, New York, NY1, UC Berkeley,
Berkeley, CA2
3:45 p.m. Hideki Ebihara. Molecular identification of the relationship between arthropodborne bunyaviruses and bats. Rocky Mountain Laboratories, NIAID, Hamilton, MT
4:00 p.m. Rebekah Kading1, R. Kityo2, E. Mossel1, J. Towner3, B. Amman3, T. Sealy3, T. Nakayiki4,
A. Gilbert5, I. Kuzmin5, B. Agwanda6, J. Kerbis7, B. Nalikka2, E. Borland1, L.
Nyakarahuka4, M. Crabtree1, J. Montgomery8, C. Rupprecht5, S. Nichol3, J. Lutwama4,
B. Miller1. Virus isolations from and arbovirus surveillance of bats in Uganda and
Kenya. CDC Division of Vector-borne Diseases, Arbovirus Diseases Branch, Fort
Collins, CO1, Makerere University, Department of Biological Sciences, Kampala,
Uganda2, CDC Division of High Consequence Pathogens and Pathology, Viral
Special Pathogens Branch, Atlanta, GA, USA3, Uganda Virus Research Institute,
Department of Arbovirology, Entebbe, Uganda4, CDC Division of High
Consequence Pathogens and Pathology, Rabies Program, Atlanta, GA5, National
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Museum of Kenya, Nairobi, Kenya6, The Field Museum, Department of Zoology,
Chicago, IL7, Centers for Disease Control and Prevention, Global Disease
Detection Program, Nairobi, Kenya8
4:15 p.m. M. Juozapaitis1, É. Aguiar Moreira1, I. Mena2, S. Giese1, D.Riegger1, A. Pohlmann3, D.
Höper3, G. Zimmer4, M. Beer3, A. García-Sastre2, Martin Schwemmle1. An infectious
bat chimeric influenza virus harboring the entry machinery of a conventional
influenza A virus. Institute for Virology, University Clinic Freiburg, Freiburg,
Germany1, Department of Microbiology, Icahn School of Medicine at Mount Sinai,
New York2, Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald3,
Institute of Virology and Immunology, Mittelhäusern, Switzerland4
4:30 p.m. N. Courtejoie, B. de Thoisy, M-C. Lise, E. Darcissac, V. Lacoste, Anne Lavergne. Virus
and bats. A permanent conflict during evolution? Institut Pasteur de la Guyane,
Cayenne, French Guiana
4:45 p.m. Open Discussion
6:00 p.m. Adjourn
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Posters
1. Kendra Alfson, A. Griffiths. Investigating the dynamics of filovirus evolution in cell culture.
Texas Biomedical Institute, San Antonio, TX
2. Ian H Mendenhall1, Maggie Skiles2, Linfa Wang1, Gavin Smith1. Virus surveillance in the bats of
Singapore: Host specificity and diversity of astroviruses. Duke University-Singapore National
University1, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD2
3. Ben Stading1, J. Osorio1, T. Rocke2. Assessment of recombinant poxviruses as vaccine
vectors for bats through in vivo imaging. University of Wisconsin1, National Wildlife Health
Center, USGS, Madison, WI2
4. Michael Weis1, L. Behner1, C. Drosten2, J. F. Drexler2, A. Maisner1. African bat henipavirus GHM74a glycoproteins have impaired fusogenic properties. Philipps-Universität Marburg1,
University of Bonn Medical Centre2
5. Olivier Pernet1, B. S. Schneider2, S. M Beaty1, M. LeBreton2, T. E. Yun3, A. Park1, T. T. Zachariah4,
T. A. Bowden5, P. Hitchens6, C. M. R. Kitchen1, P. Daszak7, J. Mazet6, A. N. Freiberg3, N. D. Wolfe2,
B. Lee1,8. Evidence for henipavirus spillover into human populations in Africa. University of
California Los Angeles1, Global Viral/Metabiota2, University of Texas Medical Branch, Galveston3,
Brevard Zoo, FL4, University of Oxford5, University of California Davis6, EcoHealth Alliance, New
York7, Icahn School of Medicine at Mount Sinai, New York, NY8
6. A. Lavergne1, B. de Thoisy2, H. Bourhy2, Vincent Lacoste3. Serological and molecular
investigation of rabies virus circulation in Amazonian bats from French Guiana. Laboratoire
des Interactions Virus-Hôtes, Institut Pasteur de la Guyane1, Centre National de Référence de la
Rage, Institut Pasteur de Paris2
7. Andres Moreira-Soto1, N. Vargas-Vargas1, B. Rodriguez-Herrera2, E. Corrales-Aguilar1. Detection
of new coronaviruses in neotropical bats suggesting wide diversity in Costa Rica. CIET,
University of Costa Rica, San Pedro1, Biology Dept., University of Costa Rica, San Pedro2
8. Terry Fei Fan Ng1, I. Steffen1, C. Driscoll2, M. P. Carlos3, A. Prioleau3, R. Schmieder4, B. Dwivedi6,
J. Wong1, Y. Cha1, S. Head5, M. Breitbart6, E. Delwart1. A New Vesiculovirus in Bats with a
History of Human Contact. Blood Systems Research Institute, San Francisco1, Maryland
Department of Natural Resources2, , Maryland Department of Health and Mental Hygiene3, San
Diego State University4, The Scripps Research Institute5, University of South Florida, FL6
9. Nidia Arechiga-Ceballos, C. Obregón-Morales, L. Perea-Martínez, A. Aguilar-Setién. Experimental
infection of Artibeus spp. bats with rabies virus. Unidad de Investigación Médica en
Inmunología, IMSS, Mexico City
10. Eric Mossel, R. C. K., M. B. Crabtree, and B. R. Miller. A newly recognized clade of nairoviruses
isolated from bats and soft ticks. Centers for Disease Control and Infection, Fort Collins, CO
11. Kerri Miazgowicz, N. van Doremalen, V. J. Munster. Determining host potential of MERS-CoV
among bat species. Rocky Mountain Laboratories, NIAID, MT
12. Blair DeBuysscher1,2, D. Scott1, H. Feldmann1 and J. Prescott1. Characterization of cell-type
specific infection of Nipah virus in vitro and in vivo. Rocky Mountain Laboratory, NIAID1,
Hamilton, MT, University of Montana, Missoula, MT2
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13. Jeff Doty, G. L. Emerson, N. F. Gallardo-Romero, R. Nordhausen, M. M. Garner, J. R. Huckabee, S.
Johnson, R. D. Wohrle, W. B. Davidson, K., Y. Li, M. Metcalfe, K. L. Karem, I. K. Damon, and D. S.
Carroll. Chiropoxvirus: Updates on a novel bat pathogen. Centers for Disease Control and
Infection, Atlanta, GA
14. Ashley Malmlov1, J. Seetahal2, C. Carrington2, V. Ramkisson2, J. Foster2, V. Munster3, S.
Quackenbush1 and T. Schountz1. Serological evidence that Tacaribe virus is circulating among
bats in Trinidad. Colorado State University1, The University of the West Indies, St. Augustine,
Republic of Trinidad and Tobago2, Rocky Mountain Laboratories, NIAID, Hamilton, MT3
15. Alvaro Aguilar Setién1, C. Obregón Morales1, L. Perea Martínez1, N. Aréchiga Ceballos1, S. Aguilar
Pierlé2. Isolation of Waddlia cocoyoc, a novel intracellular bacterium, from frugivorous bats
(Artibeus intermedius). Unidad de Investigación médica en Inmunología, IMSS, Mexico City1,
Washington State University, Pullman USA2
16. M. M. B. Moreno-Altamirano2, Edith Zenteno-López2, M. del Rosario Salinas-Tobón2, C. ObregónMorales1, Alvaro Aguilar Setién1. Comparison between human and bat (Artibeus intermedius)
monocytes and lymphocytes. Unidad de Investigación médica en Inmunología, IMSS, Mexico
City1, Escuela Nacional de Ciencias Biológicas, IPN, Mexico City2
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Presentation Abstracts
Charles H. Calisher. Are bats the source of (almost) all vertebrate viruses (and God knows what
else)? Colorado State University
Beginning in 1931 and continuing to date, well more than 140 viruses of vertebrates in 21
virus families either have been isolated from or otherwise detected in bats. These include viruses
of families Adenoviridae, Arenaviridae, Astroviridae, Bunyaviridae, Circoviridae, Coronaviridae,
Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae,
Paramyxoviridae, Parvoviridae, Picornaviridae, Polyomaviridae, Poxviridae, Reoviridae,
Retroviridae, Rhabdoviridae, and Togaviridae, as well as viruses that have not been placed in a
taxon. Many of these viruses were first recognized after they were associated with human or
livestock illnesses and deaths but most were isolated or detected coincidental to general virus
surveys or during surveillance for specific viral pathogens. Genomic sequences of some of the
viruses that have been detected in bats can be linked to the diets of bats (e.g., insects, plants,
frogs) or with the normal flora of bats (e.g., bacteria and other microorganisms). As efforts to
analyze the viromes of bats increase, it is likely that genomes of hundreds more viruses will be
detected in bats and that bats will be shown to be reservoirs of many of them. This presentation
presents a complete list of these viruses, the chronology of their discoveries, and the names of
the bats from which these viruses were first obtained. The question of significance of these
findings will also be discussed.
Susanna Lau. Bat coronaviruses: diversity, evolution and interspecies transmission. University
of Hong Kong, Hong Kong, China
Coronaviruses (CoVs) are traditionally classified into groups 1, 2 and 3, which are now
replaced by the three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with
the addition of a new genus, Deltacoronavirus. The existence of CoVs in bats was unknown until
the SARS epidemic which has boosted the search for the animal origin of SARS-CoV
(Betacoronavirus lineage B). While civet was found to be an immediate ancestor of SARS-CoV
soon after the 2003 epidemic, horseshoe bats were only identified as the natural reservoir of
SARS-related batCoVs in 2005. Such findings have awakened the world on the role of bats as an
important reservoir for CoVs. Since then, a large diversity of novel bat CoVs have been identified,
including viruses belonging to two novel subgroups, Betacoronavirus lineages C and D.
Phylogenetic analysis suggested that bats and birds are the two major hosts for the gene source
for Alphacoronavirus/Betacoronavirus and Gammacoronvirus/Deltacoronavirus respectively,
which can fuel CoV evolution and dissemination. While our understanding on bat CoVs was only
beginning to unwind, another novel CoV, MERS-CoV (Betacoronavirus lineage C), has emerged
in the Middle East since 2012, having affected >600 people with >200 deaths so far. Although
camels have recently been identified as the direct animal origin of MERS-CoV, the virus is also
closely related to lineage C betaCoVs including batCoV HKU4 and HKU5 previously discovered in
lesser bamboo bat and Japanese pipistrelle, respectively. Despite our expanded knowledge on
bat CoVs, their evolution and interspecies transmission are still poorly understood. For SARSrelated batCoVs, horseshoe bats, as the primary reservoir, can fuel recombination, and civet
SARS-CoV is likely a recombinant virus arisen from horseshoe bats of different geographical
locations. For MERS-CoV, it remains unknown if Pipistrellus bats are the natural reservoir of its
ancestor viruses, similar to the situation in SARS-CoV. For Betacoronavirus lineage D, different
genotypes of Rousettus batCoV HKU9 have been identified in the same bat individual. Moreover,
recombination events were detected between strains from different bat individuals, which may
facilitate viral evolution to generate different genotypes. As for Alphacoronavirus, evidence for
recent interspecies transmission of batCoV HKU10 has been identified between bats of different
suborders, from Leschenault's rousettes to pomona leaf-nosed bats. More studies are eagerly
awaited to unravel the evolutionary mechanisms governing the interspecies transmission of
batCoVs, and hence their potential for emergence.
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Christian Drosten. Origins, hosts and sources of MERS coronavirus. University of Bonn Medical
Centre, Bonn, Germany
Zoonotic and emerging viruses have become a growing field of research. Some remarkable
novel virus descriptions in animals have demonstrated how ignorant we are about the diversity of
viruses around us. In our efforts to delineate viral origins we may have to reassess our concept of
reservoir. In many instances, we are mixing up ecological and epidemiological implications of viral
evolution. Among the biggest challenges in this field is the integration of the concepts of virus-host
codivergence, and viral host switching. In addition, assessments of viral reservoirs with the
intention to predict future pandemic threats would have to take into account important host and
virus traits which cannot be predicted merely from virus genes. For example, we need to know
whether there are hosts which have a higher propensity to carry broader spectra or higher
concentrations of viruses, potentially without being affected. Among the viruses borne in such
reservoirs, there may be some that are more promiscuous in their choice of hosts than others,
potentially due to the conservedness of their receptor structures or the way they interfere with
conserved- or not-so-conserved immune properties. A synopsis of available approaches
demonstrates how much work needs to be done before we will be able to assess functional,
rather than genetic diversity of reservoir-borne viruses.
Vincent Munster. The ecology of Middle East respiratory syndrome coronavirus (MERS-CoV) in
a reservoir host. Rocky Mountain Laboratories, NIAID, Hamilton, MT
Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 and has
currently caused over 800 human cases with an estimated 40% case fatality rate. MERS-CoV is
phylogenetically closely related to coronaviruses detected in bats. The detection of MERS-CoV
neutralizing antibodies and MERS-CoV virus in dromedary camels points toward the involvement
of dromedary camels as intermediate reservoir in the emergence of MERS-CoV in humans.
Recently, the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific
interaction of the receptor-binding domain (RBD) of MERS-CoV spike protein and DPP4 was
determined by crystallography. Here, we link in vitro and in vivo characteristics of MERS-CoV in
primary, intermediate and end hosts.
Joseph Prescott, E. de Wit, D. Falzarano, D. Scott, H. Feldmann, V. Munster. Immunosuppression
in the rhesus macaque model of Middle East respiratory syndrome. Rocky Mountain
Laboratories, NIAID, Hamilton, MT
Middle East respiratory syndrome (MERS) is caused by a recently emerging coronavirus
(CoV). Thus far, there have been over 400 confirmed human cases, mostly in Saudi Arabia, with a
high case-fatality rate. The natural reservoir has not been identified, but the virus is suspected to
be of bat origin. Many of the reported cases have involved patients with one or more comorbidities, suggesting that perturbations in the immune response might influence the outcome of
infection. We recently developed a disease model for MERS using rhesus macaques. These
animals develop a mild-to-moderate respiratory disease, with virus replication detectible in several
tissues of the respiratory tract, as well as shedding. To determine how the immune status
influences virus replication and disease, we immunosuppressed rhesus macaques using a drug
regimen of cyclophosphamide and dexamethasone prior to inoculation with MERS-CoV.
Immunosuppression resulted in a decrease in lymphocyte counts in the blood and depletion T and
B cells and disruption of the normal tissue architecture of the spleens and mesenteric lymph
nodes. MERS-CoV replicated more efficiently and had greater dissemination throughout the
tissues in the immunosuppressed animals compared to mock immunosuppression. Additionally,
immunosuppressed animals shed higher levels of virus from oral and nasal secretions.
Histologically, the mock immunosuppressed animals showed lesions that were characterized as
multifocal, mild to marked, interstitial pneumonia frequently centered on terminal bronchioles,
abundant alveolar edema and fibrin with formation of hyaline membranes, and multifocal type II
pneumocyte hyperplasia. Despite higher virus replication in the lungs, the immunosuppressed
animals displayed fewer histologic changes associated with infection. These data suggest that
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disease caused by MERS-CoV has an immunopathogenic component and that alterations in the
immune response might play a role in disease outcome.
Supaporn Wacharapluesadee1, P. Duengkae2, A. Rodparn1, T. Kaewpom1, P. Maneeorn3, S.
Yinsakmongkon1, N. Sittidetboripat1, C. Chareesaen3, N. Khlangsap3, A. Pidthong3, K. J. Olival4,
J. H. Epstein4, P. Daszak4, P. Blair5, T. Hemachudha1. Surveillance for and diversity of
coronaviruses in bats from eastern Thailand. World Health Organization Collaborating Centre
for Research and Training on Viral Zoonoses, Faculty of Medicine, Chulalongkorn University,
Bangkok, Thailand1, Faculty of Forestry, Kasetsart University, Bangkok, Thailand2, Department of
National Parks, Wildlife and Plant Conservation, Bangkok, Thailand3, EcoHealth Alliance, New
York, USA4, Naval Medical Research Center-Asia, US Embassy Singapore, PSA Sembawang,
Singapore5
Bats are reservoirs for broad diversity of coronaviruses (CoVs), including those closely related
to human pathogens such as Severe Acute Respiratory Syndrome CoV and Middle East
Respiratory Syndrome CoV. Estimates suggest there are approximately 138 bat species in
Thailand of which 116 are insectivorous and 21 are fruit bats. Given that CoVs have potential to
spread from bats to humans and other mammal hosts, obtaining baseline surveillance data in
order to plan for public health measures is essential. In this study, we sampled bat populations
from 5 provinces in Eastern Thailand. A total of 626 bat individuals were sampled (84 faecal and
542 rectal swab) from 19 different bat species. CoV RNA was detected in 47 specimens (7.6%)
from 13 different bat species, using degenerate PCR primers designed to detect all CoVs, with a
detection rate of 1.4% to 100% per species. We identified 37 alpha (α)-CoVs; nine β-CoV group D
and one β-CoV group B (SARS-CoV related). Five of β-CoV group D belonged to a new
independent cluster closely related to HKU9 bat CoV. For the first time, we have found CoV in six
bat species that have previously not been reported to harbor CoV, namely Cynopterus sphinx,
Taphozous melanopogon, Hipposideros lekaguli, Rhinolophus shameli, Scotophilus heathii, and
Megaderma lyra. We found specimens from different bat species but from the same colony or
location were found to harbor CoVs of the same genetic lineage. Contrary to some previous
findings, our data do not support species-specific or host restriction of CoVs in bats. Knowledge of
CoV diversity and presence of cross species dissemination in bats in Thailand will help provide
more accurate data on the assessment of zoonotic potential of bat-borne CoV.
Luis Góes1, A. Cristine Campos1, G. Ambar2, A. Neto2, E. Luiz Durigon1. Comparison of 3 nested
PCR pancoronavirus detection assays in bats from Brazil. Universidade de São Paulo - USP,
São Paulo, SP, Brazil1, Universidade Estadual Paulista Júlio de Mesquita Filho - UNESP, Rio
Claro, SP, Brazil2
Coronaviruses (CoV) are enveloped positive-sense single strand RNA viruses capable to
infect birds and mammals including humans and associated with respiratory, enteric, neurological
or hepatic disease. Species of the Chiroptera order harbor the biggest number and genetic
diversity of CoV including virus phylogenetically related to emergent coronavirus that afflict
humans (CoV-SARS and CoV MERS). Despite the large number of bat species in Brazil,
approximately 15% of bats species of the world, and the great diversity of CoV in bats, studies
about the occurrence and diversity of CoV in Brazilian bats are scarce. Coronavirus detection
methodologies based on Nested PCR Pancoronavirus assays targeting ORF1ab genome
sequence, more specifically the RNA-dependent RNA polymerase (RdRp) gene has been used to
detection and diversity analysis of CoV in various species of bats around the world. Although the
methods have been successfully used in their respective studies the sensibility and scope of each
approach can be differentiated. In this study we analyzed the presence of CoV RNA in 119
intestines from 4 different Brazilian Bat species including Sturnira lilium (14), Carollia perspicillata
(32), Artibeus planirostris (2) and Artibeus lituratus (71) with 1 conventional PCR assay (Poon et
al., 2005) and 3 Nested PCR pancoronavirus assays (Chu et al., 2011; Quan et al., 2010;
Watanabe et al., 2010). Total RNA was obtained from 30mg of intestine tissue extracted in
NucliSENS® easyMAG® automatic extractor (Biomérieux). cDNA was obtained by RT-PCR using
random primers followed by PCR and Nested PCR assays. The most sensitive method was the
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Nested PCR described by Chu et al. (2011) capable to detect CoV RNA in 4 samples (3.37%),
two Alphacoronavirus (α-CoV) in Carollia perspicillata, and one Betacoronavirus (β-CoV)
(possible group C) and α-CoV in specimens of Artibeus lituratus. The 400 nucleotide sequence
obtained for the β-CoV was most closely related to CoV detected in C. perspicillata from Costa
Rica. The two α-CoV detected in C. perspicillata was most related to the BatCoV from Trinidad
Bats and from C. perspicillata from Brazil. The α-CoV detected in A. lituratus was genetic related
with BatCoV from another Bat from Costa Rica. The others methods detected just 2 of 4 samples
positive for CoV. Chu et al (2011) was the elected assay for further surveillance of novel CoVs in
Brazilian bats.
Paul Cryan. Ecology of white-nose syndrome. USGS Fort Collins Science Center, Fort Collins, CO
White-nose syndrome (WNS) is the first sustained epizootic disease to cause widespread and
high mortality in multiple species and genera of bats. First recognized 7 years ago at an index
site in New York, WNS and the causative fungus have now spread through all assemblages of
hibernating bats and their underground habitats in eastern North America. The unprecedented
impacts that WNS has exerted on populations of North American bats has likely stemmed from
the ability of the causative pathogen to exploit a vulnerability in the evolved survival strategy of
most North American hibernating bats. Proportionally few researchers with expertise in bat
ecology studied infectious diseases prior to WNS, but times are quickly changing. This talk will
focus on the importance of understanding the ecology of bats (and people who study them) when
investigating pathogens that bats host. Using WNS as an example, I will discuss several
ecological characteristics of temperate-zone bats that alone or in combination potentially make
them unique environments for pathogens. These traits include huge metabolic range, gregarious
dormancy, limited flight capabilities, consumption of vast quantities of insects, long reproductive
delays, extreme seasonal differences in behavior between the sexes, and the ability to hide so
well in dark and inaccessible places that the detailed habits and whereabouts of many species
remain unknown. Understanding the ecology of WNS and how bats evolve in its wake may help
us learn why bats might be less susceptible to microorganisms they host and to high-mortality
epizootic diseases.
David Blehert. White-nose syndrome: a deadly mycosis of hibernating bats. United States
Geological Survey, Madison, WI
White-nose syndrome (WNS) is an emergent wildlife disease that has caused unprecedented
mortality among bats of the eastern United States and Canada. Since first detected in New York
during the winter of 2006-2007, bat population declines approaching 100% have been
documented at some affected hibernacula, total estimated losses have exceeded five million bats,
and the disease has spread to 23 US states and five Canadian provinces. Prior to emergence of
WNS, such massive population declines in mammalian species due to an infectious disease were
unprecedented. Affected hibernating bats often present with visually striking white fungal growth
on their muzzles, ears, and/or wing membranes. Ongoing investigations have demonstrated that
this cutaneous fungal infection is caused by Pseudogymnoascus (formerly Geomyces)
destructans. The infection has been shown to disrupt vital physiological processes of hibernating
bats and cause behavioral disturbances. Furthermore, the pathogen is psychrophilic. While it
grows well at temperatures consistent with bat hibernation (approximately 2 to 12°C), it cannot
grow on metabolically active bats. The fungus does, however, persist throughout the year in
environmental substrates of underground bat hibernation sites, including during warm summer
months, indicating that these sites serve as persistent reservoirs for the pathogen. A growing
body of evidence suggests that P. destructans was likely introduced to North America. Thus,
WNS provides a striking example of the impacts of an exotic pathogen on naïve populations of
hosts.
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DeeAnn Reeder. The immunological response to the fungal pathogen (Pseudogymnoascus
destructans) that causes white-nose syndrome in bats. Bucknell University, Lewisburg, PA
White nose-syndrome is caused by the emerging fungal pathogen Pseudogymnoascus
desctructans (Pd). Interestingly it does not affect all species equally; for example, big brown bats
(Eptesicus fuscus) have significantly lower mortality than little brown myotis (Myotis lucifugus).
Surviving infection with Pd is presumably influenced by a number of factors, including within- and
between-species variation in thermoregulatory traits, life history and behavioral traits, and
immunological responsiveness. Although a number of traits likely favor big brown bat survival in
the face of Pd infection (including their larger body size and their preference for hibernating for
shorter periods, at colder temperatures, and solitarily or in smaller clusters), their ability to
withstand Pd infection is not truly known. We conducted captive experimental Pd infections of little
brown myotis and big brown bats, measuring multiple aspects of immune function at 3, 7, and 13
weeks post-infection. Housed in identical conditions, big brown bats developed much lower levels
of infection than little brown myotis and showed larger immune responses to infection by Pd.
Additionally, uninfected big brown bats exhibited greater immune responses in general and
appeared to maintain immune system capabilities throughout hibernation better than uninfected
little brown myotis. Most notably, big brown bats appear to be equipped to respond to infection by
Pd more quickly and effectively than little brown myotis, especially as hibernation progresses.
One interesting issue to be resolved is the degree to which immune responses to Pd are
protective or pathological during the period immediately after hibernation, when bats appear to
more actively respond to infection. To determine whether or not bats develop antibodies to the
fungus, we developed an indirect ELISA capable of detecting antibodies to Pd. We then tested
plasma from seven bat species, collected from seven eastern US states, for prevalence of Pd
antibodies. Samples were collected during spring, summer, and winter from bats in areas known
to be affected by WNS as well as unaffected areas. We found that several, but not all, species
generated antibodies to Pd, and that antibody titers were greatest several weeks after the end of
hibernation. While much remains to be discovered, our improved understanding of the
immunological response to Pd will allow us to better mitigate this disease and to make speciesspecific management decisions.
Kate Langwig1, W. Frick1, T. Kunz2, J. Foster3, A. Marm Kilpatrick1. Infection loads, not exposure,
drive impacts of white-nose syndrome among species. University of California Santa Cruz1,
Boston University2. Northern Arizona University, Flagstaff, AZ3
Disease plays an important role in structuring species communities, including driving some
species toward extinction, while others suffer relatively little impact. Disease mortality is
determined by exposure to a pathogen and the host’s susceptibility to mortality once exposed. For
microparasites, the factors determining exposure are generally thought to be most important in
determining disease impact. However, pathogen burden (loads) has recently been recognized as
being especially important. White-nose syndrome, an emerging fungal disease in bats, has
decimated populations of several species across Eastern North America, threatening several
species with extinction. In contrast, sympatric and taxonomically similar bat species experience
very little effect. We measured transmission of Pseudogymnoascus destructans, the causative
agent of WNS, and quantified fungal loads to determine why bat species suffer such large
differences in impacts from WNS. Intense transmission occurred during winter hibernation, and
by late winter almost all individuals of all species were infected, with prevalence exceeding 70% in
six species. In contrast, P. destructans loads were more variable among species, and explained
95% of the variation among species in disease impacts. Loads were, in turn, driven by the
roosting temperature of bats during hibernation, with bats roosting at warmer temperatures having
higher P. destructans loads and higher WNS impact. These results suggest that habitat selection
by bats during hibernation influences pathogen growth, which determines mortality. More broadly,
behavioral patterns that were beneficial in the absence of disease may lead to catastrophic
declines following the invasion of a novel pathogen.
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R. Sachidanandam1, O. Attie1, A. Jayaprakash1, R. S. Shabman2,3, A. Davis4 and Christopher F.
Basler2. Transcriptome profiling reveals robust interferon responses in the brain of rabies
infected Myotis lucifugus bats and identifies a novel gamma herpesvirus in a bat cell line..
Dept. Genetics, Icahn School of Medicine at Mount Sinai, New York1, Dept. Microbiology, Icahn
School of Medicine at Mount Sinai, New York2, Virology group, J. Craig Venter Institute3, Rabies
Laboratory, Wadsworth Center, New York State Department of Health, NY4
Bats have been implicated as reservoir hosts for a number of zoonotic viruses. It is therefore
critical to be able to characterize innate antiviral responses in bats and bat cells. To date,
however, there is very limited information regarding bat innate immune responses. We therefore
sought to define the innate immune transcriptome in a Myotis bat cell line, with and without
stimulation by RNA virus mimic poly(I:C) (pIC), and in the tissues of uninfected and rabies virus
(RV)-infected Myotis lucifugus animals. We identified several microRNAs and mRNAs that were
involved in the bat antiviral response. Strikingly, we identify a dramatic upregulation of interferonstimulated genes (ISGs) in the RV-infected bat brain. This robust IFN response may therefore
contribute to the disease course of RV in bats. We also identified numerous innate immune
genes in the cell line that were induced following pIC treatment. Strikingly, the cell line sequence
analysis also identified the presence of a novel gammaherpesvirus. Passage of supernatants
from the bat cell line on to Vero cells and several human cell lines demonstrated that the virus is
fully replication-competent. Vero cell grown virus allowed the sequencing of the complete
gammaherpevirus genome which encodes a variety of putative immune regulatory genes. The
database of cDNA sequences for the innate-immunity genes and bat microRNAs generated by
our study constitutes a valuable resource for research into bat immunity and can be used in
developing laboratory reagents such as PCR primers, miRNA sponges and antibodies for further
studies of the bat innate immune response to virus infection. The gammaherpesvirus may also
provide a toolkit of immune regulators that can also be used to probe the bat immune system.
Amy T. Gilbert. Lyssaviruses in bats; current knowledge and future directions. National Wildlife
Research Center, USDA, Fort Collins, CO
The number of recognized and proposed Lyssavirus species associated with bats has been
growing rapidly, with at least six viruses discovered in the last two decades. The recent
discoveries highlight bat lyssavirus diversity on nearly every continent, affecting insectivorous,
frugivorous and sanguivorous bats. All lyssaviruses demonstrate zoonotic potential and
pathogenicity in bat reservoir hosts. However, current rabies biologics do not confer crossreactivity against two of the three lyssavirus phylogroups, which are present in the Old World only
and are comprised of at least three bat-associated viruses and two viruses with an unknown
reservoir. Interestingly, Old World lyssaviruses do not appear to circulate in the diverse
assortment of reservoir hosts that is observed with rabies viruses (RABVs) among bats in the
Americas, although the diversity of Old World bat lyssavirus lineages is quite remarkable.
Furthermore, spillover infections into other mammalian hosts have been detected with many
lyssaviruses, but RABV is the only Lyssavirus that has demonstrated stable host shifts from bats
into wild carnivore populations. Despite a wealth of research on bat RABVs in the Americas, there
have been few epidemiological models that have been developed to understand natural
circulation in reservoir hosts, but existing models highlight different strategies of viral perpetuation
due to different ecologies of the focal reservoir hosts. Enhanced surveillance provides valuable
insight into the diversity and host range of lyssaviruses globally, and molecular epidemiological
studies help identify reservoir hosts and characterize complex spillover transmission among bat
communities, but the development of epidemiological models to understand perpetuation in bat
reservoir hosts would benefit from a greater investment in long-term ecological studies.
Monica Borucki1, H. Chen-Harris1, S. Messenger2, D. Wadford2, J. Allen1. Analysis of viral
populations for improved biosurveillance and prediction of viral emergence. Lawrence
Livermore National Laboratory1, California Department of Public Health, CA2
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Understanding the role of genetic variants within a viral population is a necessary step toward
predicting and treating emerging infectious diseases. The high mutation rate of RNA viruses
increases the ability of these viruses to adapt to diverse hosts and cause new human and
zoonotic diseases. The genetic diversity of a viral population within a host may allow the virus to
adapt to a diverse array of selective pressures and enable cross-species transmission events. In
2009 a large outbreak of rabies in Northern California involved a skunk rabies virus variant that
efficiently transmitted within a population of gray foxes, suggesting possible adaptation to a novel
host species. To better understand the evolution of rabies virus that enabled this host jump, we
applied deep-sequencing analysis to rabies virus samples from the outbreak. Deep-sequencing
data indicated that many of the genetic changes associated with host jump occurred prior to 2009,
and these mutations were present at very low frequencies in viral populations from samples
dating back to 1995. These results suggest deep sequencing is useful for characterization of viral
populations, and may provide insight to ancestral genomes and role of rare variants in viral
emergence.
Lisa Worledge, H. Miller. Bat disease surveillance in the UK: the role of conservation volunteers.
Bat Conservation Trust, London, UK.
Bat conservation is inherently linked to the public’s attitude towards bats. Researchers,
governmental bodies, conservation organisations, veterinary and health professionals, and
volunteers all have a role to play in making sure that there is a clear understanding of the actual
public health risks associated with diseases of bats. The UK’s press readily report on disease
stories relating to bats not just in the UK but also overseas. Often the way stories are presented
bears little resemblance to the actual research findings and the public health risks, highlighting the
importance of collaborative working and common messages. This paper discusses what can be
achieved when conservation, research and government work hand in hand and highlights the
central role that bat conservation volunteers in the UK play. As well as educating the public and
promoting best practice, volunteers play a crucial supporting role in disease surveillance. Using
the example of the long term passive surveillance programme for European Bat Lyssaviruses, we
will explain how bat conservation volunteers are engaged in disease work, their contribution, and
how their role has evolved over time, leading to new projects in other disease areas.
D. Mitzel, D. Merrill, K. Dearen, R. Russell, P. Kuehl, Robert Baker. Aerosol infection of mice with
rabies virus, and comparison to intramuscular and intranasal routes of infection. Lovelace
Respiratory Research Institute, Albuquerque, NM
Rabies virus infection occurs in humans and animals, causes significant mortality and
suffering in both, and is considered a potential bioweapon. In the Northern Hemisphere, Rabies is
endemic in various wild animals, most famously bats. While Rabies is generally considered
communicable from wild animals to humans via bites, there is some anecdotal evidence of
aerosol transmission, as well as a small number of studies in this area. Here we demonstrate that
Rabies virus can efficiently infect mice via the aerosol route, and compare this to the intranasal
(IN) and intramuscular (IM) routes. We find that a presented dose of Rabies virus of ≤2.7x104
PFU (approximate retained dose of 2.7x103) leads to uniform lethality in the aerosol infection, and
is as low or lower than that for IN or IM routes (≤3x104 and 3x103, respectively). The mean time
to death for IN or aerosol-infected animals was approximately 2 days greater than IM-infected
animals. Neural pathogenesis of Rabies virus by IN, aerosol or IM route appears similar, with
high concentrations of virus detected in neural tissues tested in moribund animals. Mean viral
titers as measured by qPCR in the spinal cord, hippocampus, cortex and cerebellum were greater
than 106 genomes/ng total RNA for IN, IM and aerosol infected mice. Virus was detected by
qPCR in multiple tissues from all groups of animals, as well as from oral swabs. Virus distribution
and quantity was similar for IN, IM and aerosol-infected mice with the exception of submandibular
lymph nodes, which had similar detection rates of Rabies for IN and IM infected mice but
decreased incidence in aerosol infected mice. Our data demonstrates that Rabies virus is highly
infectious via the aerosol route, and dramatically increases concern over its potential use as a
bioweapon.
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Lin-Fa Wang. From cell lines to bat genomes: are we ready to study virus-bat interactions in a
serious manner? Duke-National University of Singapore Graduate Medical School, Singapore
Are bats special in their ability to co-exist with viruses? This is a hot question attracting
significant scientific and media attention in the last few years. While the “yes” and “no” camps
continue to debate and search for supportive evidences, an equally important question needs to
be answered first, i.e., are we ready to scientifically address this question in a proper and serious
manner. In this presentation, I will summarise the research being conducted in our groups in
Australia and Singapore, and discuss some preliminary/promising results as well as major
stumbling blocks before we can truly address the question “are bats special?”
Peter Daszak. Linking environmental conservation with human and wildlife health. EcoHealth
Alliance, New York City, NY
While bats are known reservoir hosts for several significant zoonotic viruses (e.g. Ebola,
Nipah, SARS) and a rapidly growing number of novel viruses globally, in order to understand the
real risk that bats pose for viral spillover and human health, an ecological or One Health approach
must be taken. We argue that viral discovery in bats must be concomitant with research
investigations of environmental change (to understand the underlying drivers of spillover risk),
host ecology (e.g. roosting and foraging behaviors that create an epidemiological link with
people), and human behaviors (e.g. bushmeat hunting). Bats provide vital ecosystem services
and are increasingly threated by human activities, thus understanding these human-bat ecological
interactions is critical for conservation as well as public health. In this talk, we will highlight
EcoHealth Alliance’s ongoing research projects in Brazil and Malaysia (viral diversity and
landscape change), Bangladesh (Nipah virus ecology), China (SARS-related CoV ecology), and
other countries to describe ways to successfully link bat disease investigations and conservation.
Our approach to ecologically-focused bat disease investigations includes the development of nonlethal sampling techniques; integration of biodiversity surveys and viral surveys across an urban
to pristine forest environment; investigations of Nipah virus, foraging ecology, and cultural
practices in Bangladesh; the use of spatial ecological models to predict bat zoonoses risk; and the
application of ecological metrics developed for biodiversity science to maximize viral discovery
efforts.
Jonathan Towner. Replication of marburgviruses in their reservoir host, Egyptian fruit bats
(Rousettus aegyptiacus). U.S. Centers for Disease Control and Prevention, Atlanta, GA
Multiple ecological investigations of marburgvirus outbreaks have recently identified the
Egyptian fruit bat, Rousettus aegyptiacus as a major natural reservoir for the known
marburgviruses (MARV and RAVV) based on finding 2-5% of wild-caught bats to be actively
infected and the repeated isolation of genetically diverse marburgviruses directly from bat tissues.
Longitudinal investigations in Uganda found that infection levels in juvenile bat populations spike
on a predictable bi-annual basis, and these seasonal periods coincide with 54/65 (83%) of known
marburgvirus species-jump events, further linking R. aegyptiacus to previous MHF outbreaks. R.
aegyptiacus is a common cave-dwelling fruit bat that, in equatorial Africa, can live in massive
colonies containing over 100,000 animals. To further investigate marburgvirus replication in a
reservoir host and to establish a model system upon which studies of other virus/bat reservoir
relationships could be based, a captive breeding colony of R. aegyptiacus was established at
CDC-Atlanta that, to date, has produced over 150 bats. An initial study of juvenile bats
experimentally infected with a low passage MARV bat isolate has been completed. In that study,
three animals per day were euthanized at 3, 5-10, 12 and 28 days post-infection (dpi); controls
were euthanized at 28 dpi. Blood chemistry analyses showed a mild, statistically significant
elevation in alanine aminotransferase (ALT) at 3, 6 and 7 dpi. Liver histology revealed mild
inflammatory foci in some infected bats, similar to that in wild-caught bats. Frequency of liver foci
peaked at 7 dpi, and was significantly correlated with both ALT and hepatic viral RNA levels.
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These results contribute to the understanding of the pathogenesis of marburgvirus infection in R.
aegyptiacus, and provide support for our experimental model of this filovirus-reservoir host
system.
J. Paweska1, Petrus Jansen van Vuren1, S. McCulloch2, A. Kemp1, N. Storm2, A. Grobbelaar1, T.
Scott2, M. Geldenhuys2, M. Mortlock2, N. Moolla1, A. Coetzer2, L. Nel2, W. Markotter2. Marburg
virus infection in Egyptian fruit bats (Rousettus aegyptiacus), South Africa. Center for
Emerging and Zoonotic Diseases, National Institute for Communicable Diseases of the National
Health Laboratory Service, Sandringham, Gauteng, South Africa1, Viral Zoonosis Group,
Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences,
University of Pretoria, Pretoria, Gauteng, South Africa2
There are at least nine known colonies of Egyptian fruit bats (Rousettus aegyptiacus)
inhabiting caves throughout South Africa (SA), but their potential role in harboring Marburg virus
(MARV) has not been studied. R. aegyptiacus bats were regularly captured from June 2013 to
January 2014 at Mahune Cave located in Matlapitsi Valley, Limpopo Province, SA, and tested for
exposure to MARV. This cave contains one of the largest R. aegyptiacus colonies in the country
(up to 20, 000). Some of the bats captured were colonized and monitored for the duration of
natural active and passive immunity to MARV. There was a high overall seroprevalence of IgG
(mean 53.4%), as measured by recombinant MARV GP ELISA, with no significant difference
among adult females and males (p=0.5442), but significant difference between juveniles and
adults (p<0.001). MARV IgG antibody in adult bats was detectable in most seropositive animals
for at least one year after initial testing. Passive maternal immunity in first generation offspring
from seropositive mothers lasted 4-5 months after birth. Of 29 re-captured juveniles, 10 (34.48%)
seroconverted during the period of this study. First seroconversions were detected in JuneAugust, coinciding with the waning of maternal immunity. Of 323 pooled serum samples and 115
pooled ectoparasite samples, all were negative by Q-RT-PCR. Of 159 liver/spleen tissues, four
(2.5%) tested positive by Q-RT-PCR targeting L-polymerase gene. Partial sequencing of MARV
RNA was only possible for one of four positive bats. Phylogenetic comparison using L-polymerase
and NP sequences demonstrated that the virus circulating in Mahune cave is most closely related
to the Ozolin MARV isolate imported to SA in 1975 from Zimbabwe via an infected patient. This
study provides the first evidence of endemic circulation of MARV in Egyptian fruit bats in SA and
data on natural adaptive immunity which aid the interpretation of seroepidemiological results.
Amy Schuh, B. Amman, M. Jones, T. Sealy, L. Uebelhoer, B. Martin, S. Nichol, J. Towner.
Investigation of bat-to-bat transmission of Marburg virus in the Egyptian fruit bat,
Rousettus aegyptiacus. U.S. Centers for Disease Control and Prevention, Atlanta, GA
The Egyptian fruit bat, Rousettus aegyptiacus, was recently identified as a major natural
reservoir host for Marburg virus (MARV). Experimental inoculation of R. aegyptiacus from the
laboratory-breeding colony with MARV confirmed that this bat species is indeed the natural
reservoir host of the virus. However, the mechanisms of bat-to-bat transmission of MARV among
R. aegyptiacus remain unknown. Therefore, the objective of this study was to determine whether
bat-to-bat transmission of MARV could occur in a controlled laboratory environment through the
direct, indirect and/or airborne routes. This study used a total of 38 juvenile bats: 12 inoculated
donor (ID) bats, 24 naïve contact (NC) bats and 2 negative control (NEG CO) bats. The ID bats
were inoculated with MARV, the NC bats received no inoculation and the NEG CO bats were
inoculated with media. The ID and NC bats were housed in partitioned cages, separated by wire
mesh or solid metal partitions. Blood, urine, and oral and rectal swabs were collected from bats
daily from 0 through 25 dpi and then weekly through 56 dpi. These specimens were used to
monitor viremia and virus shedding by Q-RT-PCR and virus isolation, and to monitor the MARV
IgG antibody response. This study identified multiple routes of MARV shedding in the ID bats and
evidence of oral exposure in NC bats. Further transmission studies will be necessary to better
characterize bat-to-bat transmission of MARV in a controlled laboratory environment.
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Melinda Ng1, E. Ndungo1, M. Kaczmarek2, A. Herbert3, R. Biswas1, A. Demogines2, M. Muller5, J. H.
Kuhn4, J. M. Dye3, S. Sawyer2, K. Chandran1. Molecular evolution of the filovirus receptor
NPC1 in bats. Albert Einstein College of Medicine, Bronx, NY1, University of Texas at Austin,
Austin2, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD3, Tunnell
Consulting and NIH/NIAID Integrated Research Facility, Ford Detrick, MD4, Universitats
Klinikumbonn, Bonn, Germany5
Ebola virus and Marburg virus cause outbreaks of disease in humans and non-human
primates with high case fatality rates, for which there are no approved therapeutics or anti-virals.
The high lethality of filovirus disease in humans and non-human primates strongly suggests that
they do not naturally harbor these viruses. Indeed, infectious Marburg virus was recently isolated
and found to circulate in the cave-dwelling Egyptian rousette bat (Rousettus aegyptiacus),
providing evidence that this bat is a reservoir for Marburg virus. The evidence for fruit bats as
reservoirs for infectious EBOV is rather more circumstantial –all attempts to isolate infectious
EBOV from bats or other potential enzootic hosts have been unsuccessful. Here, we began to
investigate the possibility that the long-term coevolution of filoviruses in their reservoir hosts has
left signatures in the genomes of these hosts. As a case in point, we examined the molecular
evolution of Niemann-Pick C1 (NPC1), which encodes the essential entry receptor for filoviruses.
We reasoned that filovirus infection may have driven selection of this gene for mutations that
reduce viral entry and therefore modulate infection in the host. Indeed, we found that NPC1 has
evolved under recurrent positive selection in bats, but not in primates. To correlate this signature
of positive selection in NPC1 to its encoded protein’s function as a filovirus receptor, we examined
the capacity of NPC1 proteins derived from different bat and primate species to bind to EBOV GP.
Consistent with the results from our evolutionary analysis, sequence-dependent differences in the
EBOV-receptor interaction were observed with NPC1 proteins from bats but not primates.
Importantly, these results could fully explain differences in the susceptibility of fibroblast cell lines
derived from four of the bat species to authentic EBOV infection. Our findings reinforce a model in
which fruit bats are reservoirs for EBOV, raise the possibility that NPC1 can act as a determinant
of species-specific infection in nature, and provide the first molecular evidence that a widely
distributed African bat may be resistant to infection by EBOV.
Jon Epstein. The ecology of Nipah virus in Bangladesh. EcoHealth Alliance, New York City, NY
Nipah virus (NiV) is an emerging zoonosis that has caused near-annual outbreaks of
encephalitis in Bangladesh since 2001, with >75% mortality. Human infections are seasonal
(Dec-Apr) and almost exclusively detected within a western region of the country termed the
“Nipah Belt.” Pteropus bat species are a reservoir for henipaviruses in other parts of their range,
and Pteropus giganteus is the presumptive reservoir for Nipah virus in Bangladesh. We are
studying NiV dynamics in bats to determine how they relate to human infection patterns,
examining bat demography, ecology, movement patterns, and Nipah virus epizootiology. Here we
provide an overview of our work and insights from preliminary epidemiological and ecological data
from spatial and longitudinal Nipah virus surveillance and telemetry studies in Pteropus giganteus
in Bangladesh.
Emmie de Wit, J. Prescott, D. Falzarano, T. Bushmaker, D. Scott, H. Feldmann, V. Munster.
Modeling the zoonotic transmission cycle of Nipah virus in Bangladesh. Rocky Mountain
Laboratories, NIAID, Hamilton, MT
Since 2001, outbreaks of Nipah virus encephalitis have occurred almost every year in
Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah
virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm
sap contaminated by bats, with subsequent human-to-human transmission. To experimentally
model this proposed zoonotic transmission cycle, we first determined the viability of Nipah virus
(strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at
least 7 days, thus potentially allowing foodborne transmission. Next, we modeled foodborne Nipah
virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus.
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Drinking of 5x108 TCID50 of Nipah virus resulted in neurological disease in 5 out of 8 hamsters,
indicating that foodborne transmission of Nipah virus can indeed occur. In comparison, intranasal
(i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all
animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues
rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that
during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication
of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect
hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus
through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these
findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the
origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this
hypothesis. Finally, we showed that heating artificial palm sap containing Nipah virus to 70°C or
higher results in a rapid decrease in virus titer, thereby providing experimental evidence that
heating palm sap before consumption may aid in the prevention of zoonotic transmission of Nipah
virus from bats to humans. Moreover, the data presented here stress the importance of efforts
currently underway in Bangladesh to prevent access of bats to date palm sap collection pots,
thereby preventing contamination of date palm sap with Nipah virus and subsequent zoonotic
transmission.
Brian Ammam1, C. Albariño1, T. Sealy1, B. Bird1, A. Schuh1, S. Campbell1, U. Stroeher1, M. Jones1,
M. Vodzak2, D. Reeder2, S. Nichol1, J. Towner1. Sosuga virus (Paramyxoviridae) detected in
Egyptian fruit bats (Rousettus aegyptiacus) from Uganda. Centers for Disease Control and
Prevention, Atlanta, GA, USA1, Bucknell University, Lewisburg, PA2
In August 2012, a wildlife biologist became ill after a six week field work trip to South Sudan
and Uganda, where she caught and processed several species of bats and rodents. After
returning to the United States, she became ill and was admitted to the hospital with multiple
symptoms including fever, malaise, headache, generalized myalgia and arthralgia, stiffness in the
neck, and sore throat. After admission, she developed a maculopapular rash and oropharynx
ulcerations. The patient was released from the hospital 14 days after admission when symptoms
abated and hepatic enzyme levels and blood counts returned g to reference levels. Several
known suspect pathogens, including viral hemorrhagic fever viruses such as ebolaviruses and
marburgviruses, were ruled out through standard diagnostic testing. However, deep sequencing
and metagenomic analyses identified a novel paramyxovirus related to fruit bat borne, rubula-like
viruses. Analysis by RT-PCR of bat tissues collected during the three week period just prior to the
onset of symptoms work revealed several Rousettus aegyptiacus bats to be positive for the new
paramyxovirus, now termed Sosuga virus (Albarino et al., 2014). Further analysis of archived R.
aegyptiacus tissues collected from multiple marburgvirus investigations in Uganda strongly
implicates R. aegyptiacus as a potential natural reservoir for this novel paramyxovirus virus.
Michelle Baker. Innate antiviral immunity in the Australian black flying fox, Pteropus alecto.
Australian Animal Health Laboratory, Geelong, Australia
Bats have been identified as the natural reservoir host to an increasing number of emerging
and re-emerging viruses including Rabies, Hendra, SARS-CoV and most recently, MERS.
Although many of the viruses carried by bats are highly pathogenic in other mammals, they rarely
result in clinical signs of disease in bats. The ability of bats to coexist with viruses may be due to
the rapid control of viral replication by the innate immune response. Interferons (IFNs) provide the
first line of defence against viral infection in vertebrates resulting in the induction of an “antiviral
state” in infected and neighbouring cells. We have identified differences in the IFN production and
signalling pathway using the Australian black flying fox as a model species for studying host-virus
interactions. In contrast to other species in which IFNs are present at very low levels in
unstimulated cells, IFNA and IFN signalling molecules are expressed at a high baseline level in
unstimulated bat cells. Furthermore, stimulation of bat cells with the viral mimic, polyIC results in
high induction of IFNB but little IFNA production. Genomic analysis has revealed that bats have
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fewer IFNA genes compared to other species (three in P. alecto compared to 13 in humans) and a
number of genes within the IFN production and signalling pathway have undergone selection,
possibly due to the prolonged presence of viruses. Our studies of IFN production and signalling
molecules have provided evidence that the innate immune system of bats may be at a heightened
state of awareness which in turn may allow bats to rapidly control viruses to a very low level thus
avoiding the pathological consequences of infection.
Aaron Irving1, M. Ahn1, L. Wang1,2. Interrogating inflammasome signaling pathways in the black
flying fox, Pteropus alecto. Duke-NUS Graduate Medical School, Singapore, Singapore1,
Australian Animal Health Laboratory, CSIRO Livestock Industries, East Geelong, Victoria,
Australia2
The bat provides a unique environment for hosting virus infections. Genetic deletions of the
PYHIN gene cluster (AIM2 and IFI16), coupled with alterations in other signaling components,
lead us to believe the pathways triggered in response to infection may be different to most
mammals. To understand how regulation of viral infection is altered, and how we can control this
regulation, we are investigating the ability of viruses to activate the Inflammasome via Asc-speck
formation, oligomerization and caspase induction. In addition we will characterize other recruited
adapter molecules and compare these pathways between human/mouse and Pteropus alecto
using synthetic NLRP1/3 activators such as Nigericin, poly(dA:dT), poly(I:C), cGAMP, ATP etc.
This will allow us to compare their response to DNA from virus infection in the absence of known
Pattern Recognition Receptors (PRRs) or PRR detection of cellular/viral RNA. This will provide a
platform for understanding the lack of response bats show to most viruses and their ability to form
natural reservoirs of pathogens.
Nicole B. Glennon1, O. Jabado2, M. K. Lo3, M. L Shaw1. Characterizing the antiviral response of
the large flying fox (Pteropus vampyrus), an important ecological reservoir of
henipaviruses. Icahn School of Medicine at Mount Sinai Department of Microbiology, New York,
NY1, Icahn School of Medicine at Mount Sinai Department of Genetics and Genomic Sciences,
New York, NY2, Center for Disease Control & Prevention Viral Special Pathogens Branch, Atlanta,
GA3
Bats are important reservoirs for many viruses including, henipaviruses, SARS coronavirus,
ebola virus, and lyssaviruses. Despite being infected with a wide variety of viruses, most
infections are reported to be asymptomatic in bats. Since little is known about the bat antiviral
response we set out to profile the innate immune response of the P. vampyrus bat using next
generation sequencing. P. vampyrus is one of the reservoirs of Nipah virus (NiV) and Hendra virus
(HeV), two paramyxoviruses that can infect humans and are associated with a high mortality rate.
We obtained kidney tissue from a P. vampyrus bat and prepared primary and immortalized cells
(PVK). We characterized the activation of the innate immune response in these cells upon
infection with Newcastle disease virus (NDV), an avian paramyxovirus known to elicit a strong
innate immune response in mammalian cells. We used next generation mRNAseq technology to
determine the transcriptome of mock and NDV infected PVK cells. Bioinformatic analysis shows
that interferon and antiviral pathways are highly upregulated in NDV infected PVK cells. Genes
upregulated include well characterized members of the antiviral response, including interferon β,
RIGI, MDA5, ISG15, and IRF1 and others that are not so well characterized, such as RND1,
SERTAD1, CHAC1, and MORC3. We show that these cells induce cytokines in response to virus,
but that this cytokine response has species-specific antiviral activity. We show that PVK cells
support growth of HeV and NiV, however we fail to detect an interferon response following
infection with HeV and NiV which indicates that viral interferon antagonist mechanisms are most
likely active in bat cells.
Neeltje van Doremalen1, K. Cameron2, S. Milne-Price1, S. Olson2, A. Ondzie2, T. Bushmaker1, J-V.
Mombouli2, P. Reed2, V. Munster1. Development of a serological assay for the investigation of
emerging viruses in central African fruit bats. Laboratory of Virology, Virus Ecology Unit,
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Division of Intramural Research, NIAID, Rocky Mountain Laboratories, Hamilton, MT1, Wildlife
Conservation Society, Bronx NY2, Laboratoire National de Santé Publique, Brazzaville, Congo3
Bats have been implicated as the natural reservoir for a number of highly pathogenic
emerging viruses, including Nipah virus, Hendra virus, SARS coronavirus, MERS coronavirus,
and the Filoviruses. While these viruses are sometimes notoriously difficult to isolate from bats,
serology can be used to identify potential reservoirs and better our understanding of the
ecological dynamics within these reservoir communities. Ebola-specific antibodies have been
reported in the Central African fruit bats H. monstrosus, E. helvum, and E. franqueti, and
henipavirus-specific antibodies have been detected in E. helvum samples from Africa. We
developed assays for the serological investigation of filoviruses and henipaviruses in Central
African bat samples. His-tagged nucleoprotein from Ebola virus Zaire and Nipah virus Bangladesh
was produced via the baculovirus expression system and purified using immobilized metal chelate
affinity techniques. Purified viral nucleoprotein was then optimized to an ELISA format for the
high-throughput screening of bat serum samples. Positive control serum specific to each
recombinant protein was produced in rabbits through inoculations with adjuvanted purified protein.
In the absence of known negative and positive Central African bat serum controls, A. jamaicensis
serum samples from an experimental bat colony were used as negative controls and serum
samples from experimentally infected laboratory animals were used as positive controls. Central
African serum samples that showed high reactivity to recombinant protein in an ELISA format
were further tested with western blotting against purified recombinant viral proteins and, if enough
serum was available, by virus neutralization assay.
Elizabeth Cook1, G. Michuki2, C. Rumberia2, A. Kiyong'a2, J. Akoko2, A. Ogendo3, E. Dobson2, M.
Bronsvoort1, S. Kemp2, B. Agwanda4, E. Fevre5. A metagenomic approach identifies
plasmodium species in bats in Kenya. University of Edinburgh, Edinburgh, UK1, International
Livestock Research Institute, Nairobi, Kenya2, Kenya Field Epidemiology and Laboratory Training
Program, Nairobi, Kenya3, National Museums of Kenya, Nairobi, Kenya4, University of Liverpool,
Liverpool, UK5
Bats have been implicated in the emergence of diseases causing high human fatalities. This
project examined bats from around rural households in Kenya for zoonotic pathogens. A
concurrent cross sectional study provided information on human and domestic animal disease
within the same households. The study was conducted in the Lake Victoria Crescent region of
western Kenya between April and November 2012. A total of 89 bats were collected including
Pipistrellus sp (15), Chaerophon sp (54), Epomophorus sp (8), Scotoecus sp (10) and Taphozous
sp (2). Animals were captured from randomly selected households and anaesthetised to sample
heart blood. The animals were euthanized for collection of fresh and fixed tissues. Blood smears
were examined for haemoparasites. Total nucleic acids (DNA/RNA) were extracted from serum
and whole blood samples using the automated Roche MagnaPure LC instrument. Multiplexed
libraries were prepared for both DNA and RNA and sequenced on half a plate on the Roche 454
GS-FLX platform.Malaria-like parasites were identified in the blood smears of four bats
(Epomophorus sp). Individual libraries were prepared from 12 selected bat samples including all
Epomophorus bats and a random selection of other bat species. Among the 12 selected bat
samples 11 samples (92%) had Plasmodium species identified by sequencing. All 89 bat samples
were also sequenced in eight pools. Plasmodium species were identified in all the eight pools.
Other organisms comprising of bacteria, parasites and viruses were identified (results
unpublished). Other sequences were unclassified. Further work will be performed to confirm the
Plasmodium species. Histopathology is planned to document any pathological changes related to
disease in these animals. Ongoing work will identify pathogens in the human and domestic animal
inhabitants of these households. The identification of this Plasmodium sp and analysis of
its phylogeny may provide important information on the evolution of malaria parasites. This
organism might also give insight into the host parasite relationship that may contribute to
understanding malaria in people.
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David Morán1, D. Alvarez1, M. F. Rizzo3, Y. Bai3, L. F. Peruski2, M. Kosoy3. Geographic distribution
and diversity of bartonella in bats from Guatemala. Centro de Estudios en Salud, Universidad
del Valle de Guatemala, Guatemala, Guatemala1, Centers for Disease Control and Prevention,
Central American Region, Guatemala, Guatemala2, Centers for Disease Control and Prevention,
Division of Vector Borne Diseases, Fort Collins, CO3
Bartonella species have been found in high prevalence and high diversity in bat populations
from limited regions of Guatemala (Bai et al., 2011). We were interested in further investigating
the distribution of Bartonella spp. in bats from broader geographic areas of Guatemala. Between
August 2011 and December 2011, blood samples were obtained from 465 bats from 10
departments of Guatemala (Petén, Huehuetenango, Quiché, Alta Verapáz, Izabal,
Quetzaltenango, Retalhuleu, Suchitepéquez, Santa Rosa and Jutiapa). The bats represented 30
species from 18 genera, with the common vampire bat (Desmodus rotundus) the most prevalent
(34.2%; 159/465). Samples were cultured on blood supplemented agar and 89 isolates were
obtained from 80 bats, giving the overall prevalence of 17.2% (80/465). Bartonella prevalence
varied among bat species, (range 0% - 100%) and among study sites (range 0% - 25%).
Phylogenetic analysis of the isolates based on citrate synthase gene (gltA) revealed 43 genetic
variants, clustered into 17 phylogroups. Twelve of the phylogroups have been reported previously,
while the other five were newly identified in this study. Similarly to previously reported data, we
observed that different bat species can share the same Bartonella strains, and some individuals
may have co-infections with two Bartonella spp. Nevertheless, the four of the five new
phylogroups were associated specifically with one bat species each, which might suggest some
level of host specificity.
Ying Bai. Bartonella infections in bats. U.S. Centers for Disease Control and Prevention, Fort
Collins, CO
Previous studies of bartonella infections in bats from different regions of the world showed
these mammals are greatly susceptible to Bartonella spp.; the Bartonella spp. exhibited extremely
high heterogeneity with complex relationships to their bat hosts, which more likely relate to
multiple factors. The straw-colored fruit bat (Eidolon helvum) is widely distributed across Africa.
Early study from Kenya showed that the bats are prevalently infected with multiple but specific
Bartonella spp. We investigated Bartonella spp. in straw-colored fruit bats from Ghana, Nigeria,
and Tanzania, and compared our findings with those from Kenya. The results showed the
prevalence of bartonella infections in straw-colored fruit bats varied from 17.5% to 55.9% among
the study regions. The Bartonella detected in straw-colored fruit bats clustered into six
phylogroups (E1-E5, Ew) based on the gltA identity. To better characterize genetic diversity of
straw-colored fruit bat-associated Bartonella, we compared 79 strains isolated from the bats
collected across Africa including the above mentioned four regions and other two regions -Cameroon and island of Annobon (Equatorial Guinea) using multi-locus sequencing typing
(MLST) approach. We analyzed seven more targets (ftsZ, ribC, rpoB, nuoG, ssrA, ITS, and
16SrRNA) in addition to gltA. Resulted MLST scheme identified 46 sequence types (ST) among
the studied strains. The STs were clustered into six phylogroups that in general are in accordance
with the gltA classification. The phylogroups contained different number of STs from 2 to 14. The
group Ew, found in all regions and contained only two STs, is much more congruent compared to
others, suggesting Ew might be the most ancient Bartonella that has had long adaptation to the
straw-colored fruit bats.
Kevin Olival1, C. Weekley2. Viral discovery in bats: a quantitative review of ~100 studies from
the last seven years. EcoHealth Alliance, New York, NY1, UC Berkeley, Berkeley, CA2
Viral discovery in bats has increased dramatically in the past decade, yet a rigorous synthesis
of the published data is lacking. We extract, synthesize, and analyze data published from 94 viral
discovery studies over the last seven years since 2007, and identify specific variables of
importance to better target future discovery efforts in bats. A total of 60,018 samples from 44,322
bats comprising 17 families, 110 genera, and 340 species were taken across 94 studies. Overall,
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a total of 5,946 (9.91%) of all samples tested were found to be virus or antibody positive, with 24
viral families identified overall and >200 described as “novel” viruses. We report prevalence by
host family and sample type for each viral family. We found that 44% (19,484) of individual bats
were killed across studies, but that this factor was never significant in models that explain # of
novel viruses found or # of total viruses found. Our quantitative review has important implications
for how future viral discovery studies in bats are designed, how samples are collected, and
whether lethal sampling should be used.
Hideki Ebihara. Molecular identification of the relationship between arthropod-borne
bunyaviruses and bats. Rocky Mountain Laboratories, NIAID, Hamilton, MT
With few exceptions, biological relationships between arthropod-borne bunyaviruses and bats
have not been defined. On the basis of our bunyavirus genomics and molecular virological
studies, we have identified previously unrecognized phylogenetic relationships between
arthropod-borne viruses and viruses isolated from bats belonging to the genera Orthobunyavirus
and Phlebovirus. For instance, we have established a genetic relationship between the human
pathogenic orthobunyavirus, Nyando virus, which causes febrile illness and was isolated from
humans and mosquitoes in Africa, and uncharacterized bunyaviruses isolated from bats in Asia
and South America. In addition, we have also been focusing on the molecular characterization of
the novel bat-associated phlebovirus, Masloor virus, which was identified in India and is
genetically closely related to two novel human pathogenic tick-borne phleboviruses: Severe Fever
with Thrombocytopenia Syndrome virus (SFTSV) and Heartland virus (HRTV) that have emerged
in Eastern Asia and the United States, respectively. To evaluate the potential risk of Masloor virus
as a novel human pathogen, we characterized and compared the interferon antagonist function of
the nonstructural proteins (NSs) among tick- and bat-borne phleboviruses. Intriguingly, Masloor
virus NSs does not possess the ability to counteract interferon responses compared with those of
SFTSV and HRTV NSs, which may suggest a low potential for zoonotic transmission of this virus.
Our approach will provide new insight into the molecular evolution and ecology of arthropod-borne
bunyaviruses, and we hope that our findings will facilitate further investigation of the relationship
between bunyavirus and bats.
Rebekah Kading1, R. Kityo2, E. Mossel1, J. Towner3, B. Amman3, T. Sealy3, T. Nakayiki4, A. Gilbert5, I.
Kuzmin5, B. Agwanda6, J. Kerbis7, B. Nalikka2, E. Borland1, L. Nyakarahuka4, M. Crabtree1, J.
Montgomery8, C. Rupprecht5, S. Nichol3, J. Lutwama4, B. Miller1. Virus isolations from and
arbovirus surveillance of bats in Uganda and Kenya. CDC Division of Vector-borne Diseases,
Arbovirus Diseases Branch, Fort Collins, CO1, Makerere University, Department of Biological
Sciences, Kampala, Uganda2, CDC Division of High Consequence Pathogens and Pathology,
Viral Special Pathogens Branch, Atlanta, GA, USA3, Uganda Virus Research Institute, Department
of Arbovirology, Entebbe, Uganda4, CDC Division of High Consequence Pathogens and
Pathology, Rabies Program, Atlanta, GA5, National Museum of Kenya, Nairobi, Kenya6, The Field
Museum, Department of Zoology, Chicago, IL7, Centers for Disease Control and Prevention,
Global Disease Detection Program, Nairobi, Kenya8
Arboviruses including Rift Valley fever (RVFV), Yellow fever (YFV), West Nile (WNV),
Chikungunya (CHIKV) and Zika (ZIKV) viruses have been isolated or detected serologically from
various East African bats, however the role of bats in arbovirus transmission cycles is poorly
understood. The aim of this study was to investigate the exposure history of East African bats to
arboviruses as well as attempt virus isolation from bat tissues. Blood, tissues, or both were
obtained from 1067 bats from Uganda between 2009 and 2013, and from 449 bats from Kenya in
2011. Liver/spleen samples were mechanically homogenized in tissue culture media and virus
isolation was performed on Vero cells. Virus isolates were identified by either RT-PCR using virus
group-specific primers, or next generation sequencing. Serum samples were tested for specific
neutralizing antibodies against WNV, YFV, Dengue 2 (DENV2) virus, CHIKV, O’nyong-nyong virus
(ONNV), Babanki virus (BABV), ZIKV and RVFV by plaque reduction neutralization test. Virus
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isolates to date include Entebbe bat virus (Flaviviridae: Flavivirus) from Chaerephon pumila in
Entebbe, Fikirini rhabdovirus (novel) from Hipposideros vittatus in Kenya, and Dzifa herpesvirus
(novel) from Triaenops afer in Kenya. Rousettus aegyptiacus from Maramagambo forest in
western Uganda had specific neutralizing antibodies against CHIKV (2/303), ONNV (32/303), YFV
(3/303) and WNV (1/303). R. aegyptiacus from Mt. Elgon in eastern Uganda also contained
neutralizing antibodies against YFV (1/45). Epomophorus labiatus from the Entebbe/Kampala
area demonstrated specific neutralizing antibodies against BABV (3/52), DENV2 (1/52), and WNV
(2/52). DENV2 antibodies were also present in Chaerephon pumila (3/123) and Mops condylura
(1/36) captured around Entebbe, and Nycteris spp. (2/10) from Mt. Elgon. One C. pumila also
had a high neutralizing titer against ONNV. Testing is still in progress. Serological and virological
evidence suggest that multiple species of fruit and insectivorous bats from East Africa are
exposed to and are potential amplification hosts for arboviruses.
M. Juozapaitis1, É. Aguiar Moreira1, I. Mena2, S. Giese1, D.Riegger1, A. Pohlmann3, D. Höper3, G.
Zimmer4, M. Beer3, A. García-Sastre2, Martin Schwemmle1. An infectious bat chimeric
influenza virus harboring the entry machinery of a conventional influenza A virus. Institute
for Virology, University Clinic Freiburg, Freiburg, Germany1, Department of Microbiology, Icahn
School of Medicine at Mount Sinai, New York2, Institute of Diagnostic Virology, Friedrich-LoefflerInstitut, Greifswald3, Institute of Virology and Immunology, Mittelhäusern, Switzerland4
In 2012 the complete genomic sequence of a new and potentially harmful influenza A-like
viruses from bats (H17N10) was identified. Phylogenetic analysis revealed a close relationship of
H17N10 to influenza A viruses. Moreover, the H17N10 polymerase was shown to be fully
functional in human HEK293T cells, suggesting that these bat viruses can replicate in human
cells. However, infectious influenza virus could be neither isolated from infected bats nor
reconstituted by reverse genetic approaches, impeding further characterization of this virus. Here
we show that bat influenza virus harboring the entry machinery of a common influenza A virus
replicates well in mammalian but not in avian cells and fails to reassort with other influenza A
viruses. We found functional compatibility between bat and influenza A virus proteins that allowed
us to generate an infectious virus containing six out of the eight bat virus genes with the remaining
two genes encoding the HA and NA proteins of a prototypic influenza A virus. This engineered
virus replicated well in a broad range of mammalian cell cultures, human primary airway epithelial
cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Unlike
other influenza A viruses, the bat chimeric virus was unable to reassort with other influenza A
viruses. Although our data does not exclude the possibility of zoonotic transmission of bat
influenza viruses into the human population, they indicate that multiple barriers exist that make
this a very unlikely event.
N. Courtejoie, B. de Thoisy, M-C. Lise, E. Darcissac, V. Lacoste, Anne Lavergne. Virus and bats. A
permanent conflict during evolution? Institut Pasteur de la Guyane, Cayenne, French Guiana
Bats are described as a major reservoir of emerging viruses. Their ability to act as efficient
reservoirs and spreaders is directly linked to long-term co-evolution processes and short-term
interactions. Indeed, organisms and their pathogens are engaged in a permanent conflict that
affects the evolution of their respective genomes, more particularly the immune system genes.
More and more studies focus on bats' immunity as a key to better understand their potential as
privileged reservoir. The innate immune system, whose response is immediate and nonspecific, is
the first line of defense against pathogens. This evolutionary conserved system may play a major
role in bats, which seem to control viral replication very early. The adaptive immune response is
slower and more specific. Most immunogenetic studies have focused on genes of adaptive
immunity such as the MHC. Yet, these genes only account for a fraction of the observed
interindividual variability in susceptibility to pathogens. Thus, understanding the effect of selection
on bats’ immunity and fitness involves studying a wider range of genes, including those of innate
immunity. In this context, we characterized immunological markers involved in the innate immune
response to viral infection such as Toll-like receptors (TLR3, 7, 8, 9), RIG1-like receptors (RIG1
and MDA5) and OAS1 in several bat species present in French Guiana. We focused on four bats
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species showing differences in seroprevalence for rabies virus: Desmodus rotundus, Carollia
perspicillata, Sturnira lilium and Molossus molossus. For each of them, TLR7, RIG-1 and OAS1
have been sequenced on 50 individuals. Statistical approaches have been used to detect
selection acting on DNA and amino acid sequences. In parallel, neutral markers (Cytochrome b
and RAG 2) have been studied to give insights into the demographical history (whose signature
can mimic the one of selection). The first results, showing that the selection did not act in the
same way in every exon, enabled us to build selection patterns for each gene. Differences in
these patterns were observed between the four species. Further investigations are needed to
better understand the evolutionary history of these genes. They will provide information on the
long-term processes that have shaped the intra-specific diversity, thus leading to divergence
between species and rendering some populations sensitive or resistant to certain infections.
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Poster Abstracts
Kendra Alfson and A. Griffiths. Investigating the dynamics of filovirus evolution in cell culture.
Texas Biomedical Institute.
Filoviruses are highly lethal RNA viruses that can cause hemorrhagic fever with fatality rates
of up to 90%. No approved vaccines or therapies exist for filovirus infections. Evidence suggests
that bats may be the natural reservoir for filoviruses but they are capable of replicating in multiple
species (e.g. humans, non-human primates, and pigs). Typically, RNA viruses have high
spontaneous mutation rates due to error prone RNA-dependent RNA polymerases. A
consequence of high spontaneous mutation and replication rates is populations composed of
heterogeneous swarms of related variant sequences, often referred to as quasispecies. These
swarms have important biological consequences as they allow viruses to evolve rapidly in
response to selection pressures, which is critically relevant to viral-emergence, virulence, drugresistance, and vaccine-development. The dynamics of filovirus evolution are poorly understood
and little is known about the quasispecies present in filovirus populations. Currently, we are using
deep sequencing to assess the genetic changes associated with filovirus passage in cell culture.
These studies were performed using cultured cells derived from African green monkeys and fruit
bats. The viruses used include Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus
(MARV). We are detecting interesting adaptive changes in the consensus sequence and in the
quasispecies population associated with passaging the virus. Some of these changes appear to
be cell line and virus dependent. It is likely that the observed genomic changes can also be
correlated to phenotype and function. Our data suggest that filoviruses exhibit high genome
plasticity and are able to rapidly evolve to different environments. This could have major
implications for future filovirus research on emergence, virulence, drug-resistance, and vaccinedevelopment.
Ian H Mendenhall1, Maggie Skiles2, Linfa Wang1, Gavin Smith1. Virus surveillance in the bats of
Singapore: Host specificity and diversity of astroviruses. Duke University-Singapore National
University, Johns Hopkins Bloomberg School of Public Health.
Astroviruses are single stranded, positive sense RNA viruses that infect a wide range of
animals. These viruses are between 6.8kb – 8kb in length and have three open reading frames
encoding the protease, RNA-dependent RNA polymerase (RDRP), and capsid, which displays the
most genetic diversity. There are two groups of astroviruses, those that infect mammals
(Mamastroviruses) and birds (Avastroviruses). These viruses cause deleterious effects in a
number of species, including humans, minks, and turkeys. Bats harbor a tremendous genetic
diversity of astroviruses and there are more published sequenced from bats than all other
mammals, excluding humans. Previous studies have shown that some lineages of bat
astroviruses are host specific, there are others that appear to infect a wide variety of species. The
majority of bat astrovirus surveillance has focused on insectivorous species, while there are only
two frugivorous species with available astrovirus sequences in GenBank. Our research has
focused on virus surveillance in bats in Singapore and we have monitored several bat species
since 2011. We trapped bats using mist nets and harp traps, in addition to collecting feces and
urine from beneath roosting sites. We screened these samples for coronaviruses,
paramyxoviruses, and astroviruses with PCR and family specific primers. We have detected
astroviruses in two species, Rhinolophus lepidus and Eonycteris spelaea, the latter a frugivorous
bat in the family Pteropodidae The colony we monitor presents with a fulminant infection across
the 2.5 years of surveillance data we have. Positive PCR products were cloned into pGEM-T Easy
vector and sequenced. Sequences were aligned with reference sequences using Geneious 7.0,
manually optimized, and the best model was selected using jModel test. We did not detect
coronaviruses or paramyxoviruses in the bats we sampled. Here we present an evolutionary
analysis of the RDRP and capsid genes from Eonycteris spelaea and demonstrate the host
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specificity of Rhinolophus astroviruses. We are continuing to sample bats and screen these
samples for other viruses, such as reoviruses, herpesviruses and adenoviruses.
Ben Stading1, J. Osorio1, T. Rocke2. Assessment of recombinant poxviruses as vaccine vectors
for bats through in vivo imaging. University of Wisconsin1, National Wildlife Health Center,
USGS, Madison, WI2
Bats (Order Chiroptera) are an abundant group of mammals with tremendous ecological value
as insectivores and plant dispersers, although their role as reservoirs of significant zoonotic
diseases has become more appreciable in the last decade. Here we tested two recombinant
poxviruses, Modified Vaccinia Ankara (MVA) and Raccoonpox virus (RCN), as feasible vaccine
vectors in the Mexican Free-tailed Bat (Tadarida brasiliensis) through the use of in vivo imaging
studies. Animals were infected with recombinant poxviral vectors expressing the luciferase gene
(MVA-luc, RCN-luc) through oronasal or intramuscular routes, and sequential imaging revealed
the pattern of viral gene expression over time. No clinical illness was noted after exposure to the
vectors by either route. The imaging study revealed evidence that both viral vectors can express
the luciferase gene and are potential viable oral vaccine vectors in T. brasiliensis. To assess
immune response to this vector, data is being collected on serologic responses after vaccination
to RCN-vectored vaccination against the rabies glycoprotein (RCN-G). Poxviral vectors can
accept large insertions of foreign DNA for the production of protective antigens, and have been
shown to be effective in wildlife vaccine campaigns in the past. Orally available vaccines can be
distributed to groups of bats topically as a liquid or paste while they roost or are otherwise
congregated. By engineering recombinant RCN-based vaccines against lyssaviruses and other
important transmissible diseases, we can generate immunity in free-ranging bat populations,
offering the opportunity for “up-stream” interventions of important zoonoses and other bat
diseases.
Michael Weis1, L. Behner1, C. Drosten2, J. F. Drexler2, A. Maisner1. African bat henipavirus GHM74a glycoproteins have impaired fusogenic properties. Institute of Virology, Philipps
University Marburg, Marburg, Germany1, Institute of Virology, University of Bonn Medical Centre,
Bonn, Germany2
In recent years several studies revealed the existence of henipavirus-related viruses in African
bats. Though viral RNA has been manifold detected, live virus could never been isolated from bat
samples. Thus, the potential of new African henipaviruses to replicate in bat and non-bat cells
cannot be analyzed by infection studies. To date, this question can only be addressed by studying
the biological activity of individual viral genes. Essential steps for entry and cell-to-cell spread of
henipaviruses in any cell, is binding of the G protein to cellular ephrin receptors, and functional
expression of fusion-competent F proteins. To analyze these biological properties, we cloned the
genes for the surface glycoproteins F and G of the prototype African henipavirus GH-M74a. In
contrast to other plasmid-encoded henipavirus glycoproteins, GH-M74a-G and -F proteins did not
cause cell-to-cell fusion in many mammalian cell types readily permissive to henipavirus
infections. A limited syncytium formation was only found in HypNi/1.1 cells, a bat kidney cell line
derived from Hypsignathus monstrosus. Interestingly, the highly restricted fusion activity was
predominantly due to the F protein. While GH-M74a-G protein was found to interact with the main
henipavirus receptor ephrin-B2 and induced syncytium formation upon coexpression with
heterotypic Nipah virus F protein, GH-M74a-F did not cause evident fusion in the presence of
heterotypic Nipah virus G protein. Further analyses revealed a reduced expression of cleaved and
fusion-active GH-M74a-F protein on the cell surface, which is most likely the result of an impaired
intracellular F protein trafficking. It remains to be elucidated if this functional defect is a conserved
feature of African bat henipaviruses to downregulate fusion activity in their natural bat host.
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Olivier Pernet1, B. S. Schneider2, S. M Beaty1, M. LeBreton2, T. E. Yun3, A. Park1, T. T. Zachariah4, T.
A. Bowden5, P. Hitchens6, C. M. R. Kitchen1, P. Daszak7, J. Mazet6, A. N. Freiberg3, N. D. Wolfe2,
B. Lee1,8. Evidence for henipavirus spillover into human populations in Africa. University of
California Los Angeles1, Global Viral/Metabiota2, University of Texas Medical Branch, Galveston3,
Brevard Zoo, FL4, University of Oxford5, University of California Davis6, EcoHealth Alliance, New
York7, Icahn School of Medicine at Mount Sinai8.
Zoonotic transmission of lethal Henipaviruses (HNV) from their natural fruit bat reservoirs to
humans has only been reported in Australia and South/Southeast Asia. However, a recent study
discovered numerous and diverse HNV clades in African bat samples. To determine the potential
for HNV spillover events among humans in Africa, we examined well-curated sets of bat (Eidolon
helvum, n=44) and human (n=497) serum samples from Cameroon for Nipah virus (NiV) crossneutralizing antibodies (NiV-X-Nabs). Using a VSV-based pseudoparticle seroneutralization
assay, we detected NiV-X-Nabs in 48% and 3-4% of the bat and human samples, respectively.
The specificity, breath, and potency of anti-NiV-X-Nabs were confirmed using numerous specificity
controls unique to our infectious SN assay, including isogenic viruses pseudotyped with irrelevant
(VSV-G) or related HNV envelopes (HeV and GhV-M-74a), and follow-up confirmation with a
recombinant replication-competent Gaussia-luciferase secreting reporter NiV specifically
engineered for high sensitivity detection of anti-NiV-X-Nabs. Butchering bat meat and living in
areas undergoing deforestation were the most significant risk factors associated with
seropositivity (p= 0.0006 and 0.0136, respectively, two-tailed Fisher’s Exact test). Indeed, those
butchering bats were 29 times more likely to be seropositive than those not having contact with
bats (7/164 (4.09%) versus 0/316, respectively; OR =28.9, P=0.0002) and those living in areas of
putative deforestation were 10 times more likely to be HNV seropositive than those who were not
(3.24% vs 0.33%, respectively, OR=10.09, P=0.0088). The geographical and temporal clustering
of these seropositive cases provides evidence for recent HNV-like spillover events into the human
population in this part of Africa. Evidence for HNV spillover events warrants increased surveillance
efforts.
A. Lavergne1, B. de Thoisy2, H. Bourhy2, Vincent Lacoste3. Serological and molecular
investigation of rabies virus circulation in Amazonian bats from French Guiana. Laboratoire
des Interactions Virus-Hôtes, Institut Pasteur de la Guyane1, Centre National de Référence de la
Rage, Institut Pasteur de Paris2.
In South America, bats are the main vectors of the rabies virus. Numerous studies have been
conducted to characterize rabies virus in wild bats in Brazil, Ecuador and Chile, but in French
Guiana, information on the distribution and circulation of the virus remains fragmentary. Since
1984, 13 cases of rabies were reported in domestic animals. Genotyping of these viruses
revealed that, they were all of bat origin. In 2008, a man died of rabies in Cayenne and, once
again, virus was of bat origin. The first objective of this study was to determine the prevalence of
rabies virus in bats in French Guiana by direct and indirect screenings, to decipher how
environmental and biological parameters can explain variations of the prevalence observed
between bat communities. Then, we focused on two populations of vampire bats (Desmodus
rotundus) to monitor how the serological status varies over time. Bats were collected from 2006 to
2014, during the rainy and dry seasons, in several landscapes. Individuals coming from two
vampire bat colonies were marked by transponder for capture-mark-recapture studies. Blood
samples were collected by venipuncture for a serological survey and for molecular investigations
by RT-PCR. A total of 996 sera from 29 species were tested. The overall rate of seropositivity was
10.1%, with important interspecific variations. Multivariate logistic analysis determined 2 models to
explain seropositivity. A first model identified diet (hematophagous vs. all other diets), and pristine
forest habitats as favoring factors. A second model identified monospecific colonies and pristine
forest as favoring factors. Only 1 vampire was positive for the presence of the virus. The
monitoring of 2 vampire bat colonies revealed a high variation of seroprevalence over time. We
also got evidence of changes for serological status for some animals, switching from negative to
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positive, and vice versa on a one-year period. Serological results reported here suggest that
rabies virus actively circulates in the bat populations in French Guiana and more intensively in
forest populations. This raises the question of the contamination among species with very distinct
biological and ecological patterns. Longitudinal studies, based on the monitoring of the serological
status and presence of the virus, associated to ecological and population genetics studies have to
be pursued on D. rotundus to better understand the circulation of the virus in these bat
communities.
Andres Moreira-Soto1, N. Vargas-Vargas1, B. Rodriguez-Herrera2, E. Corrales-Aguilar1. Detection of
new coronaviruses in neotropical bats suggesting wide diversity in Costa Rica. CIET,
University of Costa Rica, San Pedro1, Biology Dept., University of Costa Rica, San Pedro2
Bats have been proposed as reservoirs of coronaviruses known to cross the host species
barrier. For example, two newly described emerging viruses SARS and MERS coronaviruses,
have been identified to have arisen from bats. Since neotropical regions are recognized hotspots
for emerging infectious diseases, we sampled bats in sites of close proximity with humans, in
fragmented sites and forests during 2012-2014. Fecal samples / intestines were collected in
different locations of Costa Rica. RNA was extracted and retrotranscribed into cDNA. RT-PCR for
a conserved region of the RdRp was done and bands were purified for sequencing. RdRp
sequences were analysed using Megalign software. Phylogenetic trees were constructed with
MrBayes software using MCMC analyses. We sampled more than 300 bats in different regions of
Costa Rica. Although detection of coronavirus was low (1.2 %), we report the detection of a novel
CoV in a Glossophaga soricina bat. Phylogenetic analyses from coronavirus found clustered
within the alphacoronavirus group, but the Glosor/batCoV had no significant homology with any
other neotropical bat CoVs found so far. In fact the highest homology (76%) was found to a CoV
found in Australia (Bat coronavirusR.meg/Australia/CoV100/2007 EU834953.1) and one found in
Costa Rica (Batcoronavirus Costa Rica strain Aju21 KC779226). The homology to the other CoV
sequences found in G. soricina in Trinidad (EU769558) was merely 70%. In the phylogenetic
analyses the Glosor/batCoV clustered in a subgroup near a CoV found in Asian bats HKU8, but
with extremely low posterior probability. Together these findings suggest that neotropical bats
could harbor more diverse coronaviruses than previously thought. It has been suggested that the
species of bat is a strong determinant in the coronavirus detected, but the finding of such a
different sequence of CoV in Glossophaga soricina might suggest that the diversity of Bat CoVs in
a given specie might be bigger than what has been found so far. It is tempting to speculate that
nectivorous bats could be more exposed to different CoVs because of their feeding habits.
Corman et al 2013 found a similar CoV in two species of nectivorous bats, suggesting this
scenario. However, more studies are needed to assess the potential epidemiological factors that
drive these diversity in bats.
Terry Fei Fan Ng1, I. Steffen1, C. Driscoll2, M. P. Carlos3, A. Prioleau3, R. Schmieder4, B. Dwivedi6, J.
Wong1, Y. Cha1, S. Head5, M. Breitbart6, E. Delwart1. A New Vesiculovirus in Bats with a
History of Human Contact. Blood Systems Research Institute, San Francisco1, Maryland
Department of Natural Resources2, , Maryland Department of Health and Mental Hygiene3, San
Diego State University4, The Scripps Research Institute5, University of South Florida6
Bats are known to carry many emerging viruses. In the United States of America (USA), dead
bats reported by civilians, or those that accidently made human contact, are routinely tested for
rabies virus. Although rabies transmission from bats to humans is rare in the USA, it remains
unanswered what other potential viruses capable of infecting humans these bats carry. As bat
habitat overlaps increasingly with human settlements, there is potential for bat viruses to spillover
to the human population, directly or through contact with pets or wildlife. In order to address this
public health concern, we investigated the tissue virome of bats with a history of human contact.
A total of 120 dead rabies-negative big brown bats (Eptesicus fuscus) were collected in Maryland
(USA), and their lungs and livers were dissected. Intact viral particles were purified and the
resulting nucleic acids were amplified and sequenced using 454 pyrosequencing and Hiseq
sequencing. From the bat tissue virome, we discovered a novel rhabdovirus, American bat
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vesiculovirus (ABVV), of the Vesiculovirus genus. Although vesiculoviruses are known to cause
zoonotic infections, little is known about vesiculovirus infections in bats. ABVV is the first
vesiculovirus identified in bats, while most other known bat rhabdoviruses belong to the
Lyssavirus genus. Phylogenetically, ABVV is close to the root of all mammalian vesiculoviruses,
suggesting early divergence from other mammalian vesiculovirus species. Using ABVV-specific
PCR, 5% (3/60) of the bats tested positive for ABVV in either the lung or the liver, confirming
active infection of ABVV in bat tissue. To further characterize this distinct lineage of bat
vesiculovirus in North America, we developed a pseudotype system for ABVV infection of different
cell lines. A vesicular stomatitis virus (VSV) construct carrying a reporter gene for firefly luciferase
(VSVΔG-Luc) and bearing the predicted synthesized ABVV glycoprotein (ABVV-G) was used to
infect Vero, BHK-21 and Tb1Lu cells. Interestingly, VSVΔG-GFP/ABVV-G pseudotypes were able
to infect BHK-21 cells, but not Vero or Tb1Lu cells. The pseudotype infection assay will be used to
establish a serum neutralization assay. We will test American bat and human serum samples for
ABVV-G neutralizing activity to determine the seroprevalence and zoonotic potential of this virus.
Nidia Arechiga-Ceballos, C. Obregón-Morales, L. Perea-Martínez, A. Aguilar-Setién. Experimental
infection of Artibeus spp. bats with rabies virus. Unidad de Investigación Médica en
Inmunología, IMSS, Mexico City
Artibeus bats have a Neotropical distribution in the Americas and are considered as
synanthropic. It has been observed that Artibeus spp. may occupy the same roosts that
Desmodus rotundus and the rabies virus variants (RABV) circulating in Artibeus spp. in Mexico
are closely related to vampire bat variants. In a study of seroprevalence of rabies antibodies in
bats our team found 6 out of 51 positive sera (11.8%) from Artibeus spp., suggesting that they
have been exposed to RABV. The aim of this work was to study the distribution of RABV in
different organs in Artibeus spp. We performed an experimental infection of Artibeus spp. with two
different vampire bat isolates (V3). High (1x105.34FFU) and low (1x103 FFU) doses of RABVand
intramuscular (IM), subcutaneous (SC) and intracranial (IC) routes, were compared. All animals
tested were lacking for anti-rabies antibodies before the infection. Only one out of 6 bats
inoculated by IC route with high doses of RABV died at day 14th post inoculation and was positive
to FAT in brain tissue, but no clinical signs were observed. None of the remaining 36 animals
inoculated with rabies SC or IM routes, high or low doses, died. The bats in this experiment do not
showed clinical signs of rabies, but high titers of antibodies were determined since 7th day postinoculation. RABV semi-nested PCR was positive in organs such as brain, stomach, liver and
heart in animals that did not show clinical signs of disease and were negative by
immunofluorescence in brain tissue. These results suggest that non-lethal pre-exposure to RABV
somehow protected the bats from rabies infection in this study, independently of doses and
inoculation routes and isolates, but the epidemiological role of frugivorous species in the
maintenance of rabies virus in the wild remains to be studied.
Eric Mossel, R. C. K., M. B. Crabtree, and B. R. Miller. A newly recognized clade of nairoviruses
isolated from bats and soft ticks. Centers for Disease Control and Infection, Fort Collins.
The nairovirus genus, family Bunyaviridae, is comprised of approximately 35 predominantly
tick-borne viruses divided into seven serogroups. The viruses can generally be sorted
phylogenetically with their tick vectors, especially regarding soft-tick (argasid) versus hard-tick
(ixodid) vectors. We recently identified through genetic sequencing three additional putative
members of the nairovirus genus from previously unassigned isolates, Kasokero virus (KASV),
Yogue virus (YOGV), and Keterah virus (KTRV). Together with Issyk-Kul virus (ISKV), recently
sequenced by Lvov and colleagues and for the first time shown to be distinct from KTRV, these
viruses form a distinct nairovirus clade most closely related to Bandia and Qalyub viruses. All four
viruses have been isolated from bats: ISKV from Nyctalus noctula in Kyrgyzstan, KASV and
YOGV from Rousettus aegyptiacus in Uganda and Senegal, respectively, and KTRV from
Scotophilus temmecki in Malaysia. KTRV and ISKV have additionally been isolated from batinfesting soft-ticks Argas pusillus and Argas vespertillonis. Human infections have been reported
with KASV and ISKV. Background data on ISKV is most voluminous, including subsequent
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isolations from additional hematophagous arthropods and other bat species. We present the
phylogenetic relationship of this group of viruses within the context of existing nairovirus
sequences and preliminary characterization studies.
Kerri Miazgowicz, N. van Doremalen, V. J. Munster. Determining host potential of MERS-CoV
among bat species. Rocky Mountain Laboratories, NIAID.
The Middle East rRespiratory syndrome coronavirus (MERS-CoV) first emerged in April, 2012.
As of May 2014, 496 MERS-CoV cases have been reported, including the first case confirmed in
the US, with a case-fatality rate of approximately 25%. The transmission dynamics of MERS-CoV
still remain unclear. Phylogenetically MERS-CoV is most closely related to coronaviruses detected
in bats, suggesting bats may act as a natural reservoir. Co-crystallization between DPP4 and
MERS-CoV spike protein has identified the critical residues of interaction required for virus
binding. Here we investigate the host potential of MERS-CoV among bat species as determined
by the receptor utilized for entry, dipeptidyl peptidase 4 (DPP4). First, we developed a RT-PCR to
obtain the DPP4 mRNA sequence from mammalian sources, e.g. Artibeus jamaicensis. From the
sequences generated and sequences available on GenBank we investigated variation of DPP4
among bat species at the critical residues of interaction. Modeling was then employed to predict
the binding energies of each species’ DPP4 to MERS-CoV spike. Finally, we began to examine
MERS-CoV susceptibility conferred by bat DPP4s in vitro. Bat cell lines derived from both
Microchiroptera and Megachiroptera suborders were tested for MERS-CoV replication.
Concurrently, bat DPP4s were transfected into non-susceptible BHKs and then tested for MERSCoV replication.
Blair DeBuysscher1,2, D. Scott1, H. Feldmann1 and J. Prescott1. Characterization of cell-type
specific infection of Nipah virus in vitro and in vivo. Rocky Mountain Laboratory, NIAID1,
University of Montana2.
Nipah virus (NiV) is a zoonotic pathogen in the family Paramyxoviridae that infects a wide
species range. The natural reservoir of NiV is the Pteropus family of fruit bats. Virus is passed
from bats to humans either from direct contact with bats, or bat secretions, or through an
intermediate host. Disease in humans is characterized by acute encephalitis and respiratory
distress. Pathogenicity in humans involves vasculitis of small blood vessels throughout the central
nervous system, lung, heart, and kidneys. Viral antigen and cytopathology is seen in endothelial
cells (ECs). Autopsies of NiV patients also show positive NiV staining in the tunica media
surrounding the affected vessels. Other than antigen positivity, little is known about smooth
muscle cell (SMC) involvement during NiV infection. In this study, we characterize infection and
pathogenesis in SMCs and ECs using the Syrian hamster and African green monkey disease
models, as well as primary human cell cultures. We found that both ECs and SMCs were positive
for viral antigen in vivo, however, no cytopathology was observed in SMCs, in contrast to ECs,
which underwent significant changes, including syncytia formation and necrosis. Infection of
human primary cells mirrored the in vivo observations. To further characterize infection of SMCs,
we followed infection of cells over time and observed low infection rates, even with a high virus
inoculation with little cell-to-cell spread and no syncytia formation. Despite the lack of
cytopathogenesis, progeny virus produced in SMCs reached high titers, similar to ECs infection.
In order to examine the resistance to infection and lack of cytopathic effect, we measured the
expression of the receptor for NiV (ephrin B2/3) on the cell surface and found that ECs had high
levels of ephrinB2/3 on their surface, while SMCs had little to no expression. The low levels of
entry receptor expression may explain NiV’s inability to cause syncytia in SMCs. SMCs could be a
location of viral replication without cytopathology. To confirm the effects of receptor on SMCs we
transfected cells with ephrin B2 and saw syncytia formation and cytopathic effect in the SMCs,
suggesting that the lack of receptor contributes to the phenotype. Through studying infection in
ECs and SMCs we can better understand effects of NiV infection in relevant cell types.
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Jeff Doty, G. L. Emerson, N. F. Gallardo-Romero, R. Nordhausen, M. M. Garner, J. R. Huckabee, S.
Johnson, R. D. Wohrle, W. B. Davidson, K., Y. Li, M. Metcalfe, K.n L. Karem, I. K. Damon, and D.
S. Carroll. Chiropoxvirus: Updates on a novel bat pathogen. Centers for Disease Control and
Infection, Atlanta.
Recent investigations have discovered new poxvirus species in bats. A wildlife hospital and
rehabilitation center in the northwestern United States received several big brown bats from 2009
to 2011 with necrosuppurative osteomyelitis in multiple joints. Although no skin or visceral lesions
were present, thin-section electron microscopy showed poxvirus particles within A-type inclusions.
Wing and joint tissues were sent to CDC for analysis and were found to be positive for poxviruses
by PCR. The virus was successfully isolated and grown in BSC-40 cells and a phylogenetic
comparison supports establishment of a new genus of Poxviridae. Since the initial report in 2013,
other bats with similar symptoms have been assessed for poxvirus infections and have been
found to be negative.
Ashley Malmlov1, J. Seetahal2, C. Carrington2, V. Ramkisson2, J. Foster2, V. Munster3, S.
Quackenbush1 and T. Schountz1. Serological evidence that Tacaribe virus is circulating
among bats in Trinidad and Tobago. Colorado State University1, The University of the West
Indies2, St. Augustine, Republic of Trinidad and Tobago, Rocky Mountain Laboratories, NIAID3
Tacaribe virus (TCRV) is a bisegmented, ambisense, RNA virus within the genus Arenavirus.
Arenaviruses are grouped into Old World lymphocytic choriomeningitis virus-Lassa virus complex
and the New World Tacaribe virus complexes. TCRV is placed within the Tacaribe complex along
with the South American hemorrhagic fever viruses: Chapare, Guanarito, Junín, Machupo, and
Sabiá viruses. The only isolates of TCRV were from 11 artibeus bats collected by investigators at
the Trinidad Regional Virology Laboratory in the Republic of Trinidad in the 1950s. TCRV has not
been isolated since, although serological data from the 1970s suggested it was circulating among
Caribbean bats. Only one isolate remains, TRVL-11573, and it has been passaged in suckling
mice and Vero cells, thus its genomic integrity is unknown. We sought to determine if TCRV is still
circulating in bat populations in Trinidad through serological investigation. We developed an
ELISA and western blot assay using His-tagged recombinant TCRV nucleocapsid antigen. Serum
from Artibeus jamaicensis that had been experimentally infected with TCRV was used as a
positive control, and serum collected from an uninfected A. jamaicensis used as a negative
control. ELISA screen of bloods from 84 bats of various species captured in Trinidad identified
several, mostly artibeus bats, as seropositive for antibodies to TCRV. Some of these were tested
by western blot. Four were negative, eight were weakly positive, and five were strongly positive.
These results suggest that TCRV or other arenaviruses continue to circulate among bats in
Trinidad.
Alvaro Aguilar Setién1, C. Obregón Morales1, L. Perea Martínez1, N. Aréchiga Ceballos1, S. Aguilar
Pierlé2. Isolation of Waddlia cocoyoc, a novel intracellular bacterium, from frugivorous bats
(Artibeus intermedius). Unidad de Investigación médica en Inmunología, IMSS, Mexico City1,
Washington State University, Pullman USA2
Artibeus intermedius, is one of the most common frugivorous bats in the tropical Americas,
ranging from the Caribbean Islands to Central America and Mexico. Several microbes of interest
have been isolated from or detected in Artibeus spp. bats, including Histoplasma capsulatum,
Trypanosoma cruzi, eastern equine encephalitis virus, Mucambo virus, Jurona virus, Catu virus,
Itaporanga virus and Tacaiuma virus. The biological characteristics of bats make them extremely
suitable hosts for disease agents. A novel chlamydia-like bacterium was isolated from an A.
intermedius bat that was collected to characterize rabies virus pathogenicity in frugivorous bats.
This animal showed emaciation, restlessness and depression. On day 20 the animal could not fly,
areas of pallor appeared on his wings. The animal died on day 28, and was negative for rabies
virus using immunofluorescence. Histopathological findings on the areas of pallor revealed
mononuclear cell infiltrates. VERO (monkey) and BHK21 (rodent) cell cultures were inoculated
with macerated biopsies from lesions. Diff-Quick staining revealed lysis and large intracytoplasmic
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vacuoles with intracellular bacteria in inoculated cell cultures. Experimentally Infected bats
showed severe lesions in lungs and spleen and pale areas on the wings. Bacteria were detected
in the lesions by immunofluorescence using hyperimmune serum generated in mice. Sequence/
phylogenetic analyses using an array of genes revealed high sequence identity to Waddlia
chondrophila, an obligate intracellular pathogen in the Chlamydiales. We describe the isolation
and characterization of this novel bacterium and propose that it be given the name Waddlia
cocoyoc since it was first isolated in Cocoyoc Morelos, Mexico.
M. M. B. Moreno-Altamirano2, Edith Zenteno-López2, M. del Rosario Salinas-Tobón2, C. ObregónMorales1, Alvaro Aguilar Setién1. Comparison between human and bat (Artibeus intermedius)
monocytes and lymphocytes. Unidad de Investigación médica en Inmunología, IMSS, Mexico
City1, Escuela Nacional de Ciencias Biológicas, IPN, Mexico City2
Bats are major reservoirs for many pathogens. However they handle infection quite efficiently
and live long lives, this is beginning to be explained in Immunological terms. Once they get
infected, they fight the pathogens off very well. Recently studies report that the immune system of
bats lack the “cytokines storm” that in other species triggers extreme and sometimes fatal
reactions to infections. Instead, bats immune system, apparently depress the inflammation, which
is in part, responsible for the host cells and tissue damage. Based on the bats capacity to support
many pathogens, it is interesting to search how their immune system works. The aim of this work
was to compare the activation capacity of macrophages and lymphocytes in Neotropical bats
Phylostomidae (Artibeus intermedius) and humans. Humans and bats macrophages, T, and B
lymphocytes were obtained by density gradient centrifugation on ficoll-hypaque solution.
Macrophages were activated with PMA/Ionomycin to test their endocytic capacity by FITC-dextran
uptake. The macrophage phenotype was assessed by staining cells with an anti-human CD14-PE
antibody. The T lymphocytes were activated with Con A. Activation was assessed by detecting
IL-2 in the supernatants, by ELISA, at 6,12,24,48 and 72 hours post activation. T Lymphocytes
were identified with an anti-human CD3-PE antibody. The B lymphocytes activation was analyzed
by detecting antibody forming cells, and IgM synthesis, on PWM- and LPS-activated B
lymphocytes. B lymphocytes phenotype was assessed by staining with an anti- human CD19-PE
antibody. All cells were observed under a confocal microscopy. Results showed that all the antihuman antibodies cross-react with bats CDs molecules and that bats lymphocytes seem to be
less activated than the human counterparts. The endocytic capacity was similar in bat and human
macrophages.
34
Infectious Diseases of Bats Symposium!
Fort Collins, CO, USA
Acknowledgements
The Organizing Committee Members
Charles H. Calisher, PhD, Emeritus Professor, Colorado State University ([email protected])
Katherine Holmes, PhD, University of Colorado Health Sciences Center ([email protected])
Richard Bowen, DVM, PhD, Colorado State University ([email protected])
Paul Cryan, PhD, US Geological Survey ([email protected])
Rebekah Kading, PhD, Centers for Disease Control and Prevention, Fort Collins ([email protected])
Edit Szalai, Colorado State University
Amy Gilbert, PhD, National Wildlife Research Center, USDA ([email protected])
Joel Rovnak, PhD, Colorado State University ([email protected])
Ashley Malmlov, DVM, Colorado State University ([email protected])
Amanda McGuire, DVM, Colorado State University ([email protected])
Danielle Adney, Colorado State University ([email protected])
Vienna Brown, Colorado State University ([email protected])
Tony Schountz, PhD, Colorado State University ([email protected])
Thanks to Ashley Malmlov for the symposium logo.
A special thanks to Lauren Ankarlo, CSU Conference Services
The Organizing Committee is grateful for the generous support of this symposium from:
Dr. Rick Lyons, Director, CSU Infectious Disease Research Complex
Dr. Mark Stetter, Dean, CSU College of Veterinary Medicine and Biomedical Sciences
Dr. Gregg Dean, Chairman, CSU Department of Microbiology, Immunology and Pathology
Dr. Alan Rudolph, CSU Vice President for Research
and
Life Technologies, Inc.
VWR, Inc.
Virology Journal
International Society for Infectious Diseases
ProMED
35
Infectious Diseases of Bats Symposium!
Fort Collins, CO, USA
Allison Hall
Wednesday reception
Thursday lunch, posters
Friday lunch
Old Town via MAX
Restaurants
Breweries
Shops
Fast-Food
Restaurants
(2 blocks)
Lory Student Center
Symposium
in North Ballroom
Campus
Housing/
Dining
MAX
Station
(Free)
Hilton Hotel (Prospect Ave.)
36
MAX
Station
Prospect
Ave
(Free)
Fort Collins, CO, USA
From Hilton
From Campus
Housing
Restaurants
Infectious Diseases of Bats Symposium!
37
Infectious Diseases of Bats Symposium!
Fort Collins, CO, USA
Name
Affiliation/Company/School
Email
Nation
Dr. Alvaro Aguilar Setién
Ms. Kendra Alfson
Mr. Jason Ambrose
Dr. Brian Amman
PhD. Nidia Arechiga
Dr. Laurie Baeten
Dr. Ying Bai
Dr. Michelle Baker
Dr. Robert Baker
Dr. Udeni Balasuriya
Dr. Christopher Basler
Mr. Daniel Becker
Ms. Laura Behner
Dr. Brian Bird
Mr. Rohan Biswas
Dr. David Blehert
Dr. Sharon Bloom
Dr. Monica Borucki
Mr. Paul William Bradley
Ms. Kelly Broussard
Dr. Gaelen Burke
Dr. Danielle Buttke
Dr. Christine Carrington
Dr. Cristina Cassetti
Dr. Douglas Causey
Dr. Jasper Fuk-Woo Chan
Ms. Elizabeth Cook
Dr. Eugenia Corrales-Aguilar
Dr. Keren Cox-Witton
Dr. Peter Daszak
Dr. April Davis
Dr. Emmie de Wit
Ms. Blair DeBuysscher
Dr. Kevin Dhondt
Dr. Samuel Dominguez
Mr. Jeffrey Doty
Dr. Christian Drosten
Dr. Hideki Ebihara
Dr. Jonathan Epstein
Dr. Ken Field
Ms. Teresa Garcia
Dr. Luis D. Giavedoni
Ms. Nicole Glennon
Dr. Luiz Góes
Mr. Yann Gomard
Miss Ana Gonzalez Reiche
Mr. Robert Grant
Dr. Ann Hawkinson
Ms. Anna Helms
Dr. Andrew Herbert
Dr. Andrew Hickey
Dr. Mike Holbrook
Dr. Leilani Hotaling
Dr. Megan Howard
Dr. Bethany Hoye
Ms. Gretchen Hume
Dr. Aaron Irving
Dr. Petrus Jansen van Vuren
Dr. Victoria Jensen
Dr. Benjamin Johnson
Dr. David Kenny
Mr. John Koprivsek
Dr. Michael Kosoy
Ms. Kirsten Kulcsar
Instituto Mexicano del Seguro Social
Texas Biomedical Research Institute
University of South Florida
Centers for Disease Control and Prevention
Institut for Epidemiologic Diagnostic and Reference
Colorado State University
Centers for Disease Control and Prevention
Australian Animal Health Laboratory, Geelong
Lovelace Respiratory Research Institute
University of Kentucky
Icahn School of Medicine at Mount Sinai
University of Georgia
Institute of Virology / Pilipps University of Marburg
Centers for Disease Control and Prevention
Albert Einstein College of Medicine
USGS National Wildlife Health Center
Emerging Infectious Diseases (Journal), CDC
Lawrence Livermore National Laboratory
Oregon State University
University of California Berkeley
University of Georgia
National Park Service
The University of the West Indies
National Institute of Allergy and Infectious Disease
University of Alaska Anchorage
The University of Hong Kong
University of Edinburgh
University of Costa Rica
Wildlife Health Australia
EcoHealth Alliance
New York State Department of Health
Rocky Mountain Laboratories NIAID
Rocky Mountain Laboratories NIAID
CIRI - U1111 INSERM
University of Colorado SOM
Centers for Disease Control and Prevention
University of Bonn Medical Centre
Rocky Mountain Laboratories NIAID
EcoHealth Alliance
Bucknell University
Colorado State University
Texas Biomedical Research Institute
Icahn School of Medicine at Mount Sinai
University of Sao Paulo
Université de La Réunion
University of Maryland
The Scientist
University of Northern Colorado
Colorado School of Public Health
USAMRIID
USPHS/BUMC
Battelle/NIAID
University of Minnesota
University of Alaska Anchorage
Centre for Integrative Ecology, Deakin University
University of Northern Colorado
Duke-NUS Graduate Medical School
National Institute for Infectious Diseases
National Biodefense Analysis & Countermeasures Ctr
BioMed Central
Denver Zoological Foundation
University of Texas HSC San Antonio
Centers for Disease Control and Prevention
Johns Hopkins University
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Infectious Diseases of Bats Symposium!
Mr. Vincent Lacoste
Dr. Sally Lahm
Mr. Eric Laing
Ms. Kate Langwig
Dr. Susanna Lau
Dr. Catherine Laughlin
Ms. Anne Lavergne
Dr. William Lee
Dr. Benhur Lee
Dr. Michael Lo
Dr. Judith Mandl
Mr. Clifton McKee
Ms. Kerri Miazgowicz
Miss Helen Miller
Dr. Chad Mire
Dr. Vikram Misra
Mr. David Morán
Mr. Andres Moreira-Soto
Dr. Eric Mossel
Dr. Vincent Munster
Dr. Terry Fei Fan Ng
Ms. Melinda Ng
Dr. Kevin Olival
Dr. Sarah Olson
Mr. Bobby Onaga
Ms. Lynn Osikowicz
Dr. Kristy Pabilonia
Dr. Eun-Chung Park
Dr. Olivier Pernet
Dr. Stephanie Petzing
Ms. Adriana Pliego-Zamora
Dr. Joseph Prescott
Dr. Krista Queen
Dr. DeeAnn Reeder
Dr. Patricia Repik
Mr. Roger Rodriguez
Dr. Ronald Rosenberg
Mr. Michael Schirmacher
Dr. Stephanie Schittone
Dr. Amy Schuh
Dr. Martin Schwemmle
Dr. Daniel Shapiro
Dr. Ben Stading
Dr. Imke Steffen
Ms. Manasi Tamhankar
Dr. Kelvin To
Dr. Suxiang Tong
Dr. Jonathan Towner
Dr. Neeltje van Doremalen
Dr. Supaporn Wacharapluesadee
Dr. Lin-Fa Wang
Mr. Michael Weis
Mrs. Christina Weller
Mrs. Lisa Worledge
Fort Collins, CO, USA
Institut Pasteur de la Guyane
George Washington University, Washington,D.C.
Uniformed Services University
University of California Santa Cruz
University of Hong Kong
National Institute of Allergy and Infectious Disease
Institut Pasteur de la Guyane
Wadsworth Center
Icahn School of Medicine at Mount Sinai
Centers for Disease Control and Prevention
National Institutes of Health
Colorado State University
Rocky Mountain Laboratories NIAID
Bat Conservation Trust
Galveston National Laboratory
University of Saskatchewan
Universidad del Valle de Guatemala
University of Costa Rica
Centers for Disease control & Prevention
Rocky Mountain Laboratories NIAID
Blood SysteMs. Research Institute/UCSF
Albert Einstein College of Medicine
EcoHealth Alliance
Wildlife Conservation Society
Colorado State University
Centers for Disease Control and Prevention
Colorado State University Veterinary Diagnostic Lab
National Institute of Allergy and Infectious Disease
University of California Los Angeles
Uniformed Services University/HMJF
Griffith University
Rocky Mountain Laboratories NIAID
Centers for Disease Control and Prevention
Bucknell University
National Institute of Allergy and Infectious Disease
Centers for Disease Control and Prevention
Bat Conservation International
Colorado College
Centers for Disease Control and Prevention
University Freiburg, Germany
University of Nevada School of Medicine
UW - Madison, School of Veterinary Medicine
Blood SysteMs. Research Institute / UCSF
UT Health Science Center at San Antonio
University of Hong Kong
Centers for Disease Control and Prevention
Centers for Disease Control and Prevention
Rocky Mountain Laboratories NIAID
Chulalongkorn University
Duke-NUS Graduate Medical School
Institute of Virology / Philipps University of Marburg
Colorado State University Veterinary Diagnostic Lab
Bat Conservation Trust
39
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[email protected]
[email protected]
a.pliego@griffith.edu.au
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isteff[email protected]
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[email protected]
[email protected]
[email protected]
Michael.Weis@staff.uni-marburg.de
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[email protected]
France
United States
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Hong Kong, China
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France
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Canada
Guatemala
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