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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 2 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 3 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 4 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 5 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 6 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 7 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 8 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 9 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 10 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 11 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 12 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 13 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 14 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 15 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 16 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 17 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 18 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 19 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 20 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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, 21 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 22 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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, 23 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 24 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 25 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 26 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 27 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 28 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 29 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 30 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 31 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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. 32 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 33 Infectious Diseases of Bats Symposium! Fort Collins, CO, USA 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 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Mexico United States United States United States Mexico United States United States Australia United States United States United States United States Germany United States United States United States United States United States United States United States United States United States Trinidad and Tobago United States United States Hong Kong, China United Kingdom Costa Rica Australia United States United States United States United States France United States United States Germany United States United States United States United States United States United States Brazil France United States United States United States United States United States United States United States United States United States Australia United States United States South Africa United States United Kingdom United States United States United States United States 38 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] 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 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] a.pliego@griffith.edu.au [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] isteff[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Michael.Weis@staff.uni-marburg.de [email protected] [email protected] France United States United States United States Hong Kong, China United States France United States United States United States United States United States United States United Kingdom United States Canada Guatemala Costa Rica United States United States United States United States United States United States United States United States United States United States United States United States Australia United States United States United States United States United States United States United States United States United States Germany United States United States United States United States Hong Kong, China United States United States United States Thailand Singapore Germany United States United Kingdom