Dissertation Bats As Reservoir Hosts
Total Page：16
File Type：pdf, Size：1020Kb
DISSERTATION BATS AS RESERVOIR HOSTS: EXPLORING NOVEL VIRUSES IN NEW WORLD BATS Submitted by Ashley Malmlov Department of Microbiology, Immunology, and Pathology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Spring 2018 Doctoral Committee: Advisor: Tony Schountz Richard Bowen Page Dinsmore Kristy Pabilonia Copyright by Ashley Malmlov 2018 All Rights Reserved ABSTRACT BATS AS RESERVOIR HOSTS: EXPLORING NOVEL VIRUSES IN NEW WORLD BATS Order Chiroptera is oft incriminated for their capacity to serve as reservoirs for many high profile human pathogens, including Ebola virus, Marburg virus, severe acute respiratory syndrome coronavirus, Nipah virus and Hendra virus. Additionally, bats are postulated to be the original hosts for such virus families and subfamilies as Paramyxoviridae and Coronavirinae. Given the perceived risk bats may impart upon public health, numerous explorations have been done to delineate if in fact bats do host more viruses than other animal species, such as rodents, and to ascertain what is unique about bats to allow them to maintain commensal relationships with zoonotic pathogens and allow for spillover. Of particular interest is data that demonstrate type I interferons (IFN), a first line defense to invading viruses, may be constitutively expressed in bats. The constant expression of type I IFNs would hamper viral infection as soon as viral invasion occurred, thereby limiting viral spread and disease. Another immunophysiological trait that may facilitate the ability to harbor viruses is a lack of somatic hypermutation and affinity maturation, which would decrease antibody affinity and neutralizing antibody titers, possibly facilitating viral persistence. In 2009 and 2010 two novel influenza A viruses (IAV) were discovered via qRT-PCR using pan- influenza primers in New World bat species. Given the unique hemagglutinin (HA) and neuraminidase (NA) glycoproteins compared to those already recognized, the two viruses were classified as H17N10 and H18N11. H17N10 IAV genome was discovered in rectal swabs collected from little yellow-shouldered bats in Guatemala, and H18N11 IAV genome was discovered in a rectal swab and gastrointestinal tract of a flat-faced fruit bat from Peru. The entire sequences for both viruses were identified using next generation and Sanger sequencing, but the virus was never isolated from wild bat populations. Both viruses differ from canonical IAVs in that the HA does not bind to the host sialic acid receptor and the ii function of NA remains undetermined. Given the divergence from other IAVs, the attention bats receive as reservoir hosts, and the lack of isolation of wild virus, H17N10 and H18N11 remain shrouded in mystery. Reverse genetics was used to rescue both viruses and we performed experimental infections in Jamaican fruit bats (Artibeus jamaicensis), drawing upon the colony housed at Colorado State University. Evidence for H17N10 infection could not be elicited after inoculation, and thus it was concluded that Jamaican fruit bats may not be susceptible. Bats inoculated with H18N11 seroconverted (determined by ELISA), had viral RNA detected in rectal but not oral swabs by qRT-PCR, and had viral RNA present in the length of the gastrointestinal tract detected by qRT-PCR. Hematoxylin and eosin stain used to characterize histopathology revealed minimal pathology that was predominately localized to the gastrointestinal tract in the form of neutrophilic, plasmacytic and lymphocytic cellular infiltration. Furthermore, two naïve transmission bats were exposed to inoculated bats and demonstrated seroconversion and viral RNA detected in rectal swabs by qRT-PCR. Results demonstrate that Jamaican fruit bats are susceptible to H18N11 and indicate transmission occurs fecal-orally. Tissue tropism is for the gastrointestinal tract. These data recapitulate transmission and tissue tropism as seen in the reservoir of IAV, water fowl, and low-pathogenic avian influence viruses in gallinaceous birds. However, this does not indicate that bats may be a reservoir for influenza viruses as H18N11 is not known to cause disease in humans and is highly divergent from other IAVs. More likely, this demonstrates an early divergence of H18N11 from other IAVs and a long-lived co-evolution between the host and the virus. Further investigation of H18N11 may provide information on relationships between bats and their viromes, which is of great importance given so many bat species harbor highly pathogenic zoonotic viruses. The impact of Zika virus (ZIKV) on the New World has been great—infecting more than 200,000 people and manifesting in some patients as severe neurological complications including microcephaly in infected fetuses and Guillain-Barré syndrome. An enzootic cycle is implicated as an important part of viral ecology, yet little is known about this cycle. Historically, different bat species demonstrated experimental susceptibility to ZIKV as they seroconvert, have neurological disease, viremia and ZIKV iii positive tissues. Jamaican fruit bats are endemic to a region that temporally overlaps with the distribution of ZIKV in the Americas and Caribbean. We sought to identify if these bats were susceptible to ZIKV and conducted a time course study to delineate progression of viral infection and pathophysiology. ELISAs were used to identify seroconversion. Quantitative RT-PCR and immunohistochemistry were used to determine tissue tropism, and hematoxylin and eosin stain was used to characterize histopathology. Jamaican fruit bats seroconverted post-inoculation with ZIKV. Evidence for virus was found by qRT-PCR in the brain and urine for ZIKV infection in some bats. IHC revealed positive testes, brain, lung, and salivary gland and a trend toward mononuclear cells, macrophage and fibroblast viral tropism. Histopathology was seen in the brain, testes and salivary gland. Based on these data it was concluded that Jamaican fruit bats could become infected with ZIKV. These bats may have a role in viral ecology. Tacaribe virus (TCRV) was isolated in the 1950s from wild artibeus bats captured in Trinidad, West Indies. The initial characterization of TCRV suggested that artibeus bats were natural reservoir hosts. However, nearly 60 years later experimental infections of Jamaican fruit bats resulted in fatal disease or clearance, suggesting this species is not a reservoir host. To further evaluate the TCRV reservoir host status of artibeus bats, we captured bats of six species in Trinidad for evidence of infection. Bats of all four frugivorous species had antibodies to TCRV nucleocapsid, whereas none of the insectivore or nectarivore species did. Many flat-faced fruit bats (A. planirostris) and great fruit bats (A. literatus) were seropositive by ELISA and western blot to TCRV nucleocapsid antigen, as were two of four Seba’s fruit bats (Carollia perspicillata) and two of three yellow-shouldered fruit bats (Sturnira lilium). Serum neutralization tests failed to detect neutralizing antibodies to TCRV from these bats. TCRV RNA was not detected in lung tissues or lung homogenates inoculated onto Vero cells. These data suggest that TCRV or a similar arenavirus continues to circulate among fruit bats of Trinidad but there is no evidence of persistent infection, suggesting artibeus bats are not reservoir hosts. iv In order to make species specific reagents and expand laboratory research on Jamaican fruit bats, de novo assembly of the Jamaican fruit bat genome was performed with Soapdenovo2. The final genome size was 1.4 kilobases with 20x coverage. v ACKNOWLEDGMENTS There is a very large community of people that have offered endless support and I would not be here without them. I would like to thank Dr. Tony Schountz for his endless patience and encouragement. I am tremendously grateful for the opportunity to research bats, and work and learn in his laboratory. I would like to thank Dr. Kristy Pabilonia for her constant mentorship. She has been an incredible advisor to me since junior year of veterinary school and I am indebted to her for all her sage advice. I would like to thank my committee members, Dr. Richard Bowen and Dr. Page Dinsmore for supporting me on this path and always providing me with guidance and advice. Thank you to my family and friends for their endless cheer and generous forgiveness for all the times I chose work and school over them. Finally, thank you to the bats. Working with them has been a wonderful experience. vi TABLE OF CONTENTS Abstract .......................................................................................................................................................... ii Acknowledgments ........................................................................................................................................ vi List of Tables ................................................................................................................................................ xi List of Figures .............................................................................................................................................. xii Chapter 1: Introduction and Literature Review ............................................................................................. 1 1.1: Introduction to Bats and Family Phyllostoma ........................................................................................ 1 1.1.1: Bat Evolution .........................................................................................................................