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Artibeus Jamaicensis) with Tacaribe Virus Ann C University of Northern Colorado Scholarship & Creative Works @ Digital UNC Dissertations Student Research 8-1-2011 Experimental infection of Jamaican fruit bats (Artibeus jamaicensis) with Tacaribe virus Ann C. Hawkinson Follow this and additional works at: http://digscholarship.unco.edu/dissertations Recommended Citation Hawkinson, Ann C., "Experimental infection of Jamaican fruit bats (Artibeus jamaicensis) with Tacaribe virus" (2011). Dissertations. Paper 150. This Text is brought to you for free and open access by the Student Research at Scholarship & Creative Works @ Digital UNC. It has been accepted for inclusion in Dissertations by an authorized administrator of Scholarship & Creative Works @ Digital UNC. For more information, please contact [email protected]. UNIVERSITY OF NORTHERN COLORADO Greeley, Colorado The Graduate School Experimental Infection of Jamaican Fruit Bats (Artibeus jamaicensis) With Tacaribe Virus A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Ann C. Hawkinson College of Natural Health Sciences School of Biological Sciences Biological Education August 2011 This Dissertation by: Ann C. Hawkinson Entitled: Experimental infection of Jamaican fruit bats (Artibeus jamaicensis) with Tacaribe virus has been approved as meeting the requirement for the Degree of Doctor of Philosophy in College of Natural Health Sciences, in School of Biological Sciences, Program of Biological Education Accepted by the Doctoral Committee William A. Schountz, Ph.D., Chair Susan M. Keenan, Ph.D., Committee Member Rick A. Adams, Ph.D., Committee Member Steven Pulos, Ph.D., Faculty Representative Date of Dissertation Defense Accepted by the Graduate School Robbyn R. Wacker, Ph.D. Assistant Vice President for Research Dean of the Graduate School & International Admissions ABSTRACT Hawkinson, Ann C. Experimental Infection of Jamaican Fruit Bats (Artibeus jamaicensis) with Tacaribe virus. Published Doctor of Philosophy dissertation, University of Northern Colorado, 2011. Virtually nothing is known about the immunology and virus-host interactions of bats (order Chiroptera). The discovery of bats as reservoirs or potential reservoirs of many important human and veterinary pathogens such as ebolaviruses and Nipah and Hendra viruses, has recently sparked an interest in bat immunology. Bats may become persistently infected with viruses without signs of pathology, while human infections often lead to severe disease and death. It is unknown how this type of virus-host interaction is established in bats. This project began to examine the immune systems and virus-host interactions in two species of bats. Tacaribe virus was isolated from two species of Artibeus bats in the early 1960s and belongs to a group of arenaviruses that cause the South American hemorrhagic fevers. Tacaribe virus is not known to cause natural human infections. It would be highly unusual for Tacaribe virus to be hosted by Artibeus bats, as it would be the only arenavirus, for which the host is known, that does not have a rodent host. Discovery of the specific immune characteristics that allow bats to become persistently infected with many pathogens may offer medically important information for the treatment of human infections. Cytokine genes from Seba’s short- tailed fruit bat (Carollia perspicillata), were cloned, sequenced, and compared to orthologous mammalian sequences. It was hypothesized that bat cytokine genes are similar to the cytokine genes of other mammals. Jamaican fruit bats (Artibeus jamaicensis) were experimentally infected with Tacaribe virus and it was hypothesized that they would become persistently infected; however, this did not occur. These results iii indicate that bat cytokine genes are highly conserved with respect to other mammalian cytokine sequences, and that Jamaican fruit bats do not become persistently infected with Tacaribe virus. iv ACKNOWLEDGEMENTS I would like to thank the many people who have helped and supported me throughout the process of pursuing my doctoral degree. First and foremost, I would like to thank my research advisor, Dr. Tony Schountz, for giving me the opportunity to do this research project. He has been an amazing mentor and I truly appreciate his guidance. I also thank my doctoral committee members, Dr. Susan Keenan, Dr. Rick Adams, and Dr. Steven Pulos, who have offered their help and support. I am extremely grateful to Dr. Richard Bowen, Department of Biomedical Sciences, Colorado State University, for his veterinary work and collaboration with experimental infections. Dr. Mitchell McGlaughlin, UNC, has been very helpful in assisting with phylogenetic analysis of cytokine gene sequences and interpretation of those results. Dr. McGlaughlin is also acknowledged for his continuous help with primer design. I would also like to acknowledge Drs. Charles Calisher, Colorado State University, Katherine V. Holmes, Department of Microbiology, University of Colorado Health Sciences Center, Aurora, Colorado, and Robert B. Tesh, Department of Pathology, and Center for Tropical Diseases, UTMB, Galveston, Texas for providing advice, Tacaribe virus stocks, and Tacaribe-specific antibody. I also thank Dr. Charles Fulhorst (UTMB) and Dr. Eric Mossel (CSU) for helpful conversations and troubleshooting with the cloning and expression of the Tacaribe nucleocapsid gene. I would like to thank Jason Shaw for his help with the capture and transport of bats from UNC to CSU facilities. I would like to thank Stephanie James for her continuous moral support and her help with virus isolations, cell culture, and IHC on this v project. I would also like to acknowledge Sara Shields for assistance in preparing cytokine alignment figures. I would like to thank several undergraduate researchers, Shira Kedmi-Feinstein, Kenny Strine, and Benjamin Lee for their assistance on RNA extraction, and cloning and sequencing of bat immune genes. I would also like to acknowledge Jeret Benet, for her assistance in virus isolation imaging, and Christina Ndaluka for her assistance with bat care. I also thank the School of Biological Sciences at the University of Northern Colorado for providing me with a teaching assistantship while earning my doctoral degree. This project was supported by the University of Northern Colorado Research Enhancement and Development Initiative, National Institutes of Health (NIH) grant AI089419, NIH Emerging Virus Diseases Unit contract AI25489 (TS, RB, CHC) and NIH grant AI089419 (TS) . Finally, I would like to thank my family and friends for their continued love and support throughout this entire process. I am especially grateful to my wonderful husband, Klint Hawkinson, for being so patient and offering an enormous amount of love and support. Thank you, to my entire family for constantly believing me. vi TALBE OF CONTENTS CHAPTER I. INTRODUCTION…………………………………………………………..…….....1 SPECIFIC AIMS AND HYPOTHESES……………………………………...4 II. REVIEW OF LITERATURE…………………………………………..…………...7 GENERAL BAT ECOLOGY……………………….....……………...……….7 BAT IMMUNOLOGY………………………….……………………………….8 PERSISTENT INFECTIONS…………….…………………………………..10 METHODS USED TO EXAMINE PERSISTENT INFECTIONS…..……..13 IMMUNOLOGIC METHODS IN BATS………..……………………...…….18 CHARACTERIZATION OF BAT IMMUNE GENES AND ANTIBODIES……………….……………………..24 BATS AS RESERVOIRS……………………......…………………………..27 BACTERIA AND OTHER MICROBES…………………………………….37 ARENAVIRUSES…………………………………………………………….39 III. MOLECULAR AND PHYLOGENETIC CHARACTERIZATION OF CYTOKINE GENES FROM SEBA’S SHORT-TAILED FRUIT BAT (Carollia perspicillata)…………………………………………………...……...46 ABSTRACT……………………………………………………………………46 INTRODUCTION…………………………………...…………………………47 MATERIALS AND METHODS…………………………………………...…48 RESULTS……………………………………………………………………...52 DISCUSSION………………………………………………………………….56 vii IV. EXPERIMENTAL INFECTION SUGGESTS JAMAICAN FRUIT BATS (Artibeus jamaicensis) ARE NOT RESERVOIRS OF TACARIBE VIRUS…………………………………………………………….….67 ABSTRACT……………………………………………………………………67 INTRODUCTION……………………………………………………………...68 MATERIALS AND METHODS………………………………………………69 RESULTS……………………………………………………………...………76 DISCUSSION………………………………………………………………….79 V. WORK TOWARD THE DEVELOPMENT OF A RAPID FIELD IMMUNOASSAY FOR DETECTING ANTIBODY TO TACARIBE VIRUS IN MAMMALS…………………………………………………….………93 ABSTRACT……………………………………………………………………93 INTRODUCTION……………………………………………………………...94 MATERIALS AND METHODS……………………………………………...96 RESULTS……………………………………………………………………...99 DISCUSSION………………………………………………………………….99 VI. CONCLUSIONS………………………………………………………………….103 REFERENCES……………………………………………………………………………108 viii LIST OF FIGURES FIGURE 1. Amino acid alignment of TNF sequences………………………………...……..59 2. Amino acid alignment of IL-10 sequences……………………….………..…....60 3. Amino acid alignment of IL-23a sequences…………………….…………..…..61 4. Amino acid alignment of GM-CSF sequences………………………………….62 5. Most parsimonious phylogenetic tree reconstructions based on cytokine gene amino acid sequences……………………….…...…63 6. Tacaribe virus is lethal to Jamaican fruit bats…………………..………….….84 7. Weight changes after inoculation with Tacaribe virus………………….....…85 8. Histology of tissues from TCRV-infected bats in initial Experiment……………………………………………………………....……86 ix LIST OF TABLES TABLE 1. Primer sequences used to clone partial cytokine cDNAs from Seba’s short-tailed fruit bat…………………………………………64 2. Race primer sequences used to amplify 5’ cDNA regions of Seba’s short-tailed fruit bat cytokine genes……………………………64 3. Polypeptide accession numbers of sequences used from NCBI…………...65 4. Oral and rectal
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