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Evolution of Equine Arteritis Virus During Persistent Infection in the Reproductive Tract of the Stallion and the Male Donkey

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Evolution of Equine Arteritis Virus During Persistent Infection in the Reproductive Tract of the Stallion and the Male Donkey University of Kentucky UKnowledge Theses and Dissertations--Veterinary Science Veterinary Science 2017 EVOLUTION OF EQUINE ARTERITIS VIRUS DURING PERSISTENT INFECTION IN THE REPRODUCTIVE TRACT OF THE STALLION AND THE MALE DONKEY Bora Nam University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2017.499 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Nam, Bora, "EVOLUTION OF EQUINE ARTERITIS VIRUS DURING PERSISTENT INFECTION IN THE REPRODUCTIVE TRACT OF THE STALLION AND THE MALE DONKEY" (2017). Theses and Dissertations-- Veterinary Science. 34. https://uknowledge.uky.edu/gluck_etds/34 This Master's Thesis is brought to you for free and open access by the Veterinary Science at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Veterinary Science by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Bora Nam, Student Dr. Udeni B. R. Balasuriya, Major Professor Dr. Daniel Howe, Director of Graduate Studies EVOLUTION OF EQUINE ARTERITIS VIRUS DURING PERSISTENT INFECTION IN THE REPRODUCTIVE TRACT OF THE STALLION AND THE MALE DONKEY THESIS A thesis submitted in partial fulfillment of the Requirements for the degree of Master of Science in the College of Agriculture, Food and Environment at the University of Kentucky By Bora Nam Lexington, Kentucky Director: Dr. Udeni B. R. Balasuriya, Professor of Virology Lexington, Kentucky 2017 Copyright © Bora Nam 2017 ABSTRACT OF THESIS EVOLUTION OF EQUINE ARTERITIS VIRUS DURING PERSISTENT INFECTION IN THE REPRODUCTIVE TRACT OF THE STALLION AND THE MALE DONKEY Equine arteritis virus (EAV) establishes persistent infection in the stallion reproductive tract, and the carrier stallion continues to shed virus in semen for weeks to years or lifelong. The objective of this study was to elucidate the intra-host evolution of EAV during persistent infection in stallions. Seven EAV seronegative stallions were experimentally infected with EAV KY84 strain and followed for 726 days post-infection, and sequential clinical samples including semen were collected for virus isolation and next-generation sequencing (NGS). In addition, archived sequential semen samples from two stallions that were naturally infected with EAV KY84 for a long-period (up to 10 years) were also sequenced by NGS. The data demonstrated genetic bottleneck event and selection during acute infection followed by intra-host quasispecies diversification during persistent infection in the stallion reproductive tract. Also, the full-length genome of a novel EAV donkey strain from Chile and a noncytopathic bovine viral diarrhea virus-1 (ncpBVDV-1) strain contaminating rabbit kidney-13 cells were also sequenced by NGS. The EAV donkey strain was genetically distinct but antigenically cross-reacted with EAV antisera, and it was phylogenetically closely related to the South African donkey strain of EAV. Genetic and phylogenetic analyses demonstrated that ncpBVDV-1 belongs to BVDV-1b group. KEYWORDS: Equine arteritis virus, equine viral arteritis, persistent infection, intra-host evolution, quasispecies, bottleneck event Bora Nam December 8, 2017 EVOLUTION OF EQUINE ARTERITIS VIRUS DURING PERSISTENT INFECTION IN THE REPRODUCTIVE TRACT OF THE STALLION AND THE MALE DONKEY By Bora Nam Udeni B.R. Balasuriya Director of Thesis Daniel Howe Director of Graduate Studies December 8, 2017 DEDICATION To God for His unconditional love and mercy upon me. To my parents and American host families for their support and belief in me. To my friends for their encouragement. To my cat Leo for always standing by me. To the horses which sacrificed for my research. ACKNOWLEDGEMENTS When I arrived Kentucky in 2009 as a pre-vet student, I walked on the campus by myself and the final destination was the Maxwell H. Gluck Equine Research Center. At that time, I whispered in my heart saying "One day, I will work here." And the dream came true, except I did not expect to stay here for eight years. Mr. Gong Seoul, a previous undergraduate research assistant, introduced me to Dr. Udeni B. R. Balasuriya's molecular virology laboratory right before he left the laboratory after completing his undergraduate studies. Mr. Seoul turned over all the general laboratory maintenance and management work such as making cell culture medium. During eight years of working as an undergraduate research assistant, a senior laboratory technician, and a graduate research assistant, I had a great and happy time working with laboratory members involved in several research projects. I felt a value to me that I was able to support the laboratory. It is incredible how the relationship influenced my life and that was how I put my first step into Gluck. The completion of my Master's degree would not have been possible without the support, patience, belief, and encouragement of many people that I have met and worked with the past. It is my honor to acknowledge everyone to give my most profound sincere appreciation. I give all my sincere heart and most profound appreciation to Dr. Udeni B. R. Balasuriya for his abundant support, supervision, and patience through my Master's degree program. He opened the door for me to join his laboratory and taught me classical virology and molecular biology techniques (virus isolation, virus titration, nucleic acid extraction, standard and real-time RT-PCR and sequencing). During the Masters program, I have obtained a fruitful understanding of equine arteritis virus, critical thinking skills iii utilized in the research and the charm out of unlimited observations in the research field by looking over Dr. Balasuriya's ambition, passion and brilliant ideas on the research project. Not only is Dr. Balasuriya my advising professor, but he is also a great mentor, friend and sometimes like a father to me. He has shown patience, generosity and thoughtful consideration of myself and helped me to complete the degree and to move forward. I am most grateful to Dr. Peter J. Timoney for his continuous trust, belief, and encouragement. Whenever I fell, he grabbed my hand to stand up and still walk through the steps of the Master's program. He always believed in my talent, strength, and value and encouraged me to be motivated. He is one of the most beautiful gentlemen I have met; with humongous cheerful energy, he is the role model that I would like to be. His broad knowledge covering epidemiology, infectious disease and sincere care for equine health and well-being in the horse industry will continuously motivate me to seek and work related to it for the horse industry. I would like to express my sincere appreciation to Dr. R. Frank Cook for accepting being part of my Masters degree committee. Within a limited period, he took a closer look at my research projects and provided me with helpful discussion and valuable input. I give my special thanks to Dr. Daniel K. Howe, the Director of Graduate Studies, for always providing me his time, thoughtful guidance and good notice while going through the Master's program. Without his consideration, I would not have been able to complete the degree by this time. I would like to show my gratitude to Dr. Ganwu Li and Ms. Ying Zheng at the Department of Veterinary Diagnostic and Production Animal Medicine, College of iv Veterinary Medicine, Iowa State University for their excellent collaboration to perform next-generation sequencing of equine arteritis virus samples and allow me to visit their laboratory to learn the complex bioinformatics analysis, to set up my computer and to perform the command line coding method. I owe my most profound gratitude to Dr. Ernest Bailey for teaching me how much fun equine genetics is and reminding me that I can be a mad scientist. His joy, encouragement, and trust always cheered me up. I give my sincere gratitude to Dr. Eugene Lyons for a great friendship. He was the first person who took me over to necropsy to collect a parasite specimen. This is a great memory that I held my breath until the necropsy was over, but then screamed and cried after I saw a big cockroach in the laboratory. I express special gratitude to Dr. Thomas W. Swerczek for sharing his story of the research foundation on Woundade and in-depth consideration in equine health.
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