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Molecular and Genomic Approaches to Understanding Host-Virus Interactions in Shaping the Outcome of Equine Arteritis Virus Infection

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Molecular and Genomic Approaches to Understanding Host-Virus Interactions in Shaping the Outcome of Equine Arteritis Virus Infection University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2011 MOLECULAR AND GENOMIC APPROACHES TO UNDERSTANDING HOST-VIRUS INTERACTIONS IN SHAPING THE OUTCOME OF EQUINE ARTERITIS VIRUS INFECTION Yun Young Go University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Go, Yun Young, "MOLECULAR AND GENOMIC APPROACHES TO UNDERSTANDING HOST-VIRUS INTERACTIONS IN SHAPING THE OUTCOME OF EQUINE ARTERITIS VIRUS INFECTION" (2011). University of Kentucky Doctoral Dissertations. 840. https://uknowledge.uky.edu/gradschool_diss/840 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations 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 and attached hereto needed written permission statements(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). I hereby grant to The University of Kentucky and its agents the non-exclusive 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 a preapproved 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 dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Yun Young Go, Student Dr. Peter J. Timoney, Major Professor Daniel Howe, Ph.D., Director of Graduate Studies MOLECULAR AND GENOMIC APPROACHES TO UNDERSTANDING HOST- VIRUS INTERACTIONS IN SHAPING THE OUTCOME OF EQUINE ARTERITIS VIRUS INFECTION DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Yun Young Go Lexington, Kentucky Co-Directors: Dr. Peter J. Timoney, Professor of Veterinary Science and Dr. Udeni B.R. Balasuriya, Professor of Virology Lexington, Kentucky 2011 Copyright © Yun Young Go 2011 ABSTRACT OF DISSERTATION MOLECULAR AND GENOMIC APPROACHES TO UNDERSTANDING HOST- VIRUS INTERACTIONS IN SHAPING THE OUTCOME OF EQUINE ARTERITIS VIRUS INFECTION Equine arteritis virus (EAV) is the causal agent of equine viral arteritis, a disease of equids. During natural outbreaks of the disease, EAV can cause abortion in pregnant mares and persistent infection in stallions. Understanding how host cellular proteins interact with viral RNA and viral proteins, as well as their role in viral infection, will enable better characterization of the pathogenesis of EAV and establishment of persistent infection in stallions. Accordingly, we hypothesized that both viral factors and host genetically related factors could influence the outcome of EAV infection in horses. To test this hypothesis, we first combined contemporary molecular biology techniques with dual color flow cytometric analysis to characterize the interactions of viral structural proteins and the equine peripheral blood mononuclear cells in vitro. Results from this study demonstrated that interactions between GP2, GP3, GP4, GP5 and M envelope proteins of EAV play a major role in determining the CD14+ monocyte tropism while the tropism of CD3+ T lymphocytes is determined by GP2, GP4, GP5 and M envelope proteins but not the GP3 protein. Secondly, a genome wide association study using SNP genotyping identified a common haplotype associated with the in vitro CD3+ T lymphocyte/resistance to EAV infection among four breeds of horses. Subsequently, these studies were extended to establish a possible correlation between the in vitro susceptibility of CD3+ T lymphocytes to EAV and establishment of persistent infection in stallions. Interestingly, carrier stallions with susceptible CD3+ T lymphocyte phenotype to EAV may represent those at higher risk of becoming persistently infected. Finally, the precise effect of EAV on the immune system of horses, innate and humoral immunity, was studied. Horses were shown to mount a strong humoral antibody response to nonstructural proteins (nsps) 2, 4, 5 and 12 of EAV, whereas nsps 1, 2 and 11 suppressed the type I interferon production. The data presented in this dissertation suggest new directions for future EAV research using genomic and proteomic approaches to study host cell factors involved in EAV attachment and entry and establishment of persistent infection in the stallions. KEYWORDS: Equine arteritis virus, Equine viral arteritis, Susceptibility/resistance, Humoral immunity, Innate immunity Yun Young Go December 10, 2011 MOLECULAR AND GENOMIC APPROACHES TO UNDERSTANDING HOST- VIRUS INTERACTIONS IN SHAPING THE OUTCOME OF EQUINE ARTERITIS VIRUS INFECTION By Yun Young Go Peter J. Timoney Co-Director of Dissertation Udeni B.R. Balasuriya Co-Director of Dissertation Daniel Howe Director of Graduate Studies December 10, 2011 To my parents for their unconditional love and support ACKNOWLEDGMENTS The completion of my doctorate would not have been possible without the support, patience and guidance of many people that I have met and worked with during my graduate studies. It is a pleasure to convey my gratitude to everyone in my humble acknowledgment. I am heartily thankful to Dr. Peter J. Timoney for his advice, supervision, and generous support throughout my studies. He opened the doors for me to join the program and has since provided support and care for me the entire time. His encouragement inspired and enriched my growth as a student, a researcher and a scientist. I truly appreciate the years that I have spent under his guidance. I owe my deepest gratitude to Dr. Udeni B.R. Balasuriya for his advice and supervision, as well as giving me an extraordinary experience throughout my studies. I was fortunate to have such an excellent mentor. The true joy and passion he has for science was motivational and served as the driving force to keep up with my research even during difficult times in the Ph.D. pursuit. I am indebted for all his contributions of time, ideas and help to make my graduate experience productive and stimulating. I sincerely appreciate his friendship and the care he provided, sometimes like a brother and a father, in my personal life as well; I am indebted to him more than he knows. I would like to express my sincere gratitude to my Ph.D. dissertation committee members: Drs. David Horohov, Neil Williams, Donald Cohen and the outside examiner, Dr. Darrell Jennings, for their time, interest, helpful comments and insightful questions during the doctoral examination. The genome-wide association study discussed in this dissertation would not have been possible without the help of Dr. Ernest Bailey. I would like to thank him for kindly sparing his time to answer my naïve questions about genetics at any time. I thank Dr. Eric Snijder at Leiden University Medical Center, The Netherlands, for inviting us to collaborate on the study identifying a new open reading frame of EAV and for fruitful discussions. It is always an honor and pleasure working with him and his graduate/postdoctoral fellows. iii I would like to show my gratitude to Dr. Susan Wong at Wadsworth Center, New York State Department of Health, whose collaborative work on developing a fluorescent microsphere immunoassay was enjoyed very much and resulted in my very first publication. It was a favorable learning experience to realize what a good collaboration is. I gratefully thank Dr. Ying Fang and her graduate student, Yanhua Li, at South Dakota State University for performing luciferase expression assays for the EAV innate immune response study. I would also like to thank Dr. Dongwan Yoo, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, for inspirational discussions regarding these experiments. I thank Dr. Kuey-Chu Chen, Department of Molecular and Biomedical Pharmacology, University of Kentucky, for introducing me to the world of pathway analysis and allowing me to use the Ingenuity Pathway Analysis software for our genetic study. I have benefited from advice and fruitful ideas offered by Dr. Frank Cook who helped me in shaping up the research and writing. I would especially like to thank Dr. Daniel Howe for his support in the role of Director of Graduate Studies. I extend my appreciation to the faculty and staff in the deparment, particularly to the laboratories of Drs. Bailey, Chambers, Cook, Horohov, Howe, MacLeod and Lear for allowing me use their equipment and showing me how to operate them. I would also like to thank both past and present fellow graduate students, in particular, Debbie Cook for performing the SNP data analysis and Stephen Coleman for sharing the equine gene annotation data with us. I would like to express my special thanks to people in the EAV labs who supported me in many respects during the completion of my project. I am particularly indebted to Jianqiang Zhang, a master of cloning, who taught me how to make complicated recombinant viruses. I thank him for his contribution to various projects, being a good companion for long nights of experiments and most importantly his friendship.
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