F'IELD AND LABORATORY BASED STUDIES ON THE TRANSMISSION AND PREVENTION OF NEW WORLD HANTAVIRUSES BY DAVID J. SAFRONETZ A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Medical Microbiology University of Manitoba Wiruripeg, Manitoba @ David J. Safronetz, 2008 THE UNIVERSITY OF MANITOBA FACULTY OF GÌ'A-DUA,TE STUDIES COPYRIGHT PERMISSION Field and Laboratory Based studies on the Transmission and prevention of New World Hantaviruses BY David Safronetz A ThesisÆracticum submitted to the Faculty of Graduate studies of rhe university of Manifoba in partial fulfillment of the requirement of the degree of Doctor of Philosophy David Safronetz O 2008 Permission has been granted to the university of Manitoba Libraries to lend a copy of this thesis/practicum, to Library and Archives canada (LAC) to tend a copy of ihis thesis/practicum, and to LAC's agent (uMlÆroeuest) to microfilm, sell copies and to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made available by authority of the copyright orvner solely for the purpose of private study and research, and may onty be reproduced and copied as permitted by copyright larvs or rvith ,"iitt.n- authorization from the copyright orvner. "*p..r, ABSTRACT The Bunyaviridae is a lalge gloup ofviruses which contains several viruses of medical importance, the majority of which are transmitted to humans by arthlopod vectors. The exception are the members of the genus -É1a ntavbus which are maintained in nature and transmitted to humans by rodent reservoirs. Unique hantavirus species have been documented essentially worldwide and are associated with two distinct diseases in humans, hemorrhagic fevel with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Cunently, treatment and prevention options for HFRS and HPS are limited and mainly consist of education programs aimed at reducing contact with rodents and their excreta. The aim ofthe studies presented here was to investigate new strategies for preventing disease in humans through i). improved understanding of viral transmission from naturally infected rodents and ii). evaluating novel vaccine candidates in a lethal HPS disease model. In an attempt to elucidate pattems of infection and transmission in rodents, field studies were conducted in southern Manitoba between August 2003 and October 2005. The findings ofthese studies provided new insight into potential mechanisms of transmission in the natural setting with respect to routes and timing ofvirus transmission in relation to the stages ofinfection and confirmed the existence ofseveral pattems observed in studies conducted elsewhere (i.e., systemic infection in rodents, age, sex and seasonal bias ofinfection). Infection status in mice was determined using serological and molecular methods. To differentiate recently infected rodents from those infected in the distant past an antibody avidity assay was developed and determined tobe 89.5%o accurate in identif,ing recently (within 30 days) infected mice based on the presence of low avidity antibodies. Initial application ofthe avidity assay revealed a greater proportion of mice with low avidity antibodies had detectable viral RNA in oropharyngeal fluid (OPF) or urine samples compared with those from mice with high avidity antibody (21% versus 6.8%), suggesting recently infected mice are more likely to transmit virus via these routes. In contrast, no diffelence in the detection ofviral RNA in whole blood samples between the low and high avidity groups was observed. Retrospective analysis of samples collected as a part ofa pilot in August 2003 demonstrated similar pattems. These data suggests, recently infected mice represent a greater risk to humans acquiring infection, however based on the low rate ofdetection of viral RNA in OPF and urine samples compared with that of blood, viral transmission among rodents may frequently occur via the blood-borne route, presumably as a part of wound to wound contact associated with aggressive behaviors. The long-term goal for the prevention ofhantaviral disease in humans is the development of effective vaccines capable of inducing complete, sterile immunity in naiVe individuals. Cunently no specific vaccine exists for HPS, and the study ofthe protective immune response has been hampered by the lack of animal models which reflect disease progression in humans. The recently described lethal hamster model of HPS was evaluated for the study of Andes virus (ANDV) induced HPS and found to be an accurate disease model which was 100% lethal within approximately 10-11 days and demonstrated several features of disease in humans including symptomolgy and pathophysiology. Two, recombinant, replication deficient, viral vector platforms (Vesicular stomatitis virus pseudo-particles bearing the ANDV glycoproteins, VSV^G*AND-GPC, and Adenovil'us constructs, recAd, individually expressing ANDV G¡, G6 orN) were evaluated as potential vaccines in the hamster model. Immunization with VSVÂG*AND-GPC was protective in approximately 50% of immunized hamsters, however survivors had detectable N antibody titers, demonstrating seroconversion and indicating the presence of replicating ANDV. By contrast, immunization with any of the recAd vectors either alone, or in combination, was 100% protective from ANDV challenge. Based on low titers ofneutralizing serum antibodies post-immunization, the protective immune response associated with the recAd vectors was likely due to a potent cellular immune response. Unlike with VSV^G*AND-GPC, complete, sterile immunity was achieved in hamsters which received both the Gn and Gc expressing recAd vectors as determined by the lack of N specific antibodies, as well as no detectable viral RNA in tissue samples al6 andg days post-challenge. The recAd vectors are useful tools for dissecting the protective immune response and wan'ant further investigation as vaccine candidates for HPS and HFRS. n AcKNoWLEDGMENTS "Knowledge is in the end based on acknowledgement." - Ludwig Wittgenstein First and foremost, I'd like to thank my family and friends for their continued support during my PhD research. I am indebted to the staff members of the NML and the Department of Medical Microbiology at the University of Manitoba who have taken time to assist me in various aspects of my research. Over the last few years I have been privileged to work with numerous people in the Zoonotic Diseases and Special Pathogens department, all of which had a hand in the work presented here. My field studies projects could not have been completed without the expertise ofDr. Robbin Lindsay. I would also like to acknowledge the help of Tyler Cote and Kai Makowski with the fieldwork and Dr'. Brian Hjelle and his laboratory (Jniversity ofNew Mexico) for numerous discussions and for providing the timed sera samples necessary for validating the avidity assay. A special debt of gratitude is also owed to the individuals who allowed me to collect deer mice fi'om their private land. I'd like to thank Dr. Heinz Feldmann for allowing me the opporlunity to carry out research in a BSL 4 laboratory as well as Daryl Dick, Ricki Feldmann, Shane Jones, and Allen Grolla for training me to do so. Hamster work was assisted by helpful discussions and emails with Drs. Jay Hooper and Victoria Wahl-Jenson (USAMRIID) and assistance from Jason Glen. The histological analysis would not have been possible without the expertise of Catherine Roberstson (NML) and Don Gardner (Rocky Mountain Laboratory). Also, I would be amiss without specifrcally acknowledging Dt. Gary Kobinger and Michael Gray (ltlMl-) for their advice and assistance with the vaccine studies, Dr. David Johnson (Oregon Health and Science University) for providing the Adenovirus constructs and the many intellectual contributions of Dr. Hideki Ebihara (University of Tokyo /NML), who for an Ebola guy knows an awfül lot about hantaviruses! During the course ofthese studies I was fortunate to be supported by studentships from the Manitoba Health Research Council and the Canadian Institutes of Health Research/lntemational Center fro Infectious Disease /lJniversity of Manitoba training program in infectious diseases with additional travel suppoú from two Faculty of Graduate studies Student travel bursaries. Finally I'd like to thank my committee members, Drs. Harvey Artsob, Michael Drebot, Heinz Feldmann, Dan Gietz, and Robbin Lindsay, for their advice and guidance over the last few years, and Dr. Corurie Schmaljohn, for providing her expert opinion as the external thesis reviewer. TABLE oF CoNTENTS Abstract... ... I Acknowledgements.............,, IV Table of Contents. ..... ... ..... VI List ofFigures.. X List of Tables... XII List of Copyrighted materials..... XIII List of Abbreviations... ..... ... XIV Chapter I: Literature Review 1. Introduction 1.1 Briefhistory 1 i.2 Taxonomy... 2 1.3 Distribution. 5 2. Hantavirus Biology 2.i Genomic organization,.. 5 2.2Yiral lifecycle...... ... 11 3. Ecology ofHantaviruses in Nature 3.1 Maintenance 15 3,2 Hantavilus infections in the natural host... ...... ...... 16 3.3 Virustransmissionbetweenrodents............ 19 4. Human Disease 4.1 Epidemio1ogy.................. 24 4.1.2 Hantavirusdisease inCanada....................... 26 4.2 Transmission to humans... .. 28 4.3 Disease...... 29 4.3.1 Hemonhagic fever with renal syndrome.......... 29 4.3.2 Hantavirus pulmonary syndrome........ 31