WILD AQUATIC BIRD AVIAN INFLUENZA VIRUS INFECTIONS IN MAMMALS: A POTENTIAL ZOONOTIC THREAT? by ELIZABETH ANN DRISKELL (Under the Direction of Elizabeth Howerth) ABSTRACT Numerous cases of direct transmission of avian H5, H7, and H9 influenza viruses from birds to humans have stimulated concern regarding a potential avian influenza pandemic. The purpose of this research is to evaluate the potential of avian influenza viruses (AIVs) from wild aquatic birds to replicate in mammals with the greater goal of assessing indirect or direct AIV transmission risk to humans. Four hundred nineteen AIV isolates from the reservoir hosts, waterfowl and shorebirds, were examined in vitro via plaquing in Madin-Darby canine kidney (MDCK) cells. Of these isolates, 93% replicated in MDCK cells and 28% of these isolates plaqued without the addition of trypsin. Twenty eight of these isolates were further examined in vivo in a BALB/c mouse model. The ability to replicate in the lower respiratory tract (lungs) of mice was highly variable and isolate dependent; however, many isolates were able to replicate to high titers in the mouse lung and induce pulmonary pathology in the absence of overt clinical disease. Two isolates that exhibited robust replication in the mouse lung were further characterized in ferrets. Both of these isolates resulted in upper and lower respiratory tract infection with nasal shedding, respiratory pathology, and seroconversion with minimal clinical disease. One of these isolates was also able to transmit from infected to naïve ferrets via direct contact. The two isolates examined in ferrets were also examined in domestic cats. Experimental infection of domestic cats with these two different shorebird AIVs did not result in disease, led to variable pharyngeal viral shedding with one of the viruses, and resulted in low levels of seroconversion of all cats. Taken together, these results indicate that wild bird influenza viruses have the ability to infect mammals without adaptation in domestic birds or other mammals; although the ability to infect mammals appears to be extremely variable based on the isolate and productive infections and transmission would likely be dependent on the magnitude of viral exposure and resultant shedding. Not only was the capacity for direct transmission demonstrated, but such events in domestic cats could lead to adaptation to or reassortment in this species. INDEX WORDS: Avian influenza virus, Cat, Ferret, Infectivity, Mammals, Mouse, Pathogenicity, Wild bird WILD AQUATIC BIRD AVIAN INFLUENZA VIRUS INFECTIONS IN MAMMALS: A POTENTIAL ZOONOTIC THREAT? by ELIZABETH ANN DRISKELL BS, Truman State University, 2001 DVM, Iowa State University, 2005 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2011 © 2011 Elizabeth Ann Driskell All Rights Reserved WILD AQUATIC BIRD AVIAN INFLUENZA VIRUS INFECTIONS IN MAMMALS: A POTENTIAL ZOONOTIC THREAT? by ELIZABETH ANN DRISKELL Major Professor: Elizabeth Howerth Committee: S. Mark Tompkins David Stallknecht Elizabeth Uhl David Swayne Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2011 DEDICATION For Jeremy. My husband, my best friend, my rock. All of my crazy ideas seem possible with you. i ACKNOWLEDGEMENTS Summing up thanks for years and years of support and inspiration in education in a few pages is pretty impossible. But here it goes… Thanks to my graduate committee. To Dr. Buffy Howerth, first and foremost, who everyone, including me, is in complete awe of. And rightfully so. You are an amazing pathologist, scientist, and mentor. The most amazing thing about you is your unending selfless support of us. Thanks for the open door; weekdays, weekends, and evenings. You have mastered more pathology than I can even dream to see. To Dr. Betsy Uhl, who shared great wisdom with me on a practical approach to balancing diagnostics, research, and teaching without compromising a gold standard. Thanks for the open door when I was frustrated or to just chat about art and life! To Dr. Mark Tompkins, who always had confidence in me and was brave enough to let me take the reins but always had an open chair on the other side of his desk for me to discuss plans and ideas. To Drs. David Stallknecht and David Swayne, who provided me with insight on the nature of avian influenza in wild birds and poultry and always asked me questions that got me thinking. Heartfelt thanks to all the residents of the UGA Pathology Department, past and present. No one can survive a combined pathology residency and PhD without co-suffering friends for support. To Drs. Holly and Justin Brown, great friends who were there when I needed a shoulder to cry on or an escape to the beach! To Dr. Julie Webb who helped me find humor in everything. Special emotional thanks to Drs. Melinda Camus, Rita McManamon, and Janildo Reis who helped me survive studying for boards. Melinda, I couldn’t have made it through gen path ii without you. Rita, your insight and optimism was crucial to prevent a breakdown. Finally, Janildo, I simply wouldn’t have passed without those countless hours we put in at Jittery Joe’s as I forced us through vet path and you quizzed me on gross path. Thanks for helping me to stay on task, keeping me from going crazy, and for introducing me to caipirinha! To all the other residents old and new…Raquel, Tomislav, Tim, Parag, Susan, Dave, Daniel, Sasmita, Leo, Shawn, Tory, Bala, Nicole, Monique, Karelma, Paola, Sabrina...though an ever-changing group, it has always been a great group of supportive friends and amazing people. Thanks to the entire UGA Pathology Department and Athens Veterinary Diagnostic Lab. From pathologists to support staff, I am the pathologist, scientist, and teacher today because I have been able to learn valuable things from all of you. Especially thanks to Dr. Harris, who is a magnificent leader and supporter, accessible to all no matter where you rank in the department. Big thanks to Glen and Bryan, necropsy couldn’t run without you and is a great place to be for residents and students because of you…thanks for always being willing to go bucket diving for me! Also special thanks to the histopathology laboratory, always willing to go the extra step with a smile so we have perfect slides. Thanks to Ginger and Becky at SCWDS who helped me master the egg. To animal resources who gave the countless mice, ferrets, and cats I used top notch care and kindness. To Merial for funding this position in the pathology department; it has been one of the best experiences of my life made possible by you. Also thanks to the CDC for funding this fantastic research project. Thanks to all my previous mentors, especially Dr. Janick-Buckner at Truman State University, had I not wanted to be a veterinarian I would have wanted to be an immunologist because of you. A big thanks to Dr. Julia Ridpath at the NADC who convinced me that I should iii do research; you are the most warm, inspiring, graceful mentor I have encountered, I would like to be just like you when I grow up! Thanks to my family, who are a never ending source of support no matter what I choose for my life. Mom and Dad, you help me ponder the right questions so I can find my path. Kelley, you help me escape the grind. To Meme and Papa, my surrogate Mom and Dad, who are a never- ending source of love and support. To my in-laws, Dan, Gerry, and Blake, how did I get so lucky to have a second set of parents and a brother that are so supportive and proud? To my son, Liam. Thanks for sharing your mom’s time with her work. You are a daily reminder of what’s important in life; your delight in living is a fresh breath of air when coming home from a day of frantic diagnostic work or unsuccessful experiments. To Jeremy, who I’d say was as close to a saint as is possible. When I decided to not only do a residency but also a PhD nearly right after vet school, you simply said “OK, if this is what you need to do.” Your patience, love and support sustain me. Thanks may cover the time you spent editing my papers, serving as my sounding board for work, covering at home when I studied for boards and even pipetting in the hood for me. But it doesn’t even come close for everything else. Don’t worry, I think I’ve run out of degrees to earn. I’m not interested in law school. Yet. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................................ ii LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION .........................................................................................................1 LITERATURE CITED ............................................................................................3 2 LITERATURE REVIEW ..............................................................................................7 INFLUENZA A: A VIRUS LOOKING FOR OPPORTUNITY ............................7 THE ECOLOGY OF AVIAN INFLUENZA ..........................................................9 THE EPIDEMIOLOGY OF INFLUENZA IN HUMANS ...................................11 DETERMINANTS OF INFLUENZA PATHOGENICITY AND HOST RANGE ......................................................................................................12