TITLE “DETECTION OF INFLUENZA A VIRUSES FROM ENVIRONMENTAL LAKE AND POND ICE” Zeynep A. Koçer A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2010 Committee: Scott O. Rogers, Advisor W. Robert Midden Graduate Faculty Representative John Castello George Bullerjahn Paul Morris ii ABSTRACT Scott O. Rogers, Advisor Environmental ice is an ideal matrix for the long-term protection of organisms due to the limitation of degradative processes. As a result of global climate change, some glaciers and polar ice fields are melting at rapid rates. This process releases viable microorganisms that have been embedded in the ice, sometimes for millions of years. We propose that viral pathogens have adapted to being entrapped in ice, such that they are capable of infecting naïve hosts after melting from the ice. Temporal gene flow, which has been termed genome recycling (Rogers et al., 2004), may allow pathogens to infect large host populations rapidly. Accordingly, we hypothesize that viable influenza A virions are preserved in lake and pond ice. Our main objective was to identify influenza A (H1-H16) from the ice of a few lakes and ponds in Ohio that have high numbers of migratory and local waterfowl visiting the sites. We developed a set of hemagglutinin subtype-specific primers for use in four multiplex RT-PCR reactions. Model studies were developed by seeding environmental lake water samples in vitro with influenza A viruses and subjecting the seeded water to five freeze-thaw cycles at -20oC and -80oC. Results demonstrated that influenza A viruses survive freeze-thaw cycles at both temperatures with a high rate of viability. We then concentrated the meltwater samples from various locations and years by ultracentrifugation and tested for viable influenza A virions using SPF (specifically pathogen free) embryonated chicken egg cultures. Matrix and hemagglutinin genes of the viruses were detected by RT-PCR (reverse transcription-polymerase chain reaction) and sequencing after several passages depending on the starting concentration of the sample. We analyzed the phylogenetic relationships of the viral strains obtained from environmental ice samples with the known strains available in GenBank based on those two different viral genes. These confirmed iii the presence of specific subtypes of influenza A viruses. The increase in positive RT-PCR amplicons indicated that the viruses were replicating in the eggs. Our results demonstrated that viable and infectious influenza A virions are preserved in environmental lake and pond ice. iv To my parents, Necmi & Mücella Koçer; To my sister, Merve Koçer; To all musicians who kept me going during all these years; and To my friends, John Tallman & Christa L. Bowen, who could not complete their degrees due to unfortunate circumstances v ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Scott O. Rogers, with great appreciation for his endless support and patience; for trusting me to take this „dark-hole‟ project; for guiding and challenging me through my PhD; and for being a great mentor by encouraging me towards to the light at the end of tunnel. I would also like to thank him for being a best friend and a father to me. There are not enough words to mention my gratitude to you, Scott. Thank you for giving me this great opportunity to work with you. I would like to thank all my committee members Dr. John Castello, Dr. Paul Morris, Dr. George Bullerjahn, and Dr. Robert Midden for giving me insightful advices, for challenging me and for being very understanding. I would like to acknowledge Dr. Robert Webster and his colleagues for giving me a great opportunity to visit St. Jude Children‟s Research Hospital facilities and for teaching me how to perform viral culture technique and for sending me the samples I needed; Dr. Yoshihiro Kawaoka and Dr. Ron Fouchier for providing me samples. I would like to thank them all for giving me great advices, endless help and for making this project possible by sending me the positive viral samples used in this study. This work was partially supported by the National Health Institute. I would like to thank all my lab mates through these years: Seung-Geuk Shin for all his help whenever I needed, Lorena Harris and Farida Sidiq for being lifetime friends and sisters to me, Ram Veerapaneni, Tom D‟Elia, Gang Zhang, Yury Shtarkman, Xing Chen, Chia-Jui Tsai, Ameria Vicol, Justin Zollar, Amal Abu-Almakarem, and the undergraduates Katie Heilman and Clarissa Polen for their great friendship and support during rough days of PhD. Thanks to my dear friends, Mike Schlais and Tamera Wales, for always being there for me. I would also like to thank all my friends in Turkey for being supportive from across the ocean and the friends in US for making vi me feel like I am home here. Thanks to the Biology Department office staff Lorraine, Linda, Chris, Steve, Deb, Marsha, Sheila and Chellie for being very friendly and very helpful through my PhD years. Last but not the least, my special thanks to my dad, Necmi, for challenging me and encouraging me to go for my dreams; to my mom, Mücella, for always being there for me and for being a great mom and a best friend and for always helping me in anyway to make my dreams come true. I would love to explain my great appreciation to both my parents for teaching me to love, to be patient, to be understanding and especially to follow your dreams. My special thanks are also to my dearest sister, Merve, for being a great friend and a great listener through all these tough years; and to my other siblings Ilknur, Gonca and Ahmet for their endless support and faith in me. THANK YOU ALL SO MUCH!! vii TABLE OF CONTENTS CHAPTER 1. INTRODUCTION ................................................................................................................. 1 1.1. Genome of influenza A viruses ..................................................................................................... 1 1.2. Genetic events leading to variations, selective pressures and evolution of influenza A ............... 4 1.2.1. Emergence of highly pathogenic avian influenza (HPAI) strains ......................................... 8 1.3. Host range of avian influenza ....................................................................................................... 9 1.4. Rationale for this dissertation ..................................................................................................... 11 1.5. Objectives ................................................................................................................................... 12 1.6. Literature cited ............................................................................................................................ 13 CHAPTER 2. PRIMER DEVELOPMENT FOR THE RAPID DETECTION OF HEMAGGLUTININ SUBTYPES OF INFLUENZA A VIRUSES FROM ENVIRONMENTAL SAMPLES ........................... 16 1. Introduction ..................................................................................................................................... 16 2. Methods........................................................................................................................................... 18 2.1. Primer Design ......................................................................................................................... 18 2.2. Sensitivity of the primers ........................................................................................................ 19 2.3. Testing for the cross-reactivity ............................................................................................... 20 2.4. Development of multiplex RT-PCR conditions ...................................................................... 20 3. Results and Discussion ................................................................................................................... 23 3.1. Primer Design ......................................................................................................................... 23 3.2. Sensitivity of the primers ........................................................................................................ 29 3.3. Testing for the cross-reactivity ............................................................................................... 29 3.4. Development of multiplex RT-PCR conditions ...................................................................... 30 4. Literature cited ................................................................................................................................ 34 CHAPTER 3. EFFECTS OF FREEZING AND THAWING ON THE VIABILITY OF INFLUENZA A VIRUSES .................................................................................................................................................... 36 1. Abstract ............................................................................................................................................... 36 viii 2. Introduction ......................................................................................................................................... 36 3. Materials and Methods ........................................................................................................................ 39 3.1. Viral strains .................................................................................................................................