A Thesis Submitted in Partial Fulfilment of the Requirements of Kingston University for the Degree of Doctor of Philosophy Evaluating Risks of Paramyxovirus and Coronavirus Emergence in China Aleksei Avery CHMURA PhD March 2017 Kingston University School of Life Sciences Faculty of Science, Engineering and Computing Kingston-upon-Thames KT1 2EE UK & EcoHealth Alliance 460 West 34th Street New York, NY 10001 USA DECLARATION I hereby declare that whilst registered for a research degree at Kingston University London I have not been a registered candidate or an enrolled student at any other academic or professional institution. I declare that the materials contained in this thesis have not been used in any other submission for an academic award. All the sources of investigation have been duly acknowledged. I have composed this thesis myself and all results presented in this thesis are from my own investigations. Aleksei Chmura 31 March 2017 i ABSTRACT Viruses such HIV, SARS Coronavirus, Ebola, and influenza A virus pose significant burdens globally to human health due to their continued emergence from wildlife reservoirs such as birds, bats, and rodents. In southern China, animal markets, wildlife trade, and human activity create unique opportunities for zoonotic emergence as wild animals frequently come into contact with domestic animals and humans. Influenza A H7N9 virus and SARS emerged from live and wild animal markets in south China where frequent mixing and high volume of species enabled rapid viral evolution and emergence. This research aims to examine zoonotic viral emergence by testing the following hypotheses: • there are novel Coronaviruses (CoVs) and Paramyxoviruses (PMVs) in wildlife reservoirs in south China • human behaviour may result in viral spillover from wildlife reservoirs • market and other wildlife trade activities may be drivers of disease emergence • CoVs and PMVs circulating in wild animal populations may be closely related with or ancestral to known pathogenic viruses. Over a five-year period, oral and anal swabs and blood samples were collected from 3,146 wild- caught bats and 559 rodents from more than 30 different species primarily focused on wildlife trade pathways in southern China. RT-PCR assays were performed to screen for CoVs and PMVs. 39 CoVs (1.2%) and 114 PMVs (3.6%) were confirmed from bat samples by sequencing. Of the latter, 80 were novel bat PMVs. No rodent samples were positive for either virus. An online survey was conducted to assess 2,238 Chinese millennials’ attitudes about wildlife consumption and perceived health-risks. The results suggest that although this population is currently the primary driver of demand for wildlife trade in China, it may also be the most effectively targeted with campaigns to educate about zoonotic emergence from wildlife reservoirs. Diverse mammalian wildlife species including two listed by IUCN as vulnerable and one on China’s endangered species list were observed to be maintained in circulation over three years in two of the largest live animal wildlife markets in south China. An overall increase in the volume of wildlife traded in the two markets was also observed. Through 87 ethnographic interviews and 685 structured interviews with rural residents observed to be exposed at some level to wildlife, strong evidence was provided that local consumption of wildlife has reduced, but exposure and awareness ii of the commensurate health-risks have not. Additionally, exposure to bats, poultry, and rodents as well as handling, hunting and raising animals were all activities positively correlated with self- reported symptoms of viral infections of unknown aetiology and potentially of zoonotic pathogens. The information garnered in this study about the current status of wildlife trade, people’s attitudes and actions, as well as the ecology of these viruses and their hosts in south China, provides data that may be used towards predicting and preventing emergence of these and other as-yet-unknown viruses. If patterns of human behaviour, wildlife trade, and viral ecology may be quantified, then a relative level of risk may be predicted and evaluated. iii ACKNOWLEDGMENTS This study was funded by the American people through the United States Agency for International Development (USAID) Emerging Pandemic Threats PREDICT project (Cooperative Agreement no. AID- OAA-A-14-00102) and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (Award Number R01AI110964) and core funding to EcoHealth Alliance in New York City. Many thanks to my Director of Studies, Prof. Mark D. Fielder and Co-Supervisor Dr. Peter Daszak for providing key oversight and guidance throughout the course of the entailed research and pushing me further than I thought possible. Although untimely abbreviated, the steady, patient, and calm direction and support of Prof. Angela Davies-Russell was an inspiration. The scope of this thesis was extensive with national and international collaborations making much of it possible. These partnerships were entered into with the utmost professional courtesy and respect and are a true testament to the dedication of each party to scientific discovery. Sincere thanks to everyone who made this thesis a reality. Much gratitude to Dr. Guangjian Zhu, Dr. Libiao Zhang, Dr. Yunzhi Zhang, Dr. Jinshuo Zhang, Jian Yang, Min Tan, Jianping Ye, and others in my field team for assistance with sample collection. Thanks also to Dr. Zhengli Shi, Dr. Xingyi Ge, Dr. Xinglou Yang, Dr. Peng Zhou, Dr. Ben Hu, Dr. Yan Zhu, Dr. Shiyue Li, Bei Ke, Huimin Huang, and others for laboratory training and assistance. Additional thanks to the laboratories at Wuhan Institute of Virology, Shanghai East China Normal University, and the Yunnan Center for Disease Control and Prevention for diagnostic testing of samples and to the Wuhan School of Public Health for logistics support for collection of human behavioural samples. Recognition to Hongying Li and Emily Hagan for providing sources of data and support with surveys. Also, thanks to Dr. Simon J. Anthony and Dr. Scott P. Lawton who provided much patient training and to Dr. Kevin J. Olival, Carlos Zambrana-Torrelio, and Alice Latinne who provided endless consultation and answers to my questions about genetics and phylogenetics. Collaborations were made possible through the generous support of Dr. Peter Daszak and EcoHealth Alliance. iv Alison Andre’s unconditional and unflappable assistance permitted me the time and space to achieve this milestone. Catherine Machalaba’s enthusiasm, cheer, and focus helped and buoyed me in untold ways during overnight paper writing sessions at the office. My sincere gratitude to the following members of my family who provided unflagging and unparalleled love, help, and support: Evelyn, Greg, Daniella, Alex, and Sofia; Gitanjali, Kaustav, Ameya, and Anoushka; Nok; Juliana and Juliana; Peter, Janet, Francesca, and Imogen; Yingying, Yumo; and Sidney. Finally, I would also like to extend my deepest love and gratitude to my incredible husband Ferry Widijanto, who has been throughout my greatest source of support, daily inspiration, patience, humor, and encouragement and without whom I would not have achieved this work. v TABLE OF CONTENTS DECLARATION ...........................................................................................................................................I ABSTRACT.................................................................................................................................................II ACKNOWLEDGMENTS ............................................................................................................................ IV TABLE OF CONTENTS .............................................................................................................................. VI LIST OF TABLES ....................................................................................................................................... X LIST OF FIGURES ..................................................................................................................................... XI LIST OF ABBREVIATIONS........................................................................................................................ XII CHAPTER 1: INTRODUCTION ....................................................................................................................1 1.1 EMERGING INFECTIOUS DISEASES ............................................................................................................2 1.2 ECONOMIC IMPACT OF EMERGING INFECTIOUS DISEASES............................................................................3 1.3 ZOONOSES, PUBLIC HEALTH, AND SECURITY .............................................................................................5 1.4 DRIVERS OF ZOONOTIC EMERGENCE ........................................................................................................6 1.5 ECOLOGY OF DISEASE EMERGENCE ..........................................................................................................8 1.6 ZOONOSES IN CHINA .......................................................................................................................... 10 1.7 CORONAVIRUSES ............................................................................................................................... 11 1.8 PARAMYXOVIRUSES ..........................................................................................................................