DETECTION AND MOLECULAR CHARACTERIZATION OF PORCINE NOROVIRUSES AND SAPOVIRUSES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Qiuhong Wang, Bachelor of Medicine, M.S. * * * * * The Ohio State University 2005 Dissertation Committee: Approved by Distinguished University Professor Linda J. Saif, Adviser Assistant Professor Julie A. Funk ____________________________ Adviser Professor John H. Hughes Graduate Program in Assistant Professor Jeffrey T. LeJeune Veterinary Preventive Medicine ABSTRACT Enteric caliciviruses are emerging pathogens that cause gastroenteritis in humans and animals. They comprise 2 genera, Norovirus and Sapovirus in the family Caliciviridae. Human noroviruses (NoV) infect people of all ages. They cause more than 90% of nonbacterial gastroenteritis outbreaks worldwide and have been listed as class B biological pathogens by the National Institutes of Health/Biodefense Program. Human sapoviruses (SaV) are associated with 1.8-9% of pediatric gastroenteritis worldwide. Whether NoVs and SaVs are zoonotic pathogens is unclear due to limited studies of animal NoVs and SaVs. However, the classic calicivirus, vesiviruses cause cross-species infections and infect marine mammals, cats, dogs, cattle and pigs. Bovine NoVs may infect humans based on a bovine NoV seroprevalence study of veterinarians in contact with cattle. Compared to cattle, pigs have a gastrointestinal tract more similar to that of humans in anatomy and physiology. Compared to bovine NoVs, porcine NoVs are genetically and antigenically more closely related to human NoVs. These data raise concerns of whether pigs may be reservoirs for the emergence of human NoVs. For animal SaVs, only 2 genetically similar porcine SaV strains and one mink SaV strain have been reported. The porcine SaV prototype Cowden strain was isolated from a diarrheic pig. It causes diarrhea and intestinal lesions in gnotobiotic (Gn) pigs. To date, no prevalence study of porcine SaVs has been reported. The main objectives of this dissertation were: 1) to investigate the genetic ii diversity of porcine NoVs and their relationship to human NoVs; 2) to investigate the genetic diversity of porcine SaVs and their relationship to human SaVs; 3) to develop sensitive and specific methods for detection of porcine NoVs and SaVs; and 4) to study the prevalence of porcine NoVs and SaVs in US swine. We screened for porcine NoVs by reverse transcription-PCR (RT-PCR) using calicivirus universal primers on 275 fecal samples from normal US adult pigs. Six samples were confirmed NoV positive by sequencing. Based on sequence analysis of the 3 kb of the genomes of 5 porcine NoVs, 3 genotypes [2 new (GII-18 and 19) and 1 confirmed (GII-11)] within GII and two potential recombinant strains were identified. One genotype (GII-18) of porcine NoVs was genetically and antigenically related to human NoVs and replicated in gnotobiotic pigs. By similar approaches, porcine SaVs were identified as genetically diverse viruses comprising at least 2 genogroups, one previously classified (GIII) and one potentially new genotype (GVI?). Two porcine SaVs were potential recombinants. One porcine SaV strain (Po/SaV/MI-QW19/2002/US) was genetically most closely related to human SaVs based on the partial RdRp sequence (286 nt). Based on the obtained sequences of porcine NoVs and SaVs, several primer pairs were designed and evaluated for the detection of porcine NoVs and SaVs, respectively. An internal control RNA was developed and used in RT-PCR to monitor for RT-PCR inhibition. A 10-fold dilution of sample RNA containing RT-PCR inhibitors usually no longer interfered in the RT-PCR reaction. Microwell hybridization assays were developed to confirm the porcine NoV- and SaV-specific amplicons. A prevalence study of porcine NoVs and SaVs in US swine was performed by the newly developed RT-PCR-hybridization assays. The porcine NoVs were detected iii exclusively from finisher pigs in 4 of 7 farms and 1 slaughterhouse with an overall prevalence of 20% in finisher pigs (range of 3-40% in the positive farms). Porcine SaVs were detected from all ages of pigs. The prevalence of porcine SaVs was 62% overall, lowest in nursing pigs (21%) and highest in post-weaning pigs (83%), and varied from 37 to 100% depending on the farm. Mixed infections of NoVs and SaVs were common in finisher pigs with an overall prevalence of 27% among NoV- or SaV-positive pigs. These findings have improved our understanding of the genetic diversity of porcine NoVs and SaVs and their relationships to human strains. Certain porcine NoVs and SaVs are genetically or antigenically related to human strains. This finding and the high prevalence of NoVs and SaVs in subclinically infected swine including their detection from slaughterhouse pigs increase the risks that pigs may be reservoirs for human NoV and SaV strains. iv Dedicated to my husband Hong Liu, son Hengyu, my parents Xushong Wang and Jie Gao, and my parents-in-law Jingqin Liu and Yongbi Tang v ACKNOWLEDGMENTS Grateful appreciation is due to my adviser, Dr Linda J. Saif, for her encouragement, challenge, guidance and support throughout my years of study. I have learned substantially from her unwavering emphasis on research quality and unlimited patience during my studies and during the preparation of manuscripts and this dissertation. I would also like to thank my current committee members, Dr. Julie A. Funk, who collected most of the field fecal samples for my studies, Dr. John H. Hughes and Dr. Jeffrey T. JeJeune, and my prior committee members (prior to their leaving OSU) Dr Srinand Sreevatsan and Dr. Qijing Zhang, for their constructive comments, advice and encouragement throughout my studies. Special thanks are due to all my colleagues working on enteric caliciviruses, Dr. Keong-Ok Chang, Dr. Mingzhang Guo, Dr. Myung Guk Han, Menira Souza, Dr. Sonia Cheetham, Veronica Costantini and Dr. Christopher Thomas for their sharing experience and knowledge, their technical assitance and their friendship throughout the years of my study. I also thank Peggy Lewis, Paul Nielsen, Wei Zhang and Ana Azevedo for their technical assistance. Many thanks are due to Dr. Marli Azevedo, Dr. Yuxin Tang, Dr. Lijuan Yuan, Dr. Kwang-il Jeong, Trang Van Nguyen and Dr. Ana Gonzalez, whose friendship has helped me through difficult times. vi I want to thank Dr. Julliete Hanson, Richard McCormick, Janette McCormick, Todd Root, Greg Meyers and Don Westfall, for technical assistance and commitment. Special thanks are to Hannah Gehman and Robin Weimer for indispensable support. vii VITA September 3, 1969 Born – Beijing, China 1989 - 1994 Bachelor of Medicine Beijing Medical University, Beijing, China 1994 -1998 Research staff Institute of Virology Chinese Academy of Preventive Medicine, Beijing, China 1998 - 2000 Master of Health Sciences The University of Tokyo, Tokyo, Japan 2000 – present Graduate Research Associate Food Animal Health Research Program Ohio Agricultural Research and Development Center Department of Veterinary Preventive Medicine The Ohio State University Wooster, Ohio PUBLICATIONS Research Publication 1. Chang, K. O., S. S. Sosnovtsev, G. Belliot, Q. Wang, L. J. Saif, and K. Y. Green. 2005. Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae. J Virol 79:1409-16. 2. Han, M. G., Q. Wang, J. R. Smiley, K. O. Chang, and L. J. Saif. 2005. Self-assembly of the recombinant capsid protein of a bovine norovirus (BoNV) into virus-like particles and evaluation of cross-reactivity of BoNV with human noroviruses. J Clin Microbiol 43:778-85. 3. Tang, Y., Q. Wang, and Y. M. Saif. 2005. Development of a ssRNA internal control template reagent for a multiplex RT-PCR to detect turkey astroviruses. J Virol Methods 126:81-6. viii 4. Wang, Q. H., J. Kakizawa, L. Y. Wen, M. Shimizu, O. Nishio, Z. Y. Fang, and H. Ushijima. 2001. Genetic analysis of the capsid region of astroviruses. J Med Virol 64:245-55. 5. Sakamoto, T., H. Negishi, Q. H. Wang, S. Akihara, B. Kim, S. Nishimura, K. Kaneshi, S. Nakaya, Y. Ueda, K. Sugita, T. Motohiro, T. Nishimura, and H. Ushijima. 2000. Molecular epidemiology of astroviruses in Japan from 1995 to 1998 by reverse transcription-polymerase chain reaction with serotype-specific primers (1 to 8). J Med Virol 61:326-31. 6. Wang, Q., and Z. Y. Fang. 1997. Molecular biology of Norwalk-like viruses (review). Foreign Medical Sciences (field: Virology ) 3:27-31 (Chinese). 7. Wang, Q., Z. Y. Fang, Z. Lu, D. Zhou, J. Zhang, and D. Wu. 1996. Detection of hepatitis A virus contamination in shellfish by antigen capture/polymerase chain reaction. Chinese Journal of virology 12:123-128 (Chinese). 8. Pan, N., Z. Y. Fang, J. Yan, Q. Wang, Z. Gong, and J. Zhang. 1995. Molecular epidemiology of group A rotaviruses during 1989-1992 in Hubei province of China. Chinese Journal of Experimental and Clinical Virology 9:250-253 (Chinese). 9. Wang, Q., and Y. Chang. 1995. The effects of chemical toxins on secretion of luteinising hormone (LH) and follicle stimulating hormone (FSH) of male pituitary (review). Foreign Medical Sciences (field: Hygiene) 3:140-144 (Chinese). 10. Chang, Y., S. Lu, X. Jin, Q. Wang, and Z. Liu. 1994. Effects of HgCl2 on setolic cells of testis in rats. Journal of Health Toxicology 8:300 (Chinese). FIELDS OF STUDY Major Field: Veterinary Preventive Medicine Studies in Molecular Virology ix TABLE OF CONTENTS Page Abstract.............................................................................................................................ii