STUDY TOWARD THE DEVELOPMENT OF ADVANCED INFLUENZA VACCINES DISSERTATION Presented in partial fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Leyi Wang B.S.,M.S. Graduate Program in Veterinary Preventive Medicine The Ohio State University 2009 Dissertation Committee: Professor Chang-Won Lee, advisor Professor Y.M. Saif, Professor Daral J. Jackwood Professor Jeffrey T. LeJeune Copyright by Leyi Wang 2009 ABSTRACT Avian influenza is one of the most economically important diseases in poultry. Since it was found in Italy in 1878, avian influenza virus has caused numerous outbreaks around the world, resulting in considerable economic losses in poultry industry. In addition to affecting poultry, different subtypes of avian influenza viruses can infect many other species, thus complicating prevention and control. Killed and fowlpox virus vectored HA vaccines have been used in the field as one of effective strategies in a comprehensive control program to prevent and control avian influenza. Live attenuated vaccines for poultry are still under development. Live attenuated vaccines can closely mimic natural infection inducing long-lasting humoral and cellular immunity. In addition, they may be used for mass vaccination. However, concerns with spread of live vaccine viruses, mutation into virulent strains from live attenuated viruses, and reassortment of vaccine and field strains prevent recommending live vaccines as poultry vaccines in the field. For this reason, there are increasing interests in the development of in ovo vaccines that can reduce the risk of spreading the vaccine virus. Therefore, in the first three parts of our study, we have explored several strategies (NS1 truncation, temperature sensitive (ts) mutations, HA substitution, and non-coding region (NCR) mutations) to attenuate viruses to reach this goal. In addition, NS1 truncation protein also can serve as a marker to differentiate infected from vaccinated animals (DIVA). In the last part of this study, we ii utilized two different strategies (NS1 protein and heterologous NA) to develop DIVA vaccines for the control of triple reassortant (TR) H3N2 influenza in turkeys. In the first part, we obtained several NS deletion genes from a parental non-stable virus, A/turkey/Oregon/71-delNS1 (H7N3 and has 10 nucleotide deletion in the NS gene) through passage in 10- or 14-day-old embryonated chicken eggs (ECE). Our results showed that most NS deletion genes are competent as far as NS2 protein, an essential structural protein, coding region is intact. Five NS1 truncation variants were obtained by traditional virus purification, followed by in vitro, in vivo and in ovo characterization. We found that NS1 truncation variants replicated well both in Vero and CEF cells, compared to wild type viruses. In western blot analysis of infected cells, NS1 proteins of A/turkey/Oregon/71-SEPRL, delNS1, Ddel-pc2, -pc4, and -pc5 could be detected while NS1 protein of A/turkey/Oregon/71-Ddel-pc1 and -pc3 could not, although there was no difference of NP protein expression among different variants. By conducting in vivo experiment in two-week-old chickens, we found that two NS1 truncation variants (A/turkey/Oregon/71-Ddel-pc3 and -pc4) were highly attenuated, induced high antibody titers and provided good protection in birds against a high dose heterologous virus challenge, which makes them suitable for potential live attenuated vaccine candidates. However, when NS1 truncation variants were used to vaccinate 18-day old ECEs in the second part of our study, the hatchability of vaccinated groups was significantly lower than that of the PBS-control group, indicating there is a need for further attenuation. Plasmid-based reverse genetics systems established for influenza A viruses make it easy to manipulate the genome of viruses. To further attenuate NS1 truncation variants, the ts mutations were then introduced into A/turkey/Oregon/71-SEPRL, and Ddel-pc3 and - iii pc4. In vitro growth analysis showed that viruses containing ts mutations exhibited clear ts phenotype in CEF cells while weak ts phenotype in MDCK cells. However, the hatchability study illustrated that ts mutations did not help to further attenuate viruses, indicating ts mutation could not work together with NS1 truncation strategy to further attenuate virus in ovo. Meanwhile, we tested whether HA substitution could improve hatchability of in ovo vaccination. Reassortant NS variant viruses with their HA genes from A/mute swan/MI/06 (H5N1) or A/turkey/OH/313053/04 (H3N2) strain and the remaining genes from two A/turkey/Oregon/71-Ddel-pc3 and -pc4 were generated by reverse genetics. Previous testing demonstrated higher hatchability of A/mute swan/MI/06 infected eggs compared to other H5 subtype strains tested. The in ovo study showed that HA substitution had a different effect on hatchability of in ovo vaccination and eggs inoculated with H3-NS1 truncation variants had higher hatchability than that of H5-NS1 truncation variants. In the third part, we explored the possibility of targeting NCRs to further attenuate NS1 truncation variants. Due to very limited NCR sequences available, we first sequenced the NCRs of seven influenza A virus strains of different host origin and varying pathogenicity using two recently developed methods. Sequence analysis showed there were not only sequence and length variations present in the segment specific NCRs among different influenza strains but also sequence variations at the fourth nucleotide of 3’ NCR of polymerase genes. To confirm that different NCR sequences could affect virus replication cycles, we first tested the role of sequence changes in the NCRs in protein expression using a green fluorescent protein (GFP) as a marker. Protein expression experiment results showed that even a single nucleotide change in the NCR of PA or PB1 iv could affect GFP protein expression. In addition, we further confirmed that mutations in NCR of PA alone or PA and PB1 combination affect virus replication by characterizing reverse genetically generated viruses in vitro. With more understanding of the role of NCR in the virus replication cycle, we speculate targeting non-conserved parts of NCR to further attenuate NS1 truncation variants is feasible. Since 2003, TR H3N2 influenza has been endemic in turkey populations in North America. Both field and experimental evidence of current available avian- and swine- origin vaccines indicates there is an urgent need to update the vaccine strain and develop an effective vaccine to control this virus infection in turkeys. Therefore, in the last part of this study, two different strategies, NS1 protein or heterologous NA, were used to generate NS-based and NA-based DIVA vaccines by the traditional reassortant method. In vivo evaluation of efficacy of DIVA vaccines was done in two-week old turkeys and breeder turkeys. The reassortant DIVA vaccines significantly reduced challenge virus shedding in the oviduct of breeder turkeys as well as trachea and cloaca of both young and breeder turkeys. Therefore, proper vaccination could effectively decrease egg production drop in the field. With an accompanying DIVA serological test, we expect those newly developed vaccines will be useful for the control of TR H3N2 influenza in turkeys. In conclusion, this study provides new information on the development of live attenuated and DIVA vaccines for the control of avian influenza. v Dedicated to my parents, Ancheng Wang and Xiurong Ding, my wife Bikun Zhou, and my daughter Sarah vi ACKNOWLEDGENTS First, I would like to thank my research advisor, Dr. Chang Won Lee, for his invaluable guidance, encouragement and support throughout the duration of my study, which made this thesis possible. Special thanks to my committee members, Dr. Y.M. Saif, Dr. Daral J. Jackwood, and Dr. Jeffrey T. LeJeune for their efforts, advice, and critical review of this manuscript. I wish to thank all the past and present members of the Dr. Lee’s laboratory for their support and scientific contributions. I especially thank Megan Strother, Keumsuk Hong, Dr. Zhuoming Qin and Dr. Mahesh Khatri for all assistance in my research. Thanks are also extended to Dr. Gireesh Rajashekara Dr. Qiuhong Wang, Dr. Hadi Yassine, and Todd Root for all their help. Special thanks are to Hannah Gehrman and Robin Weimer for their indispensable help. Finally, I want to thank my family for their love and support. vii VITA May 10, 1978………………………..Born Liaocheng, Shandong province, P.R.China 1997-2001….………………………..B.S., Veterinary Medicine, College of Animal Husbandry & Veterinary Medicine, Shandong Agricultural University, Taian, Shandong, P.R. China. 2001-2004…………………………...M.S. College of Veterinary Medicine, China Agricultural University, Beijing, P.R. China. 2004-2006…………………………...Research Associate and Lecturer, Department of Animal Science, College of Agriculture, Liaocheng University, Liaocheng, Shandong, P.R. China. 2006-present………………………...Graduate Research Associate Food Animal Health Research Program Department of Veterinary Preventive Medicine The Ohio State University, OH, U.S.A. PUBLICATIONS 1. Leyi Wang and Chang-Won Lee. 2009. Sequencing and Mutational Analysis of the Non-Coding Regions of Influenza A Virus. Veterinary Microbiology, 135: 239-247. viii 2. S. P. S. Pillai, M. Pantin-Jackwood, S. J. Jadhao, D. L. Suarez, L. Wang, H. Yassine, Y. M. Saif, C-W. Lee. 2009. Pathobiology of triple reassortant H3N2 influenza viruses in breeder turkeys and its potential implication for vaccine studies in turkeys. Vaccine. Vol. 27, No. 6: 819-824. 3. L. Wang, D. L. Suarez, M. Pantin-Jackwood, M. Mibayashi, A. García-Sastre, Y. M. Saif, C-W. Lee. 2008. Characterization of Influenza Virus Variants with Different Sizes of the Non-structural (NS) Genes and Their Potential as a Live Influenza Vaccine in Poultry. Vaccine, Vol. 26, No. 29-30: 3580-3586. 4. Chao Wang, Leyi Wang, Weitao Wang. 2006. Avian Influenza and its threat. Chinese Journal of Animal Quarantine, 2:46-47. 5. Leyi Wang, Fei Ma. 2006.