CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE VIRUSES ISOLATED FROM COMMERCIAL CHICKENS by Jacqueline Marie Harris A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the Degree of Master of Science in Animal and Food Sciences Spring 2010 Copyright 2010 Jacqueline Marie Harris All Rights Reserved i CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE VIRUSES ISOLATED FROM COMMERCIAL CHICKENS by Jacqueline Marie Harris Approved:_______________________________________________________________ Jack Gelb, Jr., Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved:_______________________________________________________________ Jack Gelb, Jr., Ph.D. Chairperson of the Department of Animal and Food Sciences Approved:_______________________________________________________________ Robin W. Morgan, Ph.D. Dean of the College of Agriculture and Natural Resources Approved:_______________________________________________________________ Debra Hess Norris, M.S. Vice Provost for Graduate and Professional Education ii ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor, Dr. Jack Gelb, Jr., for his continual guidance, encouragement, and academic support throughout my graduate education. I would like to give special thanks to the members of my graduate committee for their excellent advice and contributions during my research project, especially Dr. Daral Jackwood for his VP2 sequence analysis and Dr. Egbert Mundt for his monoclonal antibody testing. I am deeply indepted to Brian Ladman whose valuable knowledge and assistance enriched my growth as a student and researcher. I am truly grateful for all of his patience and support throughout my graduate career. In addition, I would like to thank Ruud Hein’s Intervet/Schering Plough lab group, Bruce Kingham, Joanne Kramer, Dr. Conrad Pope, Marcy Troeber, Dr. Daniel Bautista’s UD Lasher Lab group, Lauren Preskenis, Cindy Boettger, and Miguel Ruano for their time and contributions to the success and completion of this research project. I would like to express my sincerest gratitude to my family and friends for their unconditional love and encouragement. Their unwavering support and confidence in my abilities is what has shaped me into the person I am today. Most of all, I would like to thank my mom, Donna Harris, whose faith, wisdom, support, and understanding has given me the courage to face any challenge and the strength to succeed. I could not have done this without you. iii TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………vi LIST OF FIGURES……………………………………………………………………...ix ABSTRACT.......................................................................................................................x Chapter 1 INTRODUCTION………………………………………………………...…...1 2 REVIEW OF THE LITERATURE…………………………………………....6 2.1 History of Infectious Bursal Disease Virus…………………………….6 2.2 Etiology of IBDV……………………………………………………....8 2.3 Chemical Composition of IBDV……………………………………….9 2.4 Viral Proteins of IBDV………………………………………………..10 2.5 Pathogenesis and Virus Replication of IBDV………………………...12 2.6 Transmission of IBDV………………………………………………..17 2.7 Incubation Period, Clinical Signs, and Mortality……………………..17 2.8 Gross Lesions…………………………………………………………18 2.9 Microscopic Lesions…………………………………………………..20 2.10 Antigenic Variation of IBDV Strains………………………………....22 2.11 Prevention and Control of IBDV……………………………………...24 2.12 Clinical and Differential Diagnosis of IBDV…………………………28 3 MATERIALS AND METHODS………………………………………..……35 3.1 Chickens…………………………………………………………...….35 3.2 Chicken Embryos…………………………………………………..…35 3.3 Viruses………………………………………………………………...35 3.4 Virus Isolation………………………………………………………...36 3.5 Tissues and Histopathology…………………………………………...37 3.6 Viral RNA Extraction…………………………………………………38 3.7 Reverse Transcription – Polymerase Chain Reaction (RT-PCR)……………………………………………………………..38 3.8 Genome Sequencing and Analysis……………………………………40 3.9 Cultivation of Seed Stocks……………………………………...…….41 3.10 Antiserum Production…………………………………………………42 3.11 Virus Titration……………………………………………………...…43 3.12 Monoclonal Antibody Characterization………………………………44 iv 3.13 Vaccines…………………………………………………………........45 3.14 Cell Cultures…………………………………………………………..46 3.15 Virus-neutralization…………………………………………………...46 3.16 Challenge of Immunity………………………………………………..48 3.17 Progeny Challenge……………………………………………………49 3.18 Bursa to Body Weight Calculations and Statistical Analysis……..…..50 4 RESULTS…………………………………………………………………….59 4.1 VP2 Sequence Analysis…………………………………………….....59 4.2 Propagation and Pathology of Selected Viruses for Seed Stocks…………………………………………………………………60 4.3 Monoclonal Antibody Testing………………………………………...61 4.4 Monoclonal Antibody Epitope Mapping……………………………...62 4.5 Virus Neutralization in Cell Culture………………………………….63 4.6 Active Immunity – Challenge with 89/03…………………………….64 4.7 Active Immunity – Challenge with PBG98…………………………..66 4.8 Passive Immunity – Progeny Challenge……………………………....68 5 DISCUSSION………………………………………………………………...88 CONCLUSION………………………………………………………………………….94 APPENDIX A. Epitope Profiles of Infectious Bursal Disease Viruses Recognized by Monoclonal Antibodies Used in Antigen Capture Enzyme-linked Immunosorbent Assays…………………..…..96 APPENDIX B. Standard Amino Acid Abbreviations and Side Chain Properties Table……………………………………………………...…97 REFERENCES………………………………………………………………………......98 v LIST OF TABLES 3.1. Accession numbers and broiler flock information for IBDV isolation attempts………………………………………………………………...51 3.2. IBDV RT-PCR results from screening of the bursal homogenates from 3- and 4-week-old commercial broiler chickens raised on 12 farms in the Delmarva Peninsula region…………………………...................52 3.3. Propagation of Selected Field Viruses for Seed Stocks…………………………….53 3.4. Monoclonal antibody (MAb) reactivity patterns of infectious bursal disease virus field isolates using VP2 transfection and immunofluorescence assays……………………………………………………..54 3.5. Monoclonal antibody (MAb) reactivity patterns of infectious bursal disease virus field isolates and IBDV reference strains using whole virus/ELISA ssays………………………………………………….55 3.6. Vaccination-challenge of SPF leghorns using Delaware variant vaccine 89/03…………………………………………………………………….56 3.7 Vaccination-challenge of SPF leghorns using classic vaccine PBG98…………………………………………………………………………...57 3.8. Progeny challenge using progeny of 18-week-old SPF leghorn breeder chickens vaccinated with a licensed inactivated IBDV vaccine containing classic and variant strains, as well as, a strain of reovirus and challenged with IBDV field isolate 5038 and reference strain Delaware variant E…………………………………………58 4.1. Percent identity values of protein sequences of the variable region of VP2 protein of IBDV field strains isolated from Delmarva broiler farms and reference strains Delaware variant E, STC, GLS, AL-2, and RS593 as determined by multiple sequence alignment using the CLUSTAL W method……………….…69 vi 4.2. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 10 reactivity patterns for passage 1 seed stock samples of IBDV field isolates and IBDV reference strains…………………………………………………………..70 4.3. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 57 reactivity patterns for passage 1 seed stock samples of IBDV field isolates and IBDV reference strains…………………………………………………………..71 4.4. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 63 reactivity patterns for passage 1 seed stock samples of IBDV field isolates and IBDV reference strains…………………………………………………………..72 4.5. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 67 reactivity patterns for passage 1 seed stock samples of IBDV field isolates and IBDV reference strains…………………………………………………………..73 4.6. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 69 reactivity patterns for passage 1 seed stock samples of IBDV field isolates and IBDV reference strains…………………………………………………………..74 4.7. Virus-neutralization (VN) serum antibody titers of SPF leghorn chickens against IBDV strains 89/03 and PBG98 as measured by beta VN assays in chicken embryo fibroblast cells…………………………………………………………………...75 4.8. Percent protection determined by bursa to body weight ratios of one-day-old SPF leghorn chickens subcutaneously vaccinated with IBDV strain 89/03 and challenged at 21 days post-vaccination via the intraocular route with IBDV field isolates and reference strains STC and Delaware variant E……………………..76 4.9. Microscopic pathology of the bursa of Fabricius of SPF leghorn chickens vaccinated with IBDV strain 89/03 and challenged with IBDV field isolates 4813, 4947, 4955, 5038, and 5041 and IBDV reference strains STC and Delaware variant E………………………77 vii 4.10. Percent protection determined by bursa to body weight ratios of one-day-old SPF leghorn chickens subcutaneously vaccinated with IBDV strain PBG98 and challenged at 21 days post-vaccination via the intraocular route with IBDV field isolates and reference strains STC and Delaware variant E……………………..78 4.11. Microscopic pathology of the bursa of Fabricius of SPF leghorn chickens vaccinated with IBDV strain PBG98 and challenged with IBDV field isolates 4813, 4947, 4955, 5038, and 5041 and IBDV reference strains STC and Delaware variant E………………………79 4.12. Percent protection determined by bursa to body weight ratios of progeny of 18-week-old SPF leghorn breeder chickens vaccinated with a licensed inactivated IBDV vaccine