CHARACTERIZATION OF FIELD STRAINS OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) USING MOLECULAR TECHNIQUES by ALEJANDRO BANDA (Under the Direction of Pedro Villegas) ABSTRACT This study was aimed to apply different molecular techniques in the genotyping of field strains of infectious bursal disease virus (IBDV) currently present in the United States and in some other countries. The different techniques included the reverse transcription-polymerase chain reaction / restriction fragment length polymorphism (RT- PCR/RFLP), heteroduplex mobility assay (HMA), nucleotide and amino acid sequence analysis, and riboprobe in situ hybridization (ISH). From 150 samples analyzed from the United States, 80% exhibited RFLP identical to the variant Delaware E strain, other strains detected included Sal-1, D-78, Lukert, PBG-98, Delaware A, GLS IBDV standard challenge strain –like (STC-like). The analysis of the deduced amino acid sequence of the VP2 hypervariable region from six strains classified as Delaware variant E, revealed some amino acid substitutions that make them somewhat different from the original variant E strain isolated in the mid 1980s. The isolate 9109 was classified as a standard strain, but, it exhibited a unique RFLP pattern characterized by the presence of the Ssp I restriction site characteristic of the very virulent IBDV (vvIBDV) strains. The pathogenic properties of this isolate were compared to those of isolate 9865 (variant strain) and the Edgar strain. All three strains induced subclinical disease, however by in situ hybridization some differences in the tissue tropism were observed. The viral replication of the variant isolate 9865 was more restricted to the bursa of Fabricius. Isolate 9109 and the Edgar strain were also observed in thymus, cecal tonsils, spleen, kidney and proventriculus. A variety of inactivated IBDV strains received from Latin America were detected by RT-PCR/RFLP. The more interesting findings include the presence in Mexico and Venezuela of IBDV strains with unknown RFLP patterns, and the observation of RFLP patterns indicative of vvIBDV strains in Brazil and Dominican Republic. Finally, the HMA was evaluated as a method for genotyping IBDV. The HMA was able to differentiate between standard, antigenic variants and very virulent strains. Minor differences between antigenic variants were also detected. The results obtained by HMA were similar to that obtained with RFLP and phylogenetic analysis. INDEX WORDS: Infectious bursal disease, Field isolates, Molecular characterization, restriction fragment length polymorphism, Heteroduplex mobility assay, In situ hybridization CHARACTERIZATION OF FIELD STRAINS OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) USING MOLECULAR TECHNIQUES by ALEJANDRO BANDA M.V.Z. Universidad Nacional Autónoma de México, Mexico, 1992 M.C.V. Universidad Nacional Autónoma de México, Mexico, 1996 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2002 © 2002 Alejandro Banda All Rights Reserved CHARACTERIZATION OF FIELD STRAINS OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) USING MOLECULAR TECHNIQUES by Alejandro Banda Approved Mayor Professor: Pedro Villegas Committee: Corrie C. Brown Donald L. Dawe Maricarmen García Mark W. Jackwood Electronic Version Approved: Gordhan L. Patel Dean of the Graduate School The University of Georgia August 2002 iv DEDICATION To my family; my mom Lydia, my siblings Ana Lilia, Rafael, and Alfonso, and my little niece Liliana for the things we share... v ACKNOWLEDGEMENTS I am deeply thankful to my major professor, Dr. Pedro Villegas, for giving me the opportunity of being a graduate student under his direction. I would like to find a better way to express my gratitude but, I could not find it, so… THANKS!. I express also my appreciation to each professor that served on my advisory committee, Dr. Corrie C. Brown, Dr. Donald L. Dawe, Dr. Maricarmen Garcia, and Dr. Mark W. Jackwood. Thanks for your input for my research project and for your contributions to my professional development. I want to thank my good friends and colleages Dr John El-Attrache, Dr. Carlos Estevez, and Dr. Ivan Alvarado. You made my time at Dr. Villegas’ lab a quite experience. It was a real honor, I will miss all of you. I wish to thank Dr. Miguel Ruano, Dr. James Stanton, and Dr. Erika Behling- Kelly for your technical assistance. Finally I want to thank to the faculty, staff and students of the Department of Avian Medicine. I spent a wonderful time with you. Georgia and all of you will be always in my mind. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.................................................................................................v LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix CHAPTER I INTRODUCTION...................................................................................................1 Purpose of the Study ...................................................................................................1 Objectives and Originality ..........................................................................................3 II LITERATURE REVIEW......................................................................................6 Part 1: Infectious Bursal Disease ................................................................................6 Part 2: Molecular Basis of IBDV Variability ...........................................................22 Part 3: Heteroduplex Mobility Assay .......................................................................26 Part 4: In Situ Hybridization .....................................................................................29 References.................................................................................................................32 III MOLECULAR CHARACTERIZATION OF SEVEN FIELD ISOLATES OF INFECTIOUS BURSAL DISEASE VIRUS OBTAINED FROM COMMERCIAL BROILERS CHICKENS ........................56 References............................................................................................................71 vii IV MOLECULAR CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE VIRUS FROM COMMERCIAL POULTRY IN THE UNITED STATES AND LATIN AMERICA......................................83 References...........................................................................................................98 V GENOTYPING OF INFECTIOUS BURSAL DISEASE VIRUS BY HETERODUPLEX MOBILITY ASSAY......................................107 References........................................................................................................121 VI TISSUE TROPISM STUDIES OF TWO INFECTIOUS BURSAL DISEASE VIRUS FIELD ISOLATES IN COMMERCIAL BROILERS BY IN SITU HYBRIDIZATION......................133 References..........................................................................................................147 VII DISCUSSIONS AND CONCLUSIONS...........................................................157 viii LIST OF TABLES Page CHAPTER III Table 3.1 ..............................................................................................................77 Table 3.2 ..............................................................................................................78 CHAPTER IV Table 4.1 ...........................................................................................................104 CHAPTER V .............................................................................................................. Table 5.1 ..........................................................................................................126 Table 5.2 ..........................................................................................................127 CHAPTER VI Table 6.1 ..........................................................................................................151 Table 6.2 ..........................................................................................................152 Table 6.3 ..........................................................................................................153 ix LIST OF FIGURES Page CHAPTER III Figure 3.1 ............................................................................................................80 Figure 3.2 ............................................................................................................81 Figure 3.3 ............................................................................................................82 CHAPTER IV Figure 4.1 .........................................................................................................106 CHAPTER V Figure 5.1 .........................................................................................................129 Figure 5.2 .........................................................................................................130 Figure 5.3 .........................................................................................................132 CHAPTER VI Figure 6.1 .........................................................................................................155 Figure 6.2 .........................................................................................................156 CHAPTER I INTRODUCTION Purpose