Characterization of white spot syndrome virus of penaeid shrimp: Genomic cloning and sequencing, structural protein analyzing and sequencing, genetic diversity, pathology and virulence Item Type text; Dissertation-Reproduction (electronic) Authors Wang, Qiong Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 26/09/2021 11:33:44 Link to Item http://hdl.handle.net/10150/284292 INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter &ce, while others may be from any type of computer printer. 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This microform edition is protected against miauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 2 THE UNIVERSITY OF ARIZONA « GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have Oiong Fang read the dissertation prepared by_ entitled Characterization of tTIiite Spot Syndrone Virus of Penaeid Shrimp: Genomic Cloning and Sequencing, Structural Protein Analyzing and Secuencing, Genetic Diversity, Pathology and Virulence and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy Ph.i/. nn J. lus J Date lartinez Ji Hev/lett, Ph.D. Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation reqyq^Dsement. ^ Dissertation Director Donald V. Lightner Date 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation firom or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: 4 ACKNOWLEDGEMENTS My greatest gratitude is due to my mentor. Dr. Donald V. Lightner, for giving me the privilege of being your student, for your teachings, guidance and trust. You not only equip me with the priceless scientific knowledge and skills but also serve as a role model exemplifying ethic integrity and professionalism. I thank Bonnie T. Poulos, Linda M. Nunan, Rita M. Redman, and Brenda L. White for their technical assistance with the experiments and valuable discussions about my research. To my graduate advisory conunittee members. Dr. Donald V. Lightoer, Dr. Glenn J. Songer, Dr. Cornelius J. Mare, Dr. Raymond B. Nagle and Dr. Martinez J. Hewlett. Thank you for being in my committee, for willingness to give advice and for donating your precious time to give me guidance. To my colleague Kenneth W. Hasson, Carlos Pantoja, Jeffrey R. Garza, Leone L. Mohney, Kathy Tang, Wanda C. McCormack, Heidi S. Erickson, and Stephanie Durand for your friendship and cooperation. Thanks are due to my family and friends for their spiritual support. They are my motivation to be a good researcher, and a good person. I thank Wallace Clark for amino acid analysis and protein sequencing. Skip Vaught for DNA sequencing, and the Imaging Facility Laboratory at the University of Arizona for access to the electron microscope. The original isolates of WSSV were kindly provided by Dr. Paul Frelier (Department of Veterinary Pathology, College of Veterinary Medicine, Texas A & M University, College Station, Texas), Dr. Craig Browdy (South Carolina Department of Natural Resources, Waddell Mariculture Center, Bluflfton, South Carolina), Dr. Laura K. Richman (Department of Pathology, National Zoological Park, Washington D. C.), Dr. Eileen Reddington (Diagxotics, Ltd.), Dr. Ramana Murty (Andhra University, Visakhapatnam, India), and Mr. Jie Huang (Yellow Sea Fishery Research Institute, Qingdao, Shangdong, China). This research was partially funded by the Gulf Coast Research laboratory Consortium Marine Shrimp Farming Program, CREES, USDA under Grant No. 95-38808-1420, the National Sea Grant Program, USDC under the Grant No. NA56RG0617, USDC under Grant No. NA56FD0621, and a special grant from the U. S. National Fisheries Institute. 5 TABLE OF CONTENTS LIST OF FIGUEIES 7 LIST OF TABLES 8 ABSTRACT 9 1. INTRODUCTION II 1.1 Problem Definition II 1.2 Literature Review 15 1.2.1 Major Viral Diseases of Cultured Penaeid Shrimp 15 1.2.1.1 Baculoviruses 20 1.2.1.2 Parvoviruses 25 1.2.1.3 Iridoviruses 29 1.2.1.4 Picomaviruses 29 1.2.1.5 Rhabdoviruses 31 1.2.1.6 Coronaviruses 32 1.2.1.7 Togaviruses 33 1.2.1.8 Reoviruses 34 1.2.2 The White Spot Syndrome Virus 35 1.2.2.1 History of white spot syndrome disease 35 1.2.2.2 Viral characterization 38 1.2.2.3 Diagnostic techniques 42 1.2.2.4 Host range 44 1.2.2.5 Geographic distribution and dissemination 45 1.2.2.6 Economic impact and disease management methods 49 1.2.3 Baculovirus diversity and molecular biology 51 1.2.3.1 General biology of baculoviruses 51 1.2.3.2 Life cycle 52 1.2.3.3 Viral replication and regulation of gene expression 56 1.2.3.4 Baculovirus structural proteins and genes 59 1.2.3.5 Genetic relatedness of baculoviruses 62 1.3 Dissertation Format 64 2. PRESENT STUDY 67 6 TABLE OF CONTENTS - Continued APPENDIX I. PARTIAL CLONING OF THE GENOME OF THE WHITE SPOT SYNDROME VIRUS AND SEQUENCING OF CLONED Clal RESTRICTION FRAGMENTS 71 APPENDIX 2. SODIUM DODECYL SULFATE POLYACRYLAMINE GEL ELECTROPHORESIS OF THE STRUCTURAL PROTEINS OF SLX GEOGRAPHIC ISOLATES OF THE WHITE SPOT SYNDROME VIRUS AND PARTIAL AMINO ACID SEQUENCING OF THREE OF THE MAJOR STRUCTURAL POLYPEPTIDES 112 APPENDIX 3. IDENTIFICATION OF GENOMIC VARIATIONS AMONG GEOGRAPHIC ISOLATES OF WHITE SPOT SYNDROME VIRUS USING RESTRICTION ANALYSIS AND SOUTHERN BLOT HYBRIDIZATION 139 APPENDIX 4. Per os CHALLENGE OF Litopenaeus vannamei POSTLARVAE AND Farfantepenaeus duorarum JUVENILES WITH SIX GEOGRAPHIC ISOLATES OF WHITE SPOT SYNDROME VIRUS 172 REFERENCES 191 7 LIST OF FIGURES FIGURE 1. Structural components of the two baculovirus virion phenotypes: budded virus (BV), and polyhedra derived virus (PDV) 53 FIGURE 2. Cellular infection cycle of a nuclear polyhedrosis virus 54 8 LIST OF TABLES TABLE I. Major viral pathogens of cultured penaeid shrimp 18 TABLE 2. Diagnostic methods for the penaeid viral diseases 19 TABLE 3. Possible transmission routes of white spot syndrome disease M 9 ABSTRACT The purpose of this dissertation was to characterize virulence, genomic and protein composition of a newly emerged virus of penaeid shrimp: white spot syndrome virus (WSSV). A partial genomic library, covering approximately 30-50% of the genome, of WSSV isolated from crayfish Orconectes punctimanits, was constructed by digesting viral DNA with endonuclease Clal and cloning into the system pBluescript-JMl09. Three viral inserts of approximately 2.2 kb, 2.8 kb and 6.3 kb, named as QW245, CR44, and QW237 respectively, were sequenced and analyzed. Six geographic isolates of WSSV, from China, India, Thailand, South Carolina, Texas, as well as from crayfish obtained from the US National Zoo in Washington D. C. were compared by electron microscopy (TEM) and SDS-PAGE. All viral isolates contained three major polypeptides of 25, 23 and 19 kDa. A fourth major polypeptide at the 14.5 kDa position was observed in four of the viral isolates.