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Taura Syndrome Virus Taura Syndrome Virus (TSV) of Penaeid Shrimp: Infection of Penaeus monodon, Resistance of Litopenaeus vannamei and Ultrastructure of the Replication Site in Infected Cells Item Type text; Electronic Dissertation Authors Srisuvan, Thinnarat 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 01/10/2021 12:07:07 Link to Item http://hdl.handle.net/10150/194829 TAURA SYNDROME VIRUS (TSV) OF PENAEID SHRIMP: INFECTION OF Penaeus monodon, RESISTANCE OF Litopenaeus vannamei AND ULTRASTRUCTURE OF THE REPLICATION SITE IN INFECTED CELLS by Thinnarat Srisuvan ____________________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF VETERINARY SCIENCE AND MICROBIOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN PATHOBIOLOGY In the Graduate College THE UNIVERSITY OF ARIZONA 2006 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Thinnarat Srisuvan entitled Taura syndrome virus (TSV) of Penaeid Shrimp: Infection of Penaeus monodon, Resistance of Litopenaeus vannamei and Ultrastructure of the Replication Site in Infected Cells and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _______________________________________________________________________ Date: October 23, 2006 Donald V. Lightner, Ph.D. _______________________________________________________________________ Date: October 23, 2006 Carlos Reggiardo, D.V.M., Ph.D. _______________________________________________________________________ Date: October 23, 2006 David G. Besselsen, D.V.M., Ph.D. _______________________________________________________________________ Date: October 23, 2006 Kathy F. J. Tang-Nelson, Ph.D. _______________________________________________________________________ Date: October 23, 2006 Michael W. Riggs, D.V.M., Ph.D. Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies 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 requirement. ________________________________________________ Date: October 23, 2006 Dissertation Director: Donald V. Lightner 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 from 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 interest of scholarship. In all other instances, however, permission must be obtained from the author. Thinnarat Srisuvan 4 DEDICATION To my parents: Ms. Charoensri Srisuvan (นางเจริญศรี ศรีสุวรรณ) and Mr. Thaveesit Srisuvan (นายทวีสิทธิ์ ศรีสุวรรณ) กราบเทาพอแม ลูกขออุทิศความสําเร็จในการศึกษานใหี้ กับพอแม กราบขอบพระคุณที่ทานได เฝาถนอมเลี้ยงดู ใหการอบรมสั่งสอนใหลูกเปนคนดี และเปนกําลังใจในยามที่ลูกทอแท ลูกรักทานทั้ง สองและรูสํานึกในพระคุณทานเสมอมา ขออํานาจคุณพระศรีรัตนตรัยและสิ่งศักดิ์สิทธิ์ในสากล จง โปรดดลบันดาลใหทานทั้งสองมีสุขภาพแข็งแรง มีอายุยืนยาว อยเปู นรมโพธิ์รมไทรใหลูกทั้งสองคน ตลอดไป 5 TABLE OF CONTENTS Page LIST OF FIGURES__________________________________________________________________________________6 LIST OF TABLES___________________________________________________________________________________8 ABSTRACT __________________________________________________________________________________________9 CHAPTER 1. INTRODUCTION________________________________________________________________11 CHAPTER 2. EXPERIMENTAL INFECTION OF Penaeus monodon WITH TAURA SYNDROME VIRUS (TSV)____________________________________________________________18 Abstract _____________________________________________________________________________________18 Introduction_________________________________________________________________________________19 Materials and Methods ___________________________________________________________________20 Results and Discussion____________________________________________________________________23 CHAPTER 3. COMPARISON OF FOUR TAURA SYNDROME VIRUS (TSV) ISOLATES IN ORAL CHALLENGE STUDIES WITH Litopenaeus vannamei UNSELECTED OR SELECTED FOR RESISTANCE TO TSV___________________36 Abstract _____________________________________________________________________________________36 Introduction_________________________________________________________________________________37 Materials and Methods ___________________________________________________________________39 Results and Discussion ___________________________________________________________________44 CHAPTER 4. ULTRASTRUCTURE OF THE REPLICATION SITE IN TAURA SYNDROME VIRUS (TSV)-INFECTED CELLS___________________________________60 Abstract _____________________________________________________________________________________60 Introduction_________________________________________________________________________________61 Materials and Methods ___________________________________________________________________63 Results_______________________________________________________________________________________70 Discussion __________________________________________________________________________________75 REFERENCES______________________________________________________________________________________95 6 LIST OF FIGURES Figure Page 1.1 Geographic distribution of Taura syndrome virus (TSV) 15 1.2 Transmission electron micrograph of TSV 16 1.3 Schematic diagram of the genome organization of TSV 17 2.1 Photomicrographs of consecutive Penaeus monodon tissue sections 29 tested by hematoxylin and eosin (H&E) stained and in situ hybridization (ISH) tissue sections illustrating TSV lesions 2.2 Phylogenetic neighbor-joining (NJ) tree of capsid protein 2 (CP2, 31 383 amino acids) from 24 TSV isolates 2.3 Comparison of deduced amino acid sequences of CP2 from 24 32 TSV isolates with reference to UsHi94 (GenBank no. AF277675) 3.1 Survival curves of TSV-resistant (TSR) and TSV-susceptible 52 (Kona) Litopenaeus vannamei after challenge by feeding with 4 TSV isolates: Bz01, Th04, UsHi94, and Ve05 3.2 Photomicrographs of consecutive Litopenaeus vannamei tissue 53 sections tested for TSV lesions by H&E staining and ISH 3.3 SYBR-Green real-time reverse transcription polymerase chain 55 reaction (RT-PCR) 7 LIST OF FIGURES - Continued Figure Page 3.4 Means ± standard errors of TSV copy numbers µl–1 RNA within 57 pleopods of TSR and Kona Litopenaeus vannamei after challenge by feeding with 4 virus isolates: Bz01, Th04, UsHi94, and Ve05 4.1 Ultrastructural changes in cells at early stages of infection with 80 TSV 4.2 Ultrastructural changes in cells at the mid-stages of an acute phase 81 infection with TSV 4.3 Ultrastructural changes in cells at late stages of infection with TSV 82 4.4 Transmission electron micrographs of TSV in an infected cell 83 4.5 Detection of TSV with ISH in paraffin-embedded tissues by light 84 microscopy 4.6 Detection of TSV with ISH in resin-embedded tissues by light 85 microscopy 4.7 Ultrastrutural features of cells at late stages of TSV infection 86 examined by ISH using TSV-specific cDNA probes 4.8 Ultrastructural features of membrane rearrangement in TSV- 89 infected cells from gills analyzed by ISH using TSV-specific cDNA probes 4.9 Ultrastructural visualization of TSV in an infected cell tested by 93 ISH using TSV-specific cDNA probes 8 LIST OF TABLES Table Page 2.1 Taura syndrome virus (TSV) isolates used for experimental 27 bioassays and sequence analysis 2.2 Initial appearance of dead Penaeus monodon and Litopenaeus 28 vannamei and cumulative mortality after TSV infection 3.1 TSV isolates used for the oral challenge studies 51 3.2 Reproducibility of the SYBR-Green reverse transcription 56 polymerase chain reaction (RT-PCR) assay 3.3 Comparison of a conventional RT-PCR with the SYBR-Green 58 real-time RT-PCR for TSV within pleopods of TSV-resistant (TSR) and TSV-susceptible (Kona) Litopenaeus vannamei after challenge by feeding with 4 virus isolates 9 ABSTRACT Clinical signs and lesions of Taura syndrome virus (TSV) infection in Penaeus monodon were investigated by histological and in situ hybridization (ISH) analyses. Mortality among P. monodon inoculated with 2 genotypic variants of TSV (Th04Pm and Th04Lv) appeared on Day 3, with 2 out of 10 shrimp dying. Severe necrosis of cuticular epithelial cells and lymphoid organ spheroids, indicative of acute and chronic phase lesions of TSV infection, respectively, were detected in the samples. Both Th04Pm and Th04Lv belonged to a phylogenetic family of Asian TSV isolates. The results demonstrate that both mortality and histological lesions are associated with TSV infection in P. monodon. Infection with 4 genotypic variants of TSV (Bz01,
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