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Detection and Phylogenetic Analysis of Taura Syndrome Virus of Shrimp from Archived Davidson’S Fixed Paraffin Embedded Shrimp Tissue

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Detection and Phylogenetic Analysis of Taura Syndrome Virus of Shrimp from Archived Davidson’S Fixed Paraffin Embedded Shrimp Tissue Detection and Phylogenetic Analysis of Taura Syndrome Virus of Shrimp From Archived Davidson’s Fixed Paraffin Embedded Shrimp Tissue Item Type text; Electronic Thesis Authors Ochoa, Lauren 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, presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 02/10/2021 00:47:45 Link to Item http://hdl.handle.net/10150/642190 DETECTION AND PHYLOGENETIC ANALYSIS OF TAURA SYNDROME VIRUS OF SHRIMP FROM ARCHIVED DAVIDSON’S FIXED PARAFFIN EMBEDDED SHRIMP TISSUE by Lauren Ochoa ____________________________ Copyright © Lauren Ochoa 2020 A Thesis Submitted to the Faculty of the DEPARTMENT OF MICROBIOLOGY In Partial Fulfilment of the Requirements For the Degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA 2020 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the , we certify that we have read the thesis prepared by: titled: and recommend that it be accepted as fulfilling the thesis requirement for the . _________________________________________________________________ Date: ____________ _________________________________________________________________ Date: ____________ _________________________________________________________________ Date: ____________ Final approval and acceptance of this thesis final copies of the thesis to the Graduate College. I hereby certify that I have read this thesis prepared under my direction and recommend that it be accepted as fulfilling the requirement. _________________________________________________________________ Date: ____________ 3 ACKNOWLEDGEMENTS I thank the members of my committee, Dr. V.K. Viswanathan and Dr. Fiona McCarthy, for their unwavering support, assistance. and patience. Thank you to Dr. Arun K. Dhar for welcoming me into his laboratory and supporting my work. Moreover, my research mentor at the bench level, Dr. Roberto Cruz-Flores, has been an invaluable resource throughout this process. I also thank Dr. Fernando Aranguren, Jasmine Millabas, Patricia Marinez and Dari Trujillo for helping me through various research and academic endeavors. In addition, I thank every member of the Aquaculture Pathology Laboratory team and the graduate student team in ACBS for their support. Finally, I thank my family for their unconditional love and support, including, my husband, Dustin, my children, Kellan and Karson, my mother, Joy, my siblings, Kaitlyn, Mark, Danielle and Jordyn: your love and support made this possible. 4 TABLE OF CONTENTS Page LIST OF FIGURES……………………………………………………………………………....5 LIST OF TABLES………………………………………………………………………………..6 ABSTRACT………………………………………………………………………………………7 CHAPTER 1: Literature Review Origin and identification of Taura Syndrome Virus……………………………………....9 Geographic distribution and lineages of Taura Syndrome Virus………………………...11 Disease Biology……………………………………………………………………….....12 Properties of Taura Syndrome Virus and genome organization ……...…………12 Disease cycle and clinical signs………………………………………………….14 Susceptible species……………………………………………………………….18 Disease transmission……………………………………………………………..18 Current diagnostic techniques……………………………………………………………20 Disease management…………………………………………………………………......23 Biosecurity……………………………………………………………………….23 Breeding for disease resistance…………………………………………………..24 Significance……………………………………………………………………………....26 Use of formalin fixed tissue samples for studying human diseases………………………27 Davidson’s fixed paraffin embedded tissues……………………………………………..27 Rationale……………..…………………………………………………………………..29 Hypothesis and objectives………………………………………………………………..30 CHAPTER 2: Detection and phylogenetic analyses of Taura Syndrome Virus from Davidson’s fixed paraffin embedded shrimp tissue.…………………….……………………………………32 Abstract…………………………………………………………………………………..32 Introduction………………………………………………………………………………33 Materials and methods……………………………………………………………………34 Results……………………………………………………………………………………42 Discussion………………………………………………………………………………..52 CHAPTER 3: Conclusion………………………………………………………………………..54 REFERENCES…………………………………………………………………………………..56 5 LIST OF FIGURES Figure Page 1.1 Schematic diagram of the genome organization of TSV………………..…………………….13 1.2 Penaeus vannamei juveniles infected with TSV………………………….…………………..17 1.3 Routine histology of the acute, transition, and chronic phases of TSV infections in Penaeus vannamei………………………………………………………………………...……………….21 1.4 A schematic representation of the workflow in the detection and phylogenetic analysis of Taura Syndrome virus from Davidson’s fixed paraffin embedded tissue samples…………….……….31 2.1 A pictograph of RT-PCR amplification of the TSV VP1 gene for 12 representative isolates……………………………………………………………………………………………39 2.2 Quantity and quality assessment of total RNA isolated from archived TSV-infected DFPE shrimp tissues …………… …………………………………………………………..….………42 2.3 Analysis of the Ct values of TSV amplicons generated by real-time RT-PCR..………….…. 43 2.4 Representative agarose gel electrophoresis images for the detection of TSV genes and the internal control gene EF-1α…………………………………………..……………………..……45 2.5 A pictograph of RT-PCR amplification of TSV genes VP1, VP2, RdRp and shrimp internal control gene EF-1α………………………………………………………………………………..…….46 2.6 Alignment of 41 concatenated TSV sequences……………………………………………….50 2.7 Phylogenetic analysis of 41 TSV isolates …………..…………………………..……………51 6 LIST OF TABLES Table Page 2.1. Archived DFPE tissue samples selected for analyses……………………...………………...35 2.2 A summary of TSV-specific primers………………………………………………..….…….37 2.3 GenBank accessions utilized for a phylogenetic analysis of the TSV VP1 gene………….…..41 2.4 A comparison of quantity, quality, and Ct values of total RNA isolated with three RNA extraction kits…………………………………………...………………………………………..44 2.5 A comparison of the percent of successful amplifications for different regions of the TSV genome………………………………………………….………………………………………..47 2.6 A comparison of the percent of successful amplifications provided by each TSV-specific primer pair………………………………………………………………………………………..48 7 ABSTRACT Taura syndrome (TS) is an OIE-listed (World Organization for Animal Health) disease of marine shrimp that is caused by Taura syndrome virus (TSV). TSV has caused more than US $2 billion losses since the initial discovery of the disease in 1992. This work focuses on the detection and genome reconstruction of TSV from Davidson’s fixed paraffin embedded (DFPE) shrimp tissues. The data generated for this thesis demonstrate the utility of archived DFPE shrimp tissues as biological samples for detection and genetic studies in TSV. In Chapter 1, the status of TS is summarized. The review gives an overview of the diseases since it was first identified in Pacific white shrimp (Penaeus vannamei) in Ecuador in 1992. The review covers the identification of the etiologic agent, clinical signs and geographic distribution of the diseases, development of histopathological and molecular diagnostic tools. Efforts made to control the disease using biosecurity in shrimp farming, and development of genetically resistant lines of shrimp are also reviewed. Chapter 2 describes the detection of TSV from archived DFPE shrimp tissues. Twenty- nine DFPE P. vannamei shrimp tissue blocks from 2005 representing samples originating in 4 geographical locations were selected for this study. Sample screening indicated lesions of acute stages pathognomonic of TSV infection. Total RNA was isolated from DFPE blocks using three different commercial kits to compare the quality and quantity of extracted nucleic acids. The results showed that RNA isolated from Qiagen RNeasy FFPE Kit provided highest quality of RNA based on 260/280 and 260/230 ratios. Conventional and real-time RT-PCR results show that TSV nucleic acids can be detected in archived DFPE tissue samples. Sanger sequencing of representative amplicons confirmed the identity of TSV. Finally, a phylogenetic analysis based on the nucleotide sequence of the capsid proten (VP1) gene shows the genetic relationship among 8 different geographical isolates and homologous sequences of TSV isolates deposited in the GenBank database The feasibility of virus detection and genome reconstruction from archived DFPE tissues opens opportunities for the discovery of novel pathogens and will help enhance our understanding of the evolution of shrimp viruses. This study is the first of its kind in the field of shrimp pathology in using archived DFPE-derived tissue samples for pathogen detection. These results suggest that archived DFPE tissue samples can be utilized for the discovery of novel pathogens and evolutionary analyses. In addition, the findings have direct implications for disease management in shrimp. 9 CHAPTER 1: Literature Review Origin and Identification of Taura Syndrome Virus Taura Syndrome (TS), caused by Taura Syndrome Virus (TSV), first emerged in Ecuador from an unknown source in 1991, and by 1993 the disease was recognized as a major disease affecting penaeid shrimp (Jimenez 1992; Hasson et al., 1995; Lightner et al., 1995). Early reports of TS-like symptoms indicate that TS might have been present as early as 1990 (Lightner et al., 1997). Taura Syndrome spread rapidly throughout Latin America, and by 1996 the disease had spread to all shrimp-farming regions of the Americas (Lightner et al., 1997; Hasson et al., 1999). Cumulative mortality
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