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

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 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.