Taura syndrome virus (TSV), the epidemic agent of Taura syndrome (TS), was tentatively classified in the family Dicistroviridae, caused disastrous losses with high mortality rates from 60 to >90% of affected pond-cultured Litopenaeus vannamei. The amino acid sequence alignments of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of picornavirus, suggesting perhaps a similar to picornavirus polyprotein processing. Proteolytic processing of the TSV polyprotein and protease is still unclear. In order to further understand the role of protease interaction with polyprotein, several clones were constructed. In this study, we constructed several clones that expressed the TSV virus coat protein(VP-polyprotein)¡]pTSVVP¡^, putative 3C protease¡]pTSV3C¡^, protease¡ÏRdRp-polyprotein(pET3CD) and both VP+3C proteins(pTSVVP3C) in E. coli. The VP-polyprotein band was excised from SDS-polyacrylamide gels and electroeluted for antibody production in mice. Processing of the VP-polyprotein by 3C protease was carried out in the in vitro co-transcription/translation system and detected by Western blot. The results showed there were protein bands corresponding to the sizes of VP2+VP1 and VP1+VP3 indicating that the processing might have been partial or incomplete.

Taura syndrome (TS) is one of the most devastating diseases affecting the shrimp farming industry worldwide. The causative virus, Taura syndrome virus (TSV), has been identified. My work is centred on the development of monoclonal antibodies against TSV. I have also characterized a novel variant of the virus from Venezuela and evaluated the effect of hyperthermia on TSV infection. This work has resulted in 3 manuscripts, which constitute the core of this dissertation. The taxonomy throughout this dissertation is done according to Holthuis (1980).The first manuscript describes the production of a monoclonal antibody reacting with the Belize strain of TSV. The antibody, MAb 2C4, exhibits good sensitivity and specificity for TSV in immunohistochemistry (IHC) and dot blot immunoassay. MAb 2C4 reacted with the TSV-HI94, TSV-SI98 and TSV-BZ02 variants, but not with the TSV-VE05 and TSV-TH05 variants. This antibody adds and improves tools to those available for TSV diagnosis.Chapter three describes a relatively novel variant of TSV from Venezuela, which was characterized by our laboratory. By genetic sequencing, this new isolate exhibits a 94% similarity with TSV-HI94. IHC, dot blot immunoassay and bioassays were also performed. While processed samples reacted only weakly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassays, TSV-VE05 presented mortality comparable to TSV-HI94 in Penaeus vannamei. These data confirm the presence of TSV in Venezuela and that a new variant of the virus was responsible for the outbreak of TS.In chapter four, the behavior of TSV infection under hyperthermic conditions was examined. I compared the susceptibility of Kona stock P. vannamei to the infection by two variants of TSV under hyperthermic conditions (32oC). Shrimp, infected with TSV-HI94, were resistant to infection at high temperature. However, under the same hyperthermic conditions, the challenged shrimp were fully susceptible to the infection by TSV-BZ02. This susceptibility to TSV-BZ02 at higher temperatures was independent both of the route of infection and of the salinity of water. I conjecture that TSV-BZ02 might be a temperature permissible mutant of TSV.

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, Th04, UsHi94, and Ve05) in TSV-resistant (TSR) and TSV-susceptible (Kona) Litopenaeus vannamei was investigated. Survival probabilities of TSR shrimp were higher than those for Kona shrimp with all 4 variants. Th04, UsHi94, and Ve05 gave no Taura syndrome lesions with TSR shrimp. In contrast, TSR shrimp challenged with Bz01 and Kona shrimp with all 4 TSV variants exhibited severe necrosis of cuticular epithelial cells and lymphoid organ spheroids. Real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that mean TSV copy numbers in TSR shrimp infected with Bz01, Th04, and UsHi94 were significantly (p < 0.0005) lower than those in Kona shrimp. In contrast, mean TSV copy numbers in TSR and Kona shrimp infected with Ve05 were not significantly different (p > 0.4). The results show that TSR L. vannamei are susceptible to infection but give high survival rates following challenge by all 4 variants of TSV.To identify the viral replication site within shrimp infected cells, the viral RNA was located in association with virus-induced membrane rearrangement by electron microscopic ISH. Ultrastructure in the infected cells, analyzed by transmission electron microscopy, included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei were frequently seen. TSV RNA and TSV particles were found to be associated with the membranous structures. The results suggest that the proliferating membranes carry the RNA replication complex and that they are the site of nascent viral RNA synthesis.