Identification of the core sequence elements in Penaeus stylirostris densovirus promoters.

In silico analysis of three Penaeus stylirostris densovirus (PstDNV) promoters, designated P2, P11, and P61, revealed sequence motifs including the TATA box, downstream promoter element (DPE), GC- and A-rich regions, inverted repeat, activation sequence-1 like (ASL) box, and a conserved guanosine (G) at +24. To delineate the regulatory role of these motifs on promoter activity, deletion constructs were made in a promoter assay vector, pGL3 Basic, that contains a luciferase reporter gene. Luciferase assay showed that P2 had the highest promoter activity followed by P11 and P61 in Sf9 cells. The deletions of inverted repeat, DPE, and GC-rich regions in P2 had the highest negative impact on this promoter. Deletions of DPE, G at the +24, and ASL box in P11 had the highest negative impact on this promoter activity. In P61, DPE and G at +24 are the two key regulators of transcriptional activity. Identification of the key transcriptional regulators is important in understanding the PstDNV pathogenesis in shrimp. This information is also valuable in constructing shrimp viral promoter-based vectors for protein expression in insect cell culture system as well as in shrimp.

Multiplication of Taura syndrome virus in primary hemocyte culture of shrimp (Penaeus vannamei).

The propagation of Taura syndrome virus (TSV) in primary hemocyte culture of Pacific white shrimp (Penaeus vannamei) was investigated. Purified TSV was inoculated into a 24 h old primary hemocyte culture and the development of cytopathic effects was monitored. The cell morphology started changing within 6 h post-inoculation; TSV-infected hemocytes started shrinking and granular structures began to form on the cell surface. There was a gradual loss of cell viability and, by 48 h post-inoculation, most cells detached from the bottom of the 96 well microplate. The propagation of TSV during the 48 h time course studied was measured by real-time RT-PCR. TSV copy number reached the highest level by 12 h post-inoculation and then started to decrease. Using an anti-TSV polyclonal antibody, the 55 kDa VP1 capsid protein was detected by Western blot analysis. The data suggest that shrimp primary hemocyte culture supports TSV replication and could be used as a tool for the study of host-virus interactions in TSV pathogenesis.

Transcriptional analysis of Penaeus stylirostris densovirus genes.

Penaeus stylirostris densovirus (PstDNV) genome contains three open reading frames (ORFs), left, middle, and right, which encode a non-structural (NS) protein, an unknown protein, and a capsid protein (CP), respectively. Transcription mapping revealed that P2, P11 and P61 promoters transcribe the left, middle and right ORFs. NS transcript uses the D1/A1 donor/acceptor sites for splicing and has two alternate transcription termination sites (TTS) that were different from the previously predicted TTS. The transcription initiation site (TIS) and the TTS for the middle and the right ORFs conform to predicted sites. PstDNV transcript quantification in infected shrimp revealed that the NS and CP transcripts were expressed at an equivalent level and significantly higher than the middle ORF transcript. In vitro assay showed that P2 had the highest promoter activity followed by P11 and P61. Transcription mapping data provided new insights into PstDNV gene expression strategy.

Characterization of a Taura syndrome virus isolate originating from the 2004 Texas epizootic in cultured shrimp.

A comprehensive investigation of the Taura syndrome virus (TSV) isolate that caused epizootics in shrimp farms in Texas in 2004 (Texas isolate) revealed that this virus was more virulent in laboratory bioassays than the TSV reference isolate, Hawaii 1994, causing severe symptom development and rapid mortality. Histopathology of moribund animals demonstrated epithelial necrosis within the stomach, appendages, general body cuticle and gills, and the surviving animals demonstrated moderate to numerous lymphoid organ spheroids. Purified virions showed icosahedral morphology, with a diameter of 31 nm. Comparative genome analysis showed that the Texas isolate is more closely related to TSV isolates from Thailand and China than to the Hawaii isolate. The predicted tertiary structures of the inhibition of apoptosis protein (IAP) and protease domains of the Texas isolate are very similar to those of the Hawaii isolate. However, the RNA-dependent RNA polymerase (RdRp) of the Texas isolate has significant structural differences from the Hawaii isolate due to point mutation(s) in the RdRp gene. Changes in the RdRp tertiary structure might contribute to the replication fidelity, virulence and ecological adaptability of the Texas isolate.