One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.White spot syndrome virus (WSSV) is one of the most devastating shrimp pathogens, and it has caused serious damage to the worldwide shrimp culture industry. Although this virus can infect several crustacean species, including shrimp, crab, and crayfish (9,12,19,24,42), its virulence is particularly high in infected penaeid shrimp, and mortality can reach 90 to 100% within 3 to 7 days of infection (9, 46). The WSSV virion is a nonoccluded, enveloped particle of approximately 275 by 120 nm with an olive-to-bacilliform shape, and it has a nucleocapsid (300 by 70 nm) with periodic striations perpendicular to the long axis (42,43). The most prominent feature of WSSV is the presence of a tail-like extension at one end of the virion (10, 43), which gives this virus the family name Nimaviridae (27) ("nima" is Latin for "thread").The complete genome sequences of three WSSV isolates have been determined, and 532 open reading frames (ORFs) that contain Ͼ60 amino acids have been identified for the Taiwan isolate (GenBank accession number AF440570). Homology searches against the NCBInr database suggest possible roles or functions for only a few of these ORFs, and most of the ORFs posted for the Taiwan isolate show no significant similarity to other known proteins. Similar results have been reported for the other two isolates (40,44). Until recently, Ͻ5% of the ORFs had been shown to encode either nonstructural proteins (ribonucleotide reductase, protein kinase, the chimeric thymidine kinase/thymidylate kinase, and DNA polymerase [8,22,35,36]) or structural proteins...

An assay using a single-tube, 1-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the simultaneous detection of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV). Three primer sets, 9195 F/9992 R, 94 F2/R2, and ITS F/28S R, were mixed at a ratio of 3:1:1 to amplify specific fragments of the TSV, WSSV, and Penaeus vannamei genome, respectively, in the RT-PCR reaction. Shrimp samples were experimentally infected with WSSV and TSV. PCR-amplified products detected in the nucleic acid extraction of shrimp pleopods produced 4 kinds of results. With no virus infection, 1 fragment of 892 base pairs (bp) was amplified from a ribosomal RNA gene by primer set ITS F/28S R as an internal control. In samples only infected by WSSV or TSV, 2 fragments could be seen: either from WSSV (530 bp) plus the internal control or TSV (231 bp) plus the internal control, respectively. In cases of co-infection with both viruses, all 3 amplified products were detected simultaneously. This study is the first report of Penaeus vannamei specimens co-infected with WSSV and TSV being detected using a PCR method via experimental infection. KEY WORDS: RT-PCR · WSSV · TSV · Penaeid shrimp Resale or republication not permitted without written consent of the publisherDis Aquat Org 50: [9][10][11][12] 2002 wide host range among crustaceans (Flegel 1997) and distinctive clinical signs (white spots) in penaeid shrimps. The entire sequence of the double-stranded, circular DNA genome has been determined (van Hulten et al. 2001). TSV is a single-stranded RNA virus that causes serious disease in the PL, juvenile and adult stages of P. vannamei exclusively (Lightner & Redman 1998).Reverse transcription (RT) and polymerase chain reaction (PCR) technologies have proven to be powerful diagnostic tools for shrimp viral infections and for detection of viral reservoirs in asymptotic carriers. Methods for PCR diagnosis have been published for WSSV (Kimura et al. 1996, Lo et al. 1996, Takahashi et al. 1996, Kim et al. 1998, Tapay et al. 1999 and TSV (Nunan et al. 1998). This study was carried out to develop a modified method using a template comprised of total nucleic acid for simultaneous detection of WSSV and TSV in a single tube, 1-step multiplex RT-PCR. MATERIALS AND METHODS Experimental infections.Penaeus vannamei, weighing approximately 3.5 g and originating from a commercial shrimp hatchery in Tungkang, southern Taiwan, were reared from post larvae to the juvenile stage in an indoor recirculation system. Randomly selected specimens were checked using TSV RT-PCR (Nunan et al. 1998) and then WSSV PCR (Lo et al. 1996), and all were found to be PCR negative. Juveniles were stocked at a density of about 20 shrimp per 90 l aquarium tank. Inocula were prepared from patently infected (carapace with white spots) P. monodon for WSSV and from P. vannamei for TSV. Shrimp collected from cultivation ponds experiencing disease outbreaks in I-lan, north Taiwan, were proven to be virally infected by WSSV PCR ...

We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens ( Aeromonas hydrophila , Edwardsiella tarda , Flavobacterium columnare , Lactococcus garvieae , Photobacterium damselae , Pseudomonas anguilliseptica , Streptococcus iniae and Vibrio anguillarum ) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e ., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10 3 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.

A multiplex nested‐polymerase chain reaction (PCR)‐based (m‐nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae. The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non‐specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m‐nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila, 62 CFU for E. tarda, 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.

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