New genotypes of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) from the Kingdom of Saudi Arabia.

White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) are highly pathogenic to penaeid shrimp and have caused significant economic losses in the shrimp culture industry around the world. During 2010 and 2011, both WSSV and TSV were found in Saudi Arabia, where they caused severe mortalities in cultured Indian white shrimp Penaeus indicus. Most outbreaks of shrimp viruses in production facilities can be traced to the importation of infected stocks or commodity shrimp. In an attempt to determine the origins of these viral outbreaks in Saudi Arabia, we performed variable number of tandem repeat (VNTR) analyses for WSSV isolates and a phylogenetic analysis for TSV isolates. From the WSSV genome, the VNTR in open reading frames (ORFs) 125 and 94 were investigated with PCR followed by DNA sequence analysis. The genotypes were categorized as {N125, N94} where N is the number of repeat units in a specific ORF, and the subscript indicates the ORF (i.e. ORFs 125 and 94 in this case). From 15 Saudi Arabia WSSV isolates, we detected 3 genotypes: {6125, 794}, {7125, del94}, and {8125, 1394}. The WSSV genotype of {7125, del94} appears to be a new variant with a 1522 bp deletion encompassing complete coding regions of ORF 94 and ORF 95 and the first 82 bp of ORF 93. For TSV genotyping, we used a phylogenetic analysis based on the amino acid sequence of TSV capsid protein 2 (CP2). We analyzed 8 Saudi Arabian isolates in addition to 36 isolates from other areas: SE Asia, Mexico, Venezuela and Belize. The Saudi Arabian TSV clustered into a new, distinct group. Based on these genotyping analyses, new WSSV and TSV genotypes were found in Saudi Arabia. The data suggest that they have come from wild shrimp Penaeus indicus from the Red Sea that are used for broodstock.

Ultrastructural and sequence characterization of Penaeus vannamei nodavirus (PvNV) from Belize.

The Penaeus vannamei nodavirus (PvNV), which causes muscle necrosis in Penaeus vannamei from Belize, was identified in 2005. Infected shrimp show clinical signs of white, opaque lesions in the tail muscle. Under transmission electron microscopy, the infected cells exhibit increases in various organelles, including mitochondria, Golgi stacks, and rough endoplasmic reticulum. Cytoplasmic inclusions containing para-crystalline arrays of virions were visualized. The viral particle is spherical in shape and 19 to 27 nm in diameter. A cDNA library was constructed from total RNA extracted from infected shrimp. Through nucleotide sequencing from the cDNA clones and northern blot hybridization, the PvNV genome was shown to consist of 2 segments: RNA1 (3111 bp) and RNA2 (1183 bp). RNA1 contains 2 overlapped open reading frames (ORF A and B), which may encode a RNA-dependent RNA polymerase (RdRp) and a B2 protein, respectively. RNA2 contains a single ORF that may encode the viral capsid protein. Sequence analyses showed the presence of 4 RdRp characteristic motifs and 2 conserved domains (RNA-binding B2 protein and viral coat protein) in the PvNV genome. Phylogenetic analysis based on the translated amino acid sequence of the RdRp reveals that PvNV is a member of the genus Alphanodavirus and closely related to Macrobrachium rosenbergii nodavirus (MrNV). In a study investigating potential PvNV vectors, we monitored the presence of PvNV by RT-PCR in seabird feces and various aquatic organisms collected around a shrimp farm in Belize. PvNV was detected in mosquitofish, seabird feces, barnacles, and zooplankton, suggesting that PvNV can be spread via these carriers.

A quick fuse and the emergence of Taura syndrome virus.

Over the last two decades, Taura syndrome virus (TSV) has emerged as a major pathogen in penaeid shrimp aquaculture and has caused substantial economic loss. The disease was first discovered in Ecuador in 1991, and the virus is now globally distributed with the greatest concentration of infections in the Americas and Southeast Asia. To determine the evolutionary history of this virus, we constructed a phylogeny containing 83 TSV isolates from 16 countries sampled over a 16-year period. This phylogeny was inferred using a relaxed molecular clock in a Bayesian Markov chain Monte Carlo framework. We found phylogenetic evidence that the TSV epidemic did indeed originate in the Americas sometime around 1991 (1988-1993). We estimated the TSV nucleotide substitution rate at 2.37 x 10(-3) (1.98 x 10(-3) to 2.82 x 10(-3)) substitutions/site/year within capsid gene 2. In addition, the phylogeny was able to independently corroborate many of the suspected routes of TSV transmission around the world. Finally, we asked whether TSV emergence in new geographic locations operates under a quick fuse (i.e. rapid appearance of widespread disease). Using a relaxed molecular clock, we determined that TSV is almost always discovered within a year of entering a new region. This suggests that current monitoring programs are effective at detecting novel TSV outbreaks.

PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus (IHHNV) and virus-related sequences in the genome of Penaeus monodon.

We developed a PCR assay that can detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) but that does not react with IHHNV-related sequences in the genome of Penaeus monodon from Africa and Australia. IHHNV is a single-stranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in the genome of some stocks of P. monodon from Africa and Australia. These virus-related sequences have a high degree of similarity (86 and 92% identities in nucleotide sequence) to the viral genome, which has often generated false-positive reactions during PCR screening of these stocks. For this assay, a pair of IHHNV primers (IHHNV309F/R) was selected. The sequences of these primers match (100% of nucleotides) the target sequence in IHHNV, but mismatch 9 or 12 nucleotides of the genomic IHHNV-related sequences. This PCR assay was tested with various IHHNV isolates and with a number of samples of shrimp DNA that contained IHHNV-related sequences. This assay can reliably distinguish IHHNV DNA from shrimp DNA: it only detects IHHNV. Also, this pair of primers was included in a duplex PCR to detect IHHNV and simultaneously determine the presence of an IHHNV-related sequence. Using these primers, the PCR assay has a sensitivity equivalent to a PCR assay commonly used for detecting IHHNV in Litopenaeus vannamei, and can be used for routine detection.

Nonsusceptibility of primate cells to Taura syndrome virus.

Taura syndrome virus (TSV), a pathogen of penaeid shrimp and member of the family Dicistroviridae, was recently reported to have the ability to infect primate cells. We independently retested this hypothesis. Three lines of primate cells FRhK-4, MA-104, and BGMK, which are highly susceptible to infection by human picornaviruses, were challenged with TSV. Viral replication was assayed by real-time reverse transcription-polymerase chain reaction using cell media samples collected on days 0, 4, and 7 postchallenge. By day 7, genome copy numbers had decreased 25%-99%. No cytopathic effect was observed after 7 days. An in situ hybridization assay, with gene probes specific for detection of TSV, was negative for TSV in challenged cells. The infectivity of residual virus in the cell culture media at day 7 was confirmed by bioassay using TSV-free indicator shrimp (Litopenaeus vannamei). TSV did not infect the primate cells tested, and no evidence of zoonotic potential was found.