White Spot Syndrome Virus Genotypes Research Articles

Global distribution of white spot syndrome virus genotypes determined using a novel genotyping assay

White spot disease, caused by infection with white spot syndrome virus (WSSV), is a serious panzootic affecting prawn aquaculture. The disease has spread rapidly around the prawn-culturing regions of the world through a number of previously identified mechanisms. The ability to distinguish and trace strains of WSSV is of great benefit to identify, and then limit, the translocation routes of the disease. Here, we describe a novel genotyping method using 34 short tandem repeat regions of the viral genome concurrently. This technique is highly sensitive to strain differences when compared to previous methods. The efficacy of the described method is demonstrated by testing WSSV isolates from around the globe, showing regional genotypic differences. The differences in the genotypes were used to create a global minimum spanning network, and in most cases the observed relationships were substantiated with verification of transboundary movement. This novel panel of STR markers will provide a valuable epidemiological tool for white spot disease. We have applied this to an outbreak of the disease in Queensland, Australia, that occurred in 2016. While the results indicate that the source of this outbreak currently remains cryptic, the analyses have provided valuable insights with which to further study the origins of the strains involved.

Characterization and prevalence of a novel white spot syndrome viral genotype in naturally infected wild crayfish, Procambarus clarkii, in Shanghai, China.

White spot syndrome virus (WSSV) infection is commonly detected by vp28-qPCR assay in wild crayfish, Procambarus clarkii, a widespread crustacean species in the aquatic environment in China. The virions of crayfish WSSV have been isolated and purified. Based on TEM observation, they exhibited morphological structures that are identical to known WSSV. In addition, the WSSV major envelope protein VP28 was observed based on Western blot analysis of the total structural proteins of crayfish WSSV. PCR amplification and sequencing analyses of variable regions of ORF14/15, ORF23/24 and ORF94, along with viral genomic sequencing and phylogenomic analysis, indicated that the crayfish WSSV, named WSSV-CN-Pc, represents a new WSSV genotype. Intramuscular injection bioassay revealed that WSSV-CN-Pc was as virulent as the WSSV Taiwan strain. The WSSV-CN-Pc exhibited characteristics of a dominant genotype, with high infection load (107-108WSSV/mg) and high prevalence (91.7%, 110 of 120 crayfish samples) observed in the surveyed wild crayfish. WSSV-CN-Pc was also detected, with similar infection pattern as observed in crayfish, in farmed Litopenaeus vannamei shrimp that shared similar ecological niches with the sampled crayfish. Our results indicated that there was horizontal transmission of WSSV-CN-Pc between crayfish and shrimp in nature. Our findings also implicated that crayfish and shrimp farming should be integrated and managed with cautions in order to reduce the risk of spread and circulation of WSSV in the aquatic environment.

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X14/15, X23/24) (N75-N125-N94) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (595014/15, 1097123/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (595014/15, 1097123/24) (375-6125-794). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.

Novel, closely related, white spot syndrome virus (WSSV) genotypes from Madagascar, Mozambique and the Kingdom of Saudi Arabia

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the aquaculture industry around the world. During 2010 to 2012, WSSV caused severe mortalities in cultured penaeid shrimp in Saudi Arabia, Mozambique and Madagascar. To investigate the origins of these WSSV, we performed genotyping analyses at 5 loci: the 3 open reading frames (ORFs) 125, 94 and 75, each containing a variable number of tandem repeats (VNTR), and deletions in the 2 variable regions, VR14/15 and VR23/24. We categorized the WSSV genotype as {N125, N94, N75, ΔX14/15, ΔX23/24} where N is the number of repeat units in a specific ORF and ΔX is the length (base pair) of deletion within the variable region. We detected 4 WSSV genotypes, which were characterized by a full-length deletion in ORF94/95, a relatively small ORF75 and one specific deletion length in each variable region. There are 2 closely related genotypes in these 3 countries: {6125, del94, 375, Δ595014/15, Δ1097123/24} and {7125, del94, 375, Δ595014/15, Δ1097123/24}, where del is the full-length ORF deletion. In Saudi Arabia, 2 other related types of WSSV were also found: {6125, 794, 375, Δ595014/15, Δ1097123/24} and {8125, 1394, 375, Δ595014/15, Δ1097123/24}. The identical patterns of 3 loci in these 4 types indicate that they have a common lineage, and this suggests that the WSSV epidemics in these 3 countries were from a common source, possibly the environment.

Low numbers of repeat units in variable number of tandem repeats (VNTR) regions of white spot syndrome virus are correlated with disease outbreaks

White spot syndrome virus (WSSV) is the most important pathogen in shrimp farming systems worldwide including the Mekong Delta, Vietnam. The genome of WSSV is characterized by the presence of two major 'indel regions' found at ORF14/15 and ORF23/24 (WSSV-Thailand) and three regions with variable number tandem repeats (VNTR) located in ORF75, ORF94 and ORF125. In the current study, we investigated whether or not the number of repeat units in the VNTRs correlates with virus outbreak status and/or shrimp farming practice. We analysed 662 WSSV samples from individual WSSV-infected Penaeus monodon shrimp from 104 ponds collected from two important shrimp farming regions of the Mekong Delta: Ca Mau and Bac Lieu. Using this large data set and statistical analysis, we found that for ORF94 and ORF125, the mean number of repeat units (RUs) in VNTRs was significantly lower in disease outbreak ponds than in non-outbreak ponds. Although a higher mean RU number was observed in the improved-extensive system than in the rice-shrimp or semi-intensive systems, these differences were not significant. VNTR sequences are thus not only useful markers for studying WSSV genotypes and populations, but specific VNTR variants also correlate with disease outbreaks in shrimp farming systems.

White spot syndrome virus: Genotypes, Epidemiology and Evolutionary Studies

White spot syndrome virus (WSSV) is a pathogen that has emerged globally affecting shrimp populations. Comparison of WSSV genome have shown the virus to share a high genetic similarity except for a few variable genomic loci that has been employed as markers in molecular epidemiology studies for determining the origin, evolution and spread in different geographical regions. Molecular genotyping of WSSV are based on genomic deletions associated with ORF23/24 and ORF14/15 variable regions and the three variable number of tandem repeat regions, ORF75, ORF94 and ORF125. Studies show the prevalence of several genotypes for WSSV with particular genotypes being more prevalent than others in a given geographical area. Deletions associated with ORF23/24 and ORF14/15 variable regions have proven to be of evolutionary significance. Fitness and virulence studies on different genotypes of WSSV suggest that all the strains of WSSV are equally virulent, but the one with smaller genomic size is the fittest. Studies also have shown that mixed genotype infection of WSSV correlates with lower disease outbreaks. This review focuses on the genotyping studies that were undertaken in elucidating WSSV evolution and epidemiology.

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.

Indel-II region deletion sizes in the white spot syndrome virus genome correlate with shrimp disease outbreaks in southern Vietnam

Sequence comparisons of the genomes of white spot syndrome virus (WSSV) strains have identified regions containing variable-length insertions/deletions (i.e. indels). Indel-I and Indel-II, positioned between open reading frames (ORFs) 14/15 and 23/24, respectively, are the largest and the most variable. Here we examined the nature of these 2 indel regions in 313 WSSV-infected Penaeus monodon shrimp collected between 2006 and 2009 from 76 aquaculture ponds in the Mekong Delta region of Vietnam. In the Indel-I region, 2 WSSV genotypes with deletions of either 5950 or 6031 bp in length compared with that of a reference strain from Thailand (WSSV-TH-96-II) were detected. In the Indel-II region, 4 WSSV genotypes with deletions of 8539, 10970, 11049 or 11866 bp in length compared with that of a reference strain from Taiwan (WSSV-TW) were detected, and the 8539 and 10970 bp genotypes predominated. Indel-II variants with longer deletions were found to correlate statistically with WSSV-diseased shrimp originating from more intensive farming systems. Like Indel-I lengths, Indel-II lengths also varied based on the Mekong Delta province from which farmed shrimp were collected.

Longitudinal disease studies in small-holder black tiger shrimp ( Penaeus monodon) farms in Andhra Pradesh, India. III. A complex dynamic of WSSV infection and WSSV genotype distribution in farmed shrimp and wild crustaceans

A longitudinal study of white spot syndrome virus (WSSV) infection and disease was conducted from January to August 2007 in a cluster of 12 small-holder shrimp ( Penaeus monodon) ponds near Mallampudi in the Krishna District of Andhra Pradesh, India. Seven of the ponds had been sampled during the previous crop (Walker et al., 2011a); five adjacent ponds had not been sampled previously. Samples of mud were collected from the bottom of each pond at the commencement of the study. At intervals of approximately 10 days, farmed shrimp were sampled from the ponds and wild shrimp, crabs and plankton were sampled from inside ponds and from canals outside each pond. Of the 375 samples collected, 216 (57.6%) were WSSV-positive by Taqman PCR. The overall prevalence of WSSV infection varied significantly amongst sample types and was higher in farmed shrimp (76.0%) than for any other sample category (56% for crabs and plankton, 47% for wild shrimp, 25% for sediment). A wave of WSSV infection in plankton and wild crustaceans occurred across the study site, commencing at day 10, intensifying at day 20, subsiding at days 30 and 40, and passing by day 50. By day 60, only 2 ponds remained operational. WSSV-infection in plankton and wild crustacean samples collected from inside and outside these ponds again increased with a second peak at day 70. The pattern of WSSV infection in farmed shrimp appeared to follow the first wave of infection in plankton and wild crustaceans, with heavy viral genetic loads detected in most samples collected from days 30 to 80, including the final samples collected from 11 of the 12 ponds. Genotype analysis using the ORF94 variable number tandem repeat (VNTR) marker identified a very wide range of concurrently circulating WSSV genotypes (TRS1–TRS33) with multiple genotypes commonly detected in individual samples of all categories. Genotype TRS18, which had been associated with disease outbreaks in the previous crop, was not detected commonly in plankton or wild crustaceans. Although TRS18 was the most commonly detected genotype in farmed shrimp samples, it did not appear to be the cause of white spot disease outbreaks. There were several examples of simultaneous heavy infections with the same genotype in shrimp from several ponds but there was no clear pattern of association of a single WSSV genotype or a constellation of genotypes with disease outbreaks or pond abandonment.

Longitudinal disease studies in small-holder black tiger shrimp ( Penaeus monodon) ponds in Andhra Pradesh, India. II. Multiple WSSV genotypes associated with disease outbreaks in ponds seeded with uninfected postlarvae

A longitudinal study was conducted from January to August 2007 in a cluster of 61 small-holder shrimp ( Penaeus monodon) ponds at Mallampudi (16°25′29″ N, 81°19′13 ″ E) in the Krishna District of Andhra Pradesh, India. Exhaustive PCR testing of postlarvae collected from ponds at the time of seeding detected no evidence of white spot syndrome virus (WSSV) infection. However, during grow-out, disease outbreaks occurred in 42 of the ponds (68.9%) in which the mean and median crop durations were 42.8 days and 40.5 days, respectively. Only three of the 61 ponds (4.9%) were harvested after more than 120 days of culture. Of 41 ponds sampled at harvest, 35 (85.4%) were WSSV-positive by PCR, including 27 of 28 (96.4%) disease outbreak ponds, of 17 which were graded as heavy or moderate infections. Eight of 13 (61.5%) normal harvest ponds sampled were WSSV-positive at the time of harvest, of which seven (53.8%) were graded as light or very light infections. WSSV genotype analysis was conducted on samples from 35 ponds using the ORF94 variable number tandem repeat (VNTR) marker. In total, 20 different genotypes from TRS1-TRS25 (1 to 25 repeats) were detected. Multiple TRS genotypes were detected in 27 of the 35 ponds sampled (77.1%) and 73 of the 146 individual shrimp sampled (50.0%). The most evident temporal and spatial associations of WSSV genotypes with disease outbreaks were the dominance of genotype TRS18 in seven ponds located on the eastern side and genotype TRS8 in eleven ponds in the central region of the study area. The study indicated a high risk of exposure to WSSV infection during grow-out and that multiple WSSV genotypes were circulating simultaneously in the farming area. The spatial and temporal pattern of WSSV genotype distribution suggested transmission of infection within two clusters of ponds.

Genotyping of white spot syndrome virus (WSSV) geographical isolates from Brazil and comparison to other isolates from the Americas

White spot syndrome virus (WSSV) is a viral pathogen that has caused significant economic losses in shrimp farming. Variable-number tandem repeats (VNTRs) (open reading frame [ORF] 94, 125 and 75), a large deletion (ORF 23/24) and a transposase were proposed as molecular markers for genotyping. WSSV-infected shrimp Litopenaeus vannamei were collected in 2 Brazilian regions (Santa Catarina and Bahia) from 2005 to 2008. DNA was extracted and PCR of the variable regions was performed, followed by sequencing. All Santa Catarina samples showed the same number of repeats for the minisatellites analyzed. Bahia samples showed a different pattern for the regions, indicating that there are at least 2 different WSSV genotypes in Brazil. Both Brazilian isolates have an 11453 bp deletion in ORF 23/24 when compared with WSSV-TW (Taiwan), which has the full sequence for this locus. The Brazilian WSSV isolates were compared with WSSV isolates from other countries in the Americas (USA, Panama, Honduras, Mexico and Nicaragua); the repeat number patterns for the 3 VNTR regions analyzed were different between the Brazilian isolates and the other western-hemisphere isolates. This may be due to mutations in WSSV after its introduction into the different countries. Our results also show that WSSV found in Bahia and Santa Catarina very likely originated from different sources of contamination.

Genotyping of white spot syndrome virus prevalent in shrimp farms of India

DNA extracts from white spot syndrome virus (WSSV) that had infected post-larvae and juveniles of cultured shrimp, wild shrimp and crabs, which had been collected from different hatcheries and farms located along both the east and west coasts of India, revealed considerable variation in several previously identified WSSV DNA repeat regions. These include the 54 bp repeat in ORF 94, the 69 bp repeat in ORF 125 and the compound 45 and 57 bp repeat region in ORF 75. In ORF 94, 13 genotypes were observed with the number of repeats ranging from 2 to 16 units. While 7 repeat units were commonly observed (11.3%), no samples with 11 or 15 repeat units were found. In ORF 125, 11 types were found, with repeats ranging from 2 to 14 units. The most prevalent genotype displayed 4 repeat units (47.1%); no samples with 6 or 13 repeats were observed. The compound repeat region of ORF 75 displayed 6 different patterns of repeats. Samples with the same repeat pattern in one ORF did not always show identical repeat patterns in one or both of the other repeat regions. These data suggest that combined analysis of all 3 variable loci could be used to differentiate and characterize specific WSSV strains. For general epidemiological studies, the best marker with maximum variation is ORF 94, followed by ORF 125 and ORF 75. The 3 repeat regions above were used to compare WSSV genotypes from disease outbreaks on 3 sets of farms from different locations in the state of Andhra Pradesh. The genotypes within each farm set were almost identical, but differed between farm sets, suggesting that WSSV transmission occurred directly through virus carriers or water exchange between adjacent farms at each location. These findings show that genotyping can be a useful epidemiological tool for tracing the movement of WSSV within infected populations.