White Spot Syndrome Virus Isolates Research Articles

In Silico Evaluation of the PCR Performance of Different Tests for Detection of WSSV

In this study, the primers of different protocols for the detection of White Spot Syndrome Virus (WSSV) were analyzed in silico to evaluate their potential performance in PCR. As with any biological entity, this virus evolves constantly. Thus, this analysis showed that a few primers, including those recommended by the World Organization for Animal Health (WOAH), might mismatch with some isolates of WSSV, specially with isolates more recently sequenced. Furthermore, a set of primers recommended by WOAH, showed the potential to self-dimer and form hairpin loop structures, which could affect the efficiency of PCR, resulting in an inaccurate diagnostic result. From our perspective, and considering the evolutionary trajectory of this virus, it may be time for the WOAH to update the PCR protocols recommended for WSSV detection, which remains as a highly prevalent and lethal virus.

Evolutionary genomics of white spot syndrome virus

White spot syndrome virus (WSSV) has been one of the most devastating pathogens affecting the global shrimp industry since its initial outbreaks in Asia in the early 1990s. In this study, we recovered 13 complete metagenome-assembled genomes (MAGs) of Japanese WSSV isolates and 30 draft WSSV MAGs recovered from publicly available sequencing data, to investigate the genomic evolution of WSSV. Phylogenetic analysis revealed two major phylotypes, designated phylotypes I and II. Bayesian divergence time estimates placed the divergence time of the two phylotypes between 1970 and the early 1980s, with an estimated substitution rate of 1.1 × 10–5 substitutions per site per year, implying the existence of pre-pandemic genetic diversity of WSSV in Asia. Based on this scenario, phylotype I was responsible for the 1990s pandemic and spread worldwide, whereas phylotype II was localized in Asia and infiltrated Australia. Two cross-phylotype recombinant lineages were identified, which demonstrate the role of genomic recombination in generating the genetic diversity of WSSV. These results provide important insights into the evolution of WSSV and may help uncover the ultimate origins of this devastating pathogen.

Virulence and genetic differences among white spot syndrome virus isolates inoculated in Penaeus vannamei.

White spot syndrome virus (WSSV) infects several economically important aquaculture species, and has caused significant losses to the industry. This virus belongs to the Nimaviridae family and has a dsDNA genome ranging between 257 and 309 kb (more than 20 isolate genomes have been fully sequenced and published to date). Multiple routes of infection could be the cause of the high virulence and mortality rates detected in shrimp species. Particularly in Penaeus vannamei, differences in isolate virulence have been observed, along with controversy over whether deletions or insertions are associated with virulence gain or loss. The pathogenicity of 3 isolates from 3 localities in Mexico (2 from Sinaloa: 'CIAD' and 'Angostura'; and one from Sonora: 'Sonora') was evaluated in vivo in whiteleg shrimp P. vannamei infection assays. Differences were observed in shrimp mortality rates among the 3 isolates, of which Sonora was the most virulent. Subsequently, the complete genomes of the Sonora and Angostura isolates were sequenced in depth from infected shrimp tissues and assembled in reference to the genome of isolate strain CN01 (KT995472), comprising 289350 and 288995 bp, respectively. Three deletion zones were identified compared to CN01, comprising 15 genes, including 3 envelope proteins (VP41A, VP52A and VP41B), 1 non-structural protein (ICP35) and 11 other encoding proteins whose function is currently unknown. In addition, 5 genes (wsv129, wsv178, wsv204, wsv249 and wsv497) presented differences in their repetitive motifs, which could potentially be involved in the regulation of gene expression, causing virulence variations.

Susceptibility of five different sizes of pathogenfree Litopenaeus vannamei to white spot syndrome virus (WSSV) by intramuscular inoculation

White spot syndrome virus (WWSV) has become one of the most widespread causes of mortality in commercial shrimp farming. In the present study, we used PCR to determine the shrimp infectious dose 50% endpoint (SID50 ml-1) of a Chinese isolate of WSSV in 5 different sizes of pathogen-free Litopenaeus vannamei inoculated intramuscularly. The lethal dose 50% endpoint (LD50 ml-1) was also determined from the percentage of dead shrimp. The LD50 ml-1 for 2, 4, 6, 8, and 10 cm shrimp were 104.68, 105.7, 106.70, 107.75, and 108.81, respectively, and the SID50 ml-1 were 104.68, 105.70, 106.90, 107.75, and 108.94, respectively. There was no significant difference between the LD50 ml-1 and SID50 ml-1 for each shrimp size, which indicated that all infected shrimp died. The lethal and infectious titer decreased about 1 log10 as shrimp size decreased 1 grade. These data clearly indicate that adult shrimp were more susceptible to WSSV than juvenile shrimp. The horizontal comparison showed that the amount of virus in the shrimp organs increased over the experimental period. The vertical comparison showed that virus quantity was lowest in the organs of 10 cm shrimp and highest in 2 cm shrimp, which indicates that the smaller shrimp had higher levels of viral replication. Hence, the optimal size for WSSV challenge in shrimp inoculated intramuscularly was 2 cm. The determination of virus titers in different sizes of shrimp represents a step towards creating strategies to reduce the negative impacts of WSSV in the aquaculture industry.

White spot syndrome virus in decapods from Mississippi Sound, USA, and susceptibility of Palaemonetes pugio and Uca panacea to a Chinese isolate.

The presence and quantity of white spot syndrome virus (WSSV) was surveyed using TaqMan real-time PCR to assess the extent of the virus in Mississippi Sound, USA. A total of 3577 wild decapods comprising 11 species was collected between November 2012 and August 2015: WSSV was present in 10 of the 11 species. Prevalence ranged from 5.1% in Uca rapax to 38.8% in U. spinicarpa. Viral load ranged from 1.8 to 7.3 log10 copies of WSSV µg-1 total DNA. Two Gulf species, Palaemonetes pugio and U. panacea, were injected with a series of doses of a virulent WSSV isolate from China to determine relative susceptibility and virulence because continuing translocation of highly pathogenic isolates of WSSV poses risk to native species. Survival was 0-65% for P. pugio and 5-60% for U. panacea. Median survival time was lower for P. pugio than U. panacea at all doses. Mean (±SD) lethal load was 9.0 ± 8.9 log copies of WSSV µg-1 total DNA in P. pugio and 8.2 ± 8.3 in U. panacea. Mean viral load in survivors was higher in U. panacea than in P. pugio (5.8 ± 6.1 vs. 3.2 ± 3.0 log copies of WSSV µg-1 total DNA); mean viral load was lower in wild individuals of those species (2.9 ± 3.2 for P. pugio and 4.9 ± 5.0 for U. panacea). U. panacea is potentially more tolerant of WSSV than P. pugio and may serve as an important reservoir host in the community.

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.

Alternative PCR primers for genotyping of Brazilian WSSV isolates.

White spot syndrome virus (WSSV) is one of the major challenges faced by global shrimp farming in recent decades. The characterization of WSSV genetic variability has been used to determine viral dispersion and is a promising method to determine the association between genotype and virulence. The major variable regions that have been used as markers to differentiate the WSSV genomes include the VNTR loci inside ORF94, ORF75, ORF125, and insertions/deletions interspersing ORF14/15 and ORF23/24. The primers used to amplify these regions were described at least 10 years ago, but some of them do not work efficiently to identify new WSSV variants. The objective of this work was to develop improved PCR primers for WSSV genotyping based on sequence alignments that include new sequences described in recent years. We validated these new primers in a pilot study to verify the genetic variability of the WSSV in Rio Grande do Norte state (northeast Brazil), and efficiency was compared to that of other previously described primers. We confirmed that the primers we developed were more efficient for genotype Brazilian WSSV isolates, enabling us to genotype a larger number of samples. In addition, our results also introduce new data about the genetic characterization of the WSSV isolates that occur in the northeastern region of Brazil.

A new strain of white spot syndrome virus affecting Litopenaeus vannamei in Indian shrimp farms.

White spot syndrome virus (WSSV)-infected shrimp samples collected from grow-out ponds located at Nellore, Andhra Pradesh, India, showed WSSV negative and positive by PCR using primer sets specific to ORF119 and VP28 gene of WSSV, respectively. This indicated the deletion of genetic fragments in the genome of WSSV. The WSSV isolate along with lab strain of WSSV was subjected to next-generation sequencing. The sequence analysis revealed a deletion of 13,170 bp at five positions in the genome of WSSV-NS (new strain) relative to WSSV-TH and WSSV-LS (lab strain). The PCR analysis using the ORF's specific primer sets revealed the complete deletion of 10ORFs in the genome of WSSV-NS strain. The primer set was designed based on sequence covering ORF161/162/163 to amplify a product of 2,748bp for WSSV-LS and 402bp for WSSV-NS. Our surveillance programme carried out since 2002 revealed the replacement of WSSV-LS by WSSV-NS in Indian shrimp culture system.

Direct sequencing of the white spot syndrome virus from Brazil: Genome assembly and new insights on phylogeny

White spot syndrome virus (WSSV) has been the cause of great economic losses in world shrimp farming. In this work the genome of a Brazilian WSSV isolate was determined from direct sequencing of total DNA extracted from an infected whiteleg shrimp, and assembled based on a chimera template approach. Comparisons between WSSV-BR and other isolates revealed that the Brazilian virus has a relatively small genome, and is very similar to isolates from Thailand and Mexico. A phylogenetic relationship using different approaches has demonstrated that these isolates share a common evolutionary history. An analysis of conflicting phylogenetic signals also considering genomes of other isolates revealed that the evolutionary history of WSSV may be related to recombination events. We observed that these events can also be traced at some level by analyzing the homologous regions in the WSSV genome. The existence of recombination events introduces a new point of view that must be considered in the evolutionary history of WSSV.

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.

Comparative genomic analysis of three white spot syndrome virus isolates of different virulence.

Three white spot syndrome virus (WSSV) isolates of different virulence were identified in our previous study, the high-virulent strain WSSV-CN01, the moderate-virulent strain WSSV-CN02 and the low-virulent strain WSSV-CN03. In this study, the genomes of these three WSSV isolates were sequenced, annotated and compared. The genome sizes for WSSV-CN01, WSSV-CN02, and WSSV-CN03 are 309,286, 294,261, and 284,148bp, bearing 177, 164, and 154 putative protein-coding genes, respectively. The genomic variations including insertions, deletions, and substitutions were investigated. Thirty four genes show >20% variation in their sequences in WSSV-CN02 or WSSV-CN03, in comparison with WSSV-CN01, including six envelope protein genes (wsv237/vp41A, wsv238/vp52A, wsv338/vp62, wsv339/vp39, wsv077/vp36A, and wsv242/vp41B), and two immediate-early genes (wsv108 and wsv178). The genomic variations among WSSV isolates of different virulence, especially those in the coding regions, certainly provide new insight into the understanding of the molecular basis of WSSV pathogenesis.

Genotyping WSSV isolates from northwestern Mexican shrimp farms affected by white spot disease outbreaks in 2010-2012.

White spot disease (WSD) causes high mortality in cultured shrimp throughout the world. Its etiologic agent is the white spot syndrome virus (WSSV). The genomic repeat regions ORF 75, ORF 94, and ORF 125 have been used to classify WSSV isolates in epidemiological studies using PCR with specific primers and sequencing. The present study investigated the variation in nucleotide sequences from 107, 150, and 143 isolates of WSSV collected from Litopenaeus vannamei shrimp ponds with WSD outbreaks in northwestern Mexico during the period 2010-2012, in the genomic repeat regions ORFs 75, 94, and 125, respectively. The haplotypic nomenclature for each isolate was based on the number of repeat units and the position of single nucleotide polymorphisms on each ORF. We report finding 17, 43, and 66 haplotypes of ORFs 75, 94, and 125, respectively. The study found high haplotypic diversity in WSSV using the complete sequences of ORFs 94 and 125 as independent variables, but low haplotypic diversity for ORF 75. Different haplotypes of WSSV were found from region-to-region and year-to-year, though some individual haplotypes were found in different places and in more than one growing cycle. While these results suggest a high rate of mutation of the viral genome at these loci, or perhaps the introduction of new viral strains into the area, they are useful as a tool for epidemiological surveys. Two haplotypes from some of the ORFs in the same shrimp were encountered, suggesting the possibility of multiple infections.

Variations of structural protein sequences among geographical isolates of white spot syndrome virus

White spot syndrome virus (WSSV) is the causative agent of a disease that causes severe mortalities in cultured shrimp worldwide. The sequence of five structural genes of an Indian isolate of WSSV was compared with sequences from other WSSV isolates deposited in GenBank. Among the structural genes analyzed, the sequences of vp28 and vp19 had maximum divergence with nucleotide changes at ten different positions from twenty eight submissions for vp28 and six changes from seventeen submissions for vp19. The vp68, vp26, and vp281 genes were found to be highly conserved between isolates.

Stability of the WSSV ORF94 VNTR genotype marker during passage in marine shrimp, freshwater crayfish and freshwater prawns.

The white spot syndrome virus (WSSV) genome contains 3 variable number tandem repeat (VNTR) regions, located in open reading frame (ORF) 75, ORF94 and ORF125, which have been employed for molecular epizootiological studies. A previous report suggested that the ORF 94 VNTR is highly unstable, varying in the number of tandem repeats during single passages from shrimp to other crustaceans. As such rapid variations would have profound implications for the interpretation of molecular epizootiological data, we re-examined the stability of the ORF94 VNTR. Two WSSV isolates with different ORF94 VNTR genotypes (TRS5 and TRS7) were obtained from disease outbreaks in farmed black tiger shrimp Penaeus monodon in Indonesia. High titre stocks of each virus were produced by injection in specific pathogen-free (SPF) Pacific white shrimp Litopenaeus vannamei with filtered infected tissue extracts, and the genotypes were confirmed. Each stock (macerated tissue) was then used to feed SPF Pacific white shrimp, freshwater crayfish (Cherax sp.) and freshwater prawns Macrobrachium rosenbergii through 3 successive passages involving alternative hosts at each level. Taqman real-time PCR was conducted on samples from each group to confirm infection and quantify viral genetic loads. ORF94 VNTR genotype analysis conducted on samples from each of the 43 passage groups indicated no variations in the VNTR number in either genotype TRS5 or genotype TRS7. This finding is contrary to the previous report and suggests that ORF94 VNTR are stable during multiple passages in these crustaceans.

Sequence analysis of ORF94 in different White Spot Syndrome Virus (WSSV) isolates of Iran

White spot syndrome virus (WSSV) is a pathogen that causes high mortality in shrimp culture in the whole world. Sequence analysis of WSSV has shown similarity of WSSV isolates in different countries with exception of a few variable genomic loci. This study investigated the sequence variation of some Iranian WSSV isolates and previously identified isolates. Samples were collected during target surveillance and were feed, broodstock, postlarvae, artemia, crabs, and wild and cultured shrimp of northern Persian Gulf (Boushehr and Khuzestan provinces). The open reading frame (ORF) 94 sequence of different Iranian WSSV isolates were amplified using specific primers from positive samples. The ORFs 94 sequence of positive samples were sequenced and registered in the Gene Bank and then compared to other WSSV isolates. The number of repeat units in ORF94 showed that WSSV isolates were varied in number. There are SNPs (G and T) in position 48 of RUs that varies in different Boushehr and Khuzestan isolates. Also these sequences were compared to Gene Bank WSSV isolates and showed a high similarity (>90%) to Southeast Asian countries. To our knowledge this is the first report of sequence analysis in Iranian WSSV isolatesapplications.

Analysis of the complete nucleotide sequence of a white spot syndrome virus isolated from pacific white shrimp

The fourth complete genome sequence of the white spot syndrome virus (WSSV) from Pacific white shrimp (Litopenaeus vannamei) isolated from Korea (WSSV-KR) was determined. The genome is composed of 295,884 bp encompassing 515 open reading frames (ORFs) among which 90 showed no sequence homology with any known protein in BLAST searches. The remaining 425 ORFs encode functional proteins including enzymes for nucleic acid metabolism, DNA replication and transcription, and several major structural proteins. Dot plot and linear comparisons of WSSV Korean strain with other WSSV isolates showed overall similarity but with some areas of sequence difference and one large deletion area.

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.

Genotyping of white spot syndrome virus in Chinese cultured shrimp during 1998–1999

Recent studies showed that white spot syndrome virus (WSSV) isolates from different geographic locations share a high genetic similarity except the variable regions in ORF23/24 and ORF14/15, and variable number of tandem repeats (VNTR) within ORF94. In this study, genotyping was performed according to these three variable regions among WSSV isolates collected during 1998/1999 from Southern China. These WSSV isolates contain a deletion of 1168, 5657, 5898, 9316 and 11093 bp, respectively in the variable region ORF23/24 compared with WSSV-TW, and a deletion of 4749 or 5622 bp in the variable region ORF14/15 relative to TH-96-II. Four types of repeat units (RUs) (6, 8, 9 and 13 RUs) in ORF94 were detected in these isolates, with the shortest 6 RUs as the most prevalent type. Our results provide important information for a better understanding of the spatio-temporal transmission mode and the WSSV genetic evolution lineage.

Molecular cloning and recombinant expression of the VP28 carboxyl-terminal hydrophilic region from a brazilian white spot syndrome virus isolate

In the present study, a fragment of the VP28 coding sequence from a Brazilian WSSV isolate (BrVP28) was cloned, sequenced and expressed in E. coli BL21(DE3) pLysS strain in order to produce the VP28 carboxyl-terminal hydrophilic region. The expression resulted in a protein of about 21 kDa, which was purified under denaturing conditions, resulting in a final highly purified BrVP28 preparation. The recombinant protein obtained can be used in several biotechnology applications, such as the production of monoclonal antibodies which could be used in the development of diagnostic tools as well as in the studies on the characterization of white spot syndrome virus (WSSV) isolated in Brazil.

Evolutionary trajectory of white spot syndrome virus (WSSV) genome shrinkage during spread in Asia.

BackgroundWhite spot syndrome virus (WSSV) is the sole member of the novel Nimaviridae family, and the source of major economic problems in shrimp aquaculture. WSSV appears to have rapidly spread worldwide after the first reported outbreak in the early 1990s. Genomic deletions of various sizes occur at two loci in the WSSV genome, the ORF14/15 and ORF23/24 variable regions, and these have been used as molecular markers to study patterns of viral spread over space and time. We describe the dynamics underlying the process of WSSV genome shrinkage using empirical data and a simple mathematical model.Methodology/Principal FindingsWe genotyped new WSSV isolates from five Asian countries, and analyzed this information together with published data. Genome size appears to stabilize over time, and deletion size in the ORF23/24 variable region was significantly related to the time of the first WSSV outbreak in a particular country. Parameter estimates derived from fitting a simple mathematical model of genome shrinkage to the data support a geometric progression (k<1) of the genomic deletions, with k = 0.371±0.150.Conclusions/SignificanceThe data suggest that the rate of genome shrinkage decreases over time before attenuating. Bioassay data provided support for a link between genome size and WSSV fitness in an aquaculture setting. Differences in genomic deletions between geographic WSSV isolates suggest that WSSV spread did not follow a smooth pattern of geographic radiation, suggesting spread of WSSV over long distances by commercial activities. We discuss two hypotheses for genome shrinkage, an adaptive and a neutral one. We argue in favor of the adaptive hypothesis, given that there is support for a link between WSSV genome size and fitness.