White Spot Syndrome Virus Genome Research Articles

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3–48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

White spot syndrome virus endogenous viral elements (EVE) revealed by circular viral copy DNA (cvcDNA) in shrimp

Circular, viral copy DNA (cvcDNA) can reveal the existence of endogenous viral elements (EVE) in shrimp genomic DNA. Here we describe isolation and sequencing of cvcDNA from a breeding stock of the whiteleg shrimp Penaeus vannamei. The stock was developed by onward breeding and selection for white spot syndrome virus (WSSV)-free individual survivors of white spot disease outbreaks. The stock exhibits high tolerance to WSSV. A pool of DNA extracted from 10 shrimp from this stock was subjected to cvcDNA isolation and amplification followed by high throughput sequencing. This revealed DNA fragments corresponding to locations covering much of the 300,000 bp WSSV genome. However, high frequency-read-fragments (HFRF) mapped to a surprisingly small region of approximately 1400 bp. We hypothesized that the HFRF reflected their selection due to provision of tolerance to WSSV. Four PCR primer sets were designed to cover the 1365 bp mapped region. One pair (Set 1) targeted the whole 1365 bp mapped region, while another 3 primer sets (Set 2–4), targeted regions within the 1365 bp target. All 4 primer sets were used with DNA samples from each of 36 shrimp from the same breeding stock (including the 10 used for cvcDNA preparation). Individual positive PCR results varied from shrimp to shrimp, ranging from only 1 primer set up to 4 primer sets. Only 1 specimen gave an amplicon of 1365 bp, while others gave single to multiple positive amplicons in both continuous and discontinuous regions. This indicated that the amplicons did not arise from contiguous targets and might vary in insertion length. The results also confirmed that none of the shrimp were infected with WSSV. Sequencing of the PCR amplicons of expected sizes revealed sequence identity to extant WSSV genomes. This data will be used to screen the breeding stock for EVE that provide WSSV tolerance.

Single-cell RNA-seq revealed heterogeneous responses and functional differentiation of hemocytes against white spot syndrome virus infection in Litopenaeus vannamei.

Current knowledge of shrimp hemocyte classification mainly comes from morphology, which hinder in-depth characterization of cell lineage development, functional differentiation, and different immune response of hemocyte types during pathogenic infections. Here, single-cell RNA sequencing was used for mapping hemocytes during white spot syndrome virus (WSSV) infection in Litopenaeus vannamei, identifying 16 cell clusters and evaluating their potential antiviral functional characteristics. We have described the dynamic balance between viral infestation and hemocyte immunity. And the functional differentiation of hemocytes under WSSV stimulation was further characterized. Our results provided a comprehensive transcriptional landscape and revealed the heterogeneous immune response in shrimp hemocytes during WSSV infection.

Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa.

White spot syndrome virus is a highly contagious pathogen affecting shrimp farming worldwide. The host range of this virus is primarily limited to crustaceans, such as shrimps, crabs, prawns, crayfish, and lobsters; however, several species of non-crustaceans, including aquatic insects, piscivorous birds, and molluscs may serve as the vectors for ecological dissemination. The present study was aimed at studying the faecal virome of domestic chickens (Gallus gallus domesticus) in Makhanda, Eastern Cape, South Africa. The cloacal swab specimens (n = 35) were collected from domestic chickens in December 2022. The cloacal swab specimens were pooled-each pool containing five cloacal swabs-for metagenomic analysis using a sequence-independent single-primer amplification protocol, followed by Nanopore MinION sequencing. While the metagenomic sequencing generated several contigs aligning with reference genomes of animal viruses, one striking observation was the presence of a White spot syndrome virus genome in one pool of cloacal swab specimens. The generated White spot syndrome virus genome was 273,795 bp in size with 88.5% genome coverage and shared 99.94% nucleotide sequence identity with a reference genome reported in China during 2018 (GenBank accession: NC_003225.3). The Neighbour-Joining tree grouped South African White spot syndrome virus genome with other White spot syndrome virus genomes reported from South East Asia. To our knowledge, this is the first report of a White spot syndrome virus genome generated from domestic chickens. The significance of White spot syndrome virus infection in domestic chickens is yet to be determined.

Pathogenicity of white spot syndrome virus (WSSV) after multiple passages in mud crab, Scylla olivacea

White spot syndrome virus (WSSV) is a highly virulent shrimp pathogen with a broad host range. Among the hosts, though mud crab, Scylla olivacea is reported to be more susceptible to WSSV than S. serrata and S. paramamosain, a detailed study on the pathogenicity and genome stability of the virus after multiple passages has yet to be reported. Firstly, to test the pathogenicity of the virus, WSSV was intramuscularly injected into healthy shrimp, Penaeus vannamei. Experimentally infected P. vannamei showed the first mortality at 36 h post-injection (hpi), followed by 100 % cumulative mortality in 7 days post-injection (dpi). However, S. olivacea injected with the WSSV inoculum derived from infected shrimp showed the first mortality at 48 hpi and a cumulative mortality of 70 % at the end of the ten days experiment. Subsequently, WSSV was sequentially passaged five times in Scylla olivacea to find out any change in the virulence of the virus in each passage. S. olivacea groups injected with 1st, second, third and fourth passages derived from the crab recorded the first mortality between 48 and 56 hpi and the cumulative mortality of 60 to 70 % at the end of the ten days experiment. Injection of WSSV inoculum in P. vannamei derived from multiple passages in S. olivaceae revealed the retention of the pathogenicity of the virus. Shrimp groups injected with WSSV derived from different passages showed first mortality between 24 and 36 hpi and cumulative mortality of 100 % between 6 and 7 dpi. The average viral load in the shrimp groups injected with WSSV inoculum derived from shrimp was 3.6 × 108, whereas in shrimp injected with the inoculum derived from 1st, third and fifth passages from crab showed 4.0 × 108, 4.7 × 108 and 4.3 × 108 copies per 100 ng DNA. Histological examination of the gill and stomach tissue of shrimp injected with inoculum prepared from shrimp as well as the inoculum derived from 1st, third and fifth passages in S. olivacea revealed characteristic pathological manifestations of the WSSV infection in gill and stomach tissues such as hypertrophied nuclei, Cowdry A-type inclusions as well as massive basophilic intranuclear inclusions. Further, to study the genome stability, the primers targeting highly variable regions of the WSSV genome (ORF94, ORF125, ORF75, variable region (VR) 14/15 and VR 23/24) were used to amplify WSSV derived from different passages and the amplified PCR products were sequenced. The sequence analysis revealed the WSSV genome stability after multiple passages in mud crab, S. olivacea.

Identification of Various InDel-II Variants of the White Spot Syndrome Virus Isolated from Frozen Shrimp and Bivalves Obtained in the Korean Commercial Market.

White spot syndrome virus (WSSV) poses a significant threat to the global shrimp industry. We investigated the presence of WSSV in frozen shrimp (n = 86) and shellfish (n = 185) from the Korean market (2010-2018). The detection rate of first-step polymerase chain reaction (PCR) in domestic shrimp was 36.8% (7/19), whereas that in imported shrimp was 0.01% (1/67). Furthermore, the WSSV genome was amplified from domestic bivalve mollusks by first- and second-step PCR with accuracies of 3.4% (5/147) and 15.6% (23/147), respectively. The genetic relatedness of InDel-II regions among WSSVs detected in domestic shrimp groups revealed four variants (777, 5649, 11,070 and 13,046 bp insertion or deletion), and imported shrimp groups had four variants (10,778, 11,086, 11,500 and 13,210 bp) compared with the putative ancestor WSSV strain. The 5649 bp variant was the dominant type among the WSSV variants detected in domestic shrimp (54.5%, 6/11). Notably, bivalve mollusks exhibited six variants (777, 5649, 5783, 5876, 11,070 and 13,046 bp), including four variants detected in shrimp, indicating that bivalve mollusks could facilitate WSSV tracking. In a challenge test, whiteleg shrimp (Litopenaeus vannamei) exhibited varying mortality rates, indicating a link between InDel-II deletion and viral replication. These findings highlight the complexity of WSSV transmission.

Cinnamaldehyde, a major component of Cinnamomum cassia Presl ethanol extract, has the potential to unlock the outbreak of WSSV

The white spot syndrome virus (WSSV) is rapid onset and high mortality, which has caused a devastating blow to shrimp culture around the world. However, there are no effective measures to prevent and control the WSSV. Thus, it is urgent to find a safe and efficient measures to prevent the outbreak of WSSV. Here, the anti-WSSV activities of 20 Chinese herbal medicine ethanol extracts were evaluated in Litopenaeus vannamei. Among these, the ethanol extract of cinnamon (Cinnamomum cassia Presl) had the highest inhibition rate of 91% on WSSV genome replication. Surprisingly, cinnamaldehyde, the main component of cinnamon extract, has a simple structure and could reduce the copy level of viral genome in shrimp, with the strongest inhibitory effect of 93% at 72 h, and more than 40% of diseased shrimp survived. Further experiments showed that cinnamon extracts and cinnamaldehyde could prolong the cumulative survival time of WSSV-infected larvae, and continuous treatment with cinnamaldehyde could maintain the replication of WSSV genome at a low level. In addition, cinnamon extracts and cinnamaldehyde could significantly inhibit the horizontal transmission of virions at 72 h by 71% and 42%, respectively. Obviously, this further reduces the risk of WSSV outbreak. Similarly, cinnamaldehyde pre-incubated shrimp and virions could improve the tolerance of shrimp to WSSV and reduce the infectivity of virions. Taken together, cinnamon extract and cinnamaldehyde could inhibit WSSV genome replication and reduce mortality rate of WSSV-infected shrimp. The results provided a scientific basis for the use of cinnamon extract and cinnamaldehyde to unlock the outbreak of WSSV, and were worthy of further study and consideration.

Discovery of a substance that enhances the immune response in Litopenaeus vannamei against WSSV: Baicalein, a major component of Scutellaria baicalensis

White spot syndrome virus (WSSV) is a pathogen that can infect many crustaceans, and that has caused huge economic losses in the shrimp industry. No effective prevention and control measures are currently available. In the present study, the ethanolic extract of Scutellaria baicalensis (SBEE) at 200 mg/L provided >50% protection for WSSV-infected shrimp larvae, and baicalein at 100 μM, one of the main anti-WSSV active substances in S. baicalensis, was 66.7% effective. When shrimp larvae were infected with WSSV after 8 h of SBEE and baicalein preincubation, the respective copy numbers of the viral genome were decreased by 31% and 46% versus the control group. Similarly, WSSV genome copy number reductions of 49% and 55% were observed in WSSV-infected shrimp larvae after 72 h of SBEE and baicalein immersion, respectively. In addition, SBEE or baicalein continuous treatment significantly reduced the viral loads in vivo, effectively blocking the horizontal transmission of WSSV between hosts. SBEE or baicalein also enhanced the expression of Toll4, Dorsal and antimicrobial peptides (AMPs) to restrict WSSV infection. In conclusion, S. baicalensis and baicalein are promising feed additives and immune enhancers for the control of WSSV infection.

Creation of Standard DNA for Detection and Quantification of White Spot Syndrome Virus in Shrimps by Real-time PCR (qPCR)

White spot syndrome virus (WSSV) is a double stranded DNA virus that causes WSS diseases for many crustaceans including the Penaeidae family shrimp. In this study, standard DNA for detection and quantification of WSSV by real-time polymerase chain reaction (qPCR) method was created. A specific gene fragment of 126 bp from WSSV genome was successfully amplified by PCR and cloned into pGEM-T vector. qPCR calibration curves using the created recombinant pGEM vector harboring 126 bp gene fragment as the standard DNA at concentrations of 3x102 to 3x108 copies/mL showed to have good linear regression with an efficiency of 99.1%, correlation coefficient R2 of 0.998 and the slope of -3.344. The recombinant vector was also used as a positive standard for detection and quantification of WSSV in a number of WSSV-infected shrimp samples and the values ranging from 3.69x103 copies/mL to 1.25x108 copies/mL WSSV were found in the collected samples.

Exosomes from WSSV-infected shrimp contain viral components that mediate virus infection.

Exosomes have been described as vesicles that mediate intercellular communication and thus affect normal and pathological processes. Furthermore, many viruses have been reported to deliver viral components to host cells through exosomes. However, the roles of exosomes in invertebrates response to virus infection are poorly understood. In this study, we found that exosomes purified from white spot syndrome virus (WSSV)-infected hemocytes of shrimp could promote viral replication. These exosomes contained WSSV genomic DNA and nucleocapsid protein VP15, suggesting that exosomes can transfer viral genetic materials between cells, although the exosomes did not have similar infection ability to viruses. Remarkably, in exosomes WSSV DNA was bound to VP15 protein, and moreover VP15 silencing significantly suppressed WSSV infection and reduced the WSSV genome fragments in exosomes, indicating that the presence of VP15 is required for the packing of WSSV DNA inside the exosomes and thereby assists virus to complete immune escape. The above results not only contribute to elucidation of the infection and transmission mechanisms of WSSV, but are also of great significance for further study of virus-host interaction and reasonable prevention measures. Taken together, our findings provide a novel insight into the regulation of virus transmission via exosomes and highlight potential therapeutic strategies.

Three Draft Genome Sequences of White Spot Syndrome Virus from India.

ABSTRACTWhite spot syndrome virus (WSSV) is a pathogen causing significant economic losses to shrimp aquaculture worldwide. Previously, five genome sequences of the virus from farmed shrimp (Penaeus vannamei and Penaeus monodon) in India were reported, all originating from farms located on the east coast of the country. Here, we report three new and distinct WSSV genome sequences, two from shrimp (P. vannamei) farmed on the west coast of India and the third from the east coast.

Genome assembly and annotation of the white spot syndrome virus - infected pacific white shrimp (Ltopenaeus vannamei) in Vietnam

Pacific white shrimp (Penaeus vannamei or Litopenaeus vannamei) is native to South America, high economic value, and widely cultivated in the world and Vietnam. Over the last two decades, viral diseases have seriously threatened the shrimp aquaculture industry. Among the viral diseases, white spot syndrome virus (WSSV) is the most important viral pathogens of shrimp farming. WSSV causes a cumulative mortality can reach 100% within 3–10 days. Genome sequencing and assembly has been an important step for deciphering molecular mechanisms and accelerating genetic improvements of traits of interest in economically important species. This study aims at constructing and annotating the genome of white spot syndrome virus - infected Pacific white shrimp in Vietnam. The whole genome sequencing data was de novo assembled using SOAP denovo2 to obtained draft genome of WSSV- infected L. vannamei shrimp. The draft genome contained 3,180,049 scaffolds (genome size ~1.67 Gb) with the length arranging from 200 bp to 137,569 bp and with N50 as 616 bp. Applying gene prediction method, we have been able to identify 187,948 putative genes. The results have shown that 33,611 genes were annotate in NT database and 133,548 genes were annotated in UniProtKB/Swissprot database. These results are only the initial information about white spot syndrome virus - infected Pacific white shrimp but they are really important for future studies relating to white spot syndrome virus – resistance L. vannamei shrimp in Vietnam.

G-Quadruplex Formation in a Putative Coding Region of White Spot Syndrome Virus: Structural and Thermodynamic Aspects.

White spot disease (WSD) is one of the most devastating viral infections of crustaceans caused by the white spot syndrome virus (WSSV). A conserved sequence WSSV131 in the DNA genome of WSSV was found to fold into a polymorphic G‐quadruplex structure. Supported by two mutant sequences with single G→T substitutions in the third G4 tract of WSSV131, circular dichroism and NMR spectroscopic analyses demonstrate folding of the wild‐type sequence into a three‐tetrad parallel topology comprising three propeller loops with a major 1 : 3 : 1 and a minor 1 : 2 : 2 loop length arrangement. A thermodynamic analysis of quadruplex formation by differential scanning calorimetry (DSC) indicates a thermodynamically more stable 1 : 3 : 1 loop isomer. DSC also revealed the formation of additional highly stable multimeric species with populations depending on potassium ion concentration.

In Litopenaeus vannamei, the cuticular chitin-binding proteins LvDD9A and LvDD9B retard AHPND pathogenesis but facilitate WSSV infection

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication.

Genome reconstruction of white spot syndrome virus (WSSV) from archival Davidson\u2019s-fixed paraffin embedded shrimp (Penaeus vannamei) tissue

Formalin-fixed paraffin-embedded (FFPE) tissues are a priceless resource for diagnostic laboratories worldwide. However, DNA extracted from these tissues is often not optimal for most downstream molecular analysis due to fragmentation and chemical modification. In this study, the complete genome of white spot syndrome virus (WSSV) was reconstructed from ~ 2-year-old archived Davidson’s-fixed paraffin-embedded (DFPE) shrimp tissue using Next Generation Sequencing (NGS). A histological analysis was performed on archived DFPE shrimp tissue and a sample showing a high level of WSSV infection was selected for molecular analysis. The viral infection was further confirmed by molecular methods. DNA isolated from DFPE and fresh frozen (FF) tissues were sequenced by NGS. The complete genome reconstruction of WSSV (~ 305 kbp) was achieved from both DFPE and FF tissue. Single nucleotide polymorphisms, insertion and deletions were compared between the genomes. Thirty-eight mutations were identified in the WSSV genomes from the DFPE and FF that differed from the reference genome. This is the first study that has successfully sequenced the complete genome of a virus of over 300 kbp from archival DFPE tissue. These findings demonstrate that DFPE shrimp tissue represents an invaluable resource for prospective and retrospective studies, evolutionary studies and opens avenues for pathogen discovery.

WSV152 induces apoptosis and promotes viral replication in Litopenaeus vannamei

Previous studies have indicated that white spot syndrome virus (WSSV) infection induces apoptosis in many shrimp organs. However, the mechanism by which WSSV causes host apoptosis remains largely unknown. In this study, we demonstrated the function of wsv152, the first mitochondrial protein identified as encoded by WSSV. Glutathione S-transferase pulldown and co-immunoprecipitation analysis revealed that wsv152 interacts with the shrimp mitochondrial protein cytochrome c oxidase 5a (COX5a), a subunit of the COX complex. We also found that wsv152 expression significantly increased the rate of apoptosis, suggesting a role of wsv152 in WSSV-induced apoptosis in shrimp. Knockdown of wsv152 in vivo led to downregulation of several apoptosis-related shrimp genes, including cytochrome c, apoptosis-inducing factor and caspase-3. Suppression of wsv152 also resulted in significant reductions in the number of WSSV genome copies in tissues and in the mortality of WSSV-infected shrimp. Together, these results suggest that wsv152 targets host COX5a and is associated with the expression profiles of apoptosis-related shrimp genes. Wsv152 is likely also involved in WSSV-induced apoptosis, thereby facilitating virus infection and playing a complex role in WSSV pathogenesis.

White spot syndrome virus down-regulates expression of histones H2A and H4 of Penaeus vannamei to promote viral replication.

The white spot syndrome virus (WSSV) is a highly lethal and contagious pathogen to most cultivated shrimp species. The WSSV genome contains the ICP11 gene and its encoded protein acts as a multifunctinal DNA mimic protein that disrupts the nucleosome assembly by binding to the histone proteins H2A and H3. In addition, WSSV provokes severe nuclear hypertrophy and DNA damage. However, little is known about the influence of WSSV on the expression of the host's genes encoding for histones. Therefore, we investigated the effect of WSSV infection on the expression of the genes encoding histones in shrimp Penaeus vannamei. An RT-qPCR assay was performed to evaluate the temporal expression of H2A and H4 transcripts in the shrimp. Significant changes were observed in the expression of these genes, which coincided with the dynamics of replication of the virus. H2A reached its maximum expression levels at 12 hpi. Thus, it may be suggested that this is a viral strategy to evade the host's immune response in order to promote viral replication.

Glutamine Metabolism in Both the Oxidative and Reductive Directions Is Triggered in Shrimp Immune Cells (Hemocytes) at the WSSV Genome Replication Stage to Benefit Virus Replication

White spot syndrome virus (WSSV) is the causative agent of a shrimp disease that has caused huge global economic losses. Although its pathogenesis remains poorly understood, it has been reported that in the shrimp immune cells (hemocytes) targeted by WSSV, the virus triggers both the Warburg effect and glutamine metabolism at the WSSV genome replication stage (12 h post infection). Glutamine metabolism follows two pathways: an oxidative pathway mediated by α-KGDH (α-ketoglutarate dehydrogenase) and an alternative reductive pathway mediated by IDH1 and IDH2 (isocitrate dehydrogenase 1 and 2). Here we used isotopically labeled glutamine ([U-13C]glutamine and [1-13C]glutamine) as metabolic tracers to show that, at the replication stage, both the oxidative and reductive glutamine metabolic pathways were activated. We further show that the mRNA expression levels of α-KGDH and IDH1 were increased in WSSV-infected shrimps and that silencing of α-KGDH, IDH1, and IDH2 with their respective dsRNAs led to a decrease in WSSV gene expression and WSSV replication. Taken together, our findings provide new evidence for WSSV-induced metabolic reprogramming in hemocytes and demonstrate its importance in virus replication.

Characterization of putative proteins encoded by variable ORFs in white spot syndrome virus genome

BackgroundWhite Spot Syndrome Virus (WSSV) is an enveloped double-stranded DNA virus which causes mortality of several species of shrimp, being considered one of the main pathogens that affects global shrimp farming. This virus presents a complex genome of ~ 300 kb and viral isolates that present genomes with great identity. Despite this conservation, some variable regions in the WSSV genome occur in coding regions, and these putative proteins may have some relationship with viral adaptation and virulence mechanisms. Until now, the functions of these proteins were little studied. In this work, sequences and putative proteins encoded by WSSV variable regions were characterized in silico.ResultsThe in silico approach enabled determining the variability of some sequences, as well as the identification of some domains resembling the Formin homology 2, RNA recognition motif, Xeroderma pigmentosum group D repair helicase, Hemagglutinin and Ankyrin motif. The information obtained from the sequences and the analysis of secondary and tertiary structure models allow to infer that some of these proteins possibly have functions related to protein modulation/degradation, intracellular transport, recombination and endosome fusion events.ConclusionsThe bioinformatics approaches were efficient in generating three-dimensional models and to identify domains, thereby enabling to propose possible functions for the putative polypeptides produced by the ORFs wsv129, wsv178, wsv249, wsv463a, wsv477, wsv479, wsv492, and wsv497.

White spot syndrome virus (WSSV) in cultured juvenile blue crabs Callinectes sapidus: oral versus injection exposure, and feeding frequency effects.

The efficacy of oral versus injection exposure and the effect of feeding frequency on the transmission of white spot syndrome virus (WSSV) in cultured juvenile blue crabs Callinectes sapidus were investigated. Crabs in Group 1 (G-1, n = 48) were exposed once orally to 100 mg of WSSV-infected shrimp tissue mg-1 of body weight (BW). The oral inoculum contained 2.6 × 109 WSSV genome copies mg-1 tissue. Group 2 (G-2, n = 46) received the same dosage once weekly for 5 wk. Group 3 (G-3, n = 12) was injected with 0.01 ml (2.6 × 107 genome copies 0.01 ml-1) WSSV inoculum g-1 BW. Group 4 (G-4, n = 12) was injected with 0.01 ml WSSV-negative shrimp serum and saline mixture g-1 BW. Dead and moribund animals were frozen at -80°C. After 37 d, all remaining crabs were frozen. Genomic DNA from gill tissue was evaluated for the presence and quantity of WSSV using TaqMan real-time PCR. All G-3 animals died and tested positive. No G-4 animals died or tested positive. In the fed groups, WSSV prevalence was approximately 16%, but viral load was higher and survival was lower in G-2 compared to G-1. Injected animals carried a higher viral load than fed animals, and dead animals had higher viral loads than live animals. Blue crab juveniles are susceptible to WSSV, but oral exposure does not efficiently transmit WSSV in juvenile blue crabs. Some animals can die from WSSV if repeatedly exposed.