White Spot Syndrome Virus DNA Research Articles

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.

Heat shock protein 90 of Pacific white shrimp (Litopenaeus vannamei) is possibly involved in promoting white spot syndrome virus infection

Viruses cause up to 60% of disease-associated losses in shrimp aquaculture, and the white spot syndrome virus (WSSV) is a major viral pathogen in shrimp. Heat shock proteins (HSPs) are host chaperones that help promote many viral infections. We investigated the involvement of Litopenaeus vannamei (Lv) HSP90 in WSSV infections. Expression of LvHSP90 at the transcript and protein levels were upregulated after WSSV infection. Silencing LvHSP90 resulted in the increased cumulative mortality rate and the reduction of circulating hemocytes. The inhibition of LvHSP90 also induced the expression of apoptosis-related genes which indicated the induction of apoptotic pathway and might lead to shrimp death. However, lower the number of WSSV-infected cells and viral copy numbers were detected in the LvHSP90-silenced shrimp compared with those of the controls, corresponding with significantly decreased expressions of viral genes, including the immediate-early genes WSV083 and WSV249 and viral DNA polymerase. Conversely, injecting shrimp with WSSV that had been co-incubated with a recombinant LvHSP90 (rLvHSP90) promoted WSSV infection as evidenced by an increased cumulative mortality rate and viral copy numbers at 40–48 h post infection (hpi). Subcellular localization of LvHSP90 in WSSV-infected hemocytes at 3, 6 and 12 hpi demonstrated increased expression and translocation of LvHSP90 into the nucleus where WSSV DNA can replicate. Thus, LvHSP90 might be involved in the WSSV pathogenesis by promoting WSSV replication.

Validation of qPCR from a crude extract for the rapid detection of white spot syndrome virus.

White spot disease (WSD) has posed a serious threat to the China and the global shrimp aquaculture. In order to diagnose white spot syndrome virus (WSSV) early and prevent the spread and outbreak of WSD, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay from the crude extract of samples heated lysis was established, which was further compared with the universal qPCR assay to verify the shrimp samples. The limit of detection (LOD) of pre-amplification qPCR assay and universal qPCR assay was 2.80 copies and 20.57 copies per reaction at 95% CI, respectively. It had good WSSV specificity and did not show cross-detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreas necrosis disease (AHPND), necrotizing hepatopancreatitis bacteria (NHPB), and decapod iridescent virus 1 (DIV1). A total of 36 shrimp samples were detected as WSSV DNA positive by pre-amplification qPCR with crude extract from samples heated lysis and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5 ~ 99.9%, 95% CI) and 100% (81.5 ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10499-022-00920-9.

Application of iron flocculation to concentrate white spot syndrome virus in seawater

The iron flocculation method, which comprises the Fe-virus flocculate formation-filtration-resuspension steps, is extensively used to concentrate and precipitate viruses distributed in water. To apply this method to concentrate white spot syndrome virus (WSSV) in seawater, viral genomic and infective recovery yields were compared between polyethylene sulfone (PES) and polycarbonate (PC) membrane filters and two types of resuspension buffers (oxalate and ascorbate). Viral genome quantitation was determined above a 95 % limit of detection (11.48 viral DNA copies/μL) using quantitative real-time PCR. From WSSV-spiked seawater (100–106 viral DNA copies/mL), the viral genomic recovery yields of the PES-Oxalate, PC-Oxalate, PES-Ascorbate, and PC-Ascorbate conditions were 78.67 % ± 12.90 %, 84.53 % ± 24.30 %, 85.59 % ± 16.98 %, and 93.74 % ± 7.44 %, respectively. The detectable Fe-virus flocculates collected by the PC membrane were approximately 101 WSSV DNA copies/mL of seawater, a value more than 10-fold higher than that compared to the PES membrane filter (102 WSSV DNA copies/mL), regardless of the resuspension buffer types. WSSV resuspended with oxalate buffer caused mass mortality among whiteleg shrimp (Litopenaeus vannamei), inducing the expression of the virus envelope protein, VP28, similar to that of a native virus, suggesting stable viral activity during the resuspension process. Based on the PES-Ascorbate, WSSV particles could be successfully concentrated in seawater from shrimp farms with white spot disease outbreaks (approximately 102 WSSV DNA copies/mL). Collectively, these findings indicate that the simple and efficient method of iron flocculation is sufficient to concentrate WSSV in seawater and could be used as a non-invasive approach and one of the reasonable diagnostic processes for white spot disease surveillance.

First report of White Spot Syndrome Virus (WSSV) DNA in red swamp crayfish (Procambarus clarkii) in Germany

This work describes the first detection of WSSV genomic DNA in decapods introduced into Germany via food for human consumption. A batch of cooked, frozen, packed and labelled red swamp crayfish (Procambarus clarkii) was imported into an EU member state and subsequently moved to Germany. Some individual crayfish of this batch sampled as a labelled consumer package during an official routine food control showed signs of White spot disease at food inspection. To confirm the presence or absence of White spot syndrome virus (WSSV) DNA, PCR analysis was performed. All samples of affected red swamp crayfish tested positive for WSSV DNA. The food batch was immediately blocked by the competent veterinary authority and destroyed. Although the introduction of infectious WSSV into the EU could be ruled out this case indicates the risk of global trading and the importance of appropriate border controls in order to prevent the introduction of listed or emerging aquatic animal diseases via consignments of products of aquatic animal origin.

White spot syndrome virus-encoded microRNA promotes viral replication by maintaining viral early gene expression

Disease especially caused by White spot syndrome virus (WSSV) has a huge impact on the shrimp production industry. An understanding of shrimp-WSSV interaction could lead to an effective approach to control WSSV infection. MicroRNA (miRNA) is a short small RNA which functions in post-transcriptional gene regulation and is involved in the WSSV infection. Previously, WSSV-encoded miRNAs were identified from infected P. monodon. We investigated wsv-miR-9 which showed the highest expression in WSSV-infected gill tissue. The expression of wsv-miR-9 was up-regulated upon WSSV infection and reduced in moribund shrimp. Antisense inhibition using anti-miRNA oligonucleotides (AMO) resulted in a complete inhibition of wsv-miR-9 expression. An in vitro functional study demonstrated that mimic of wsv-miR-9 could function in translational suppression by decreasing the reporter protein activity when compared to the mutant target. In shrimp, loss of wsv-miR-9 by AMO9 resulted in a delay of viral replication after 6–12 h post WSSV challenge. A reduction of WSSV early genes, ie1 and DNA polymerase mRNA expressions was observed within 6 h. These results reveal that wsv-miR-9 is important in maintaining early viral gene expression.

WSSV proteins and DNA genome released by ultrasonic rupture can infect crayfish as effectively as intact virions

Proteins and nucleic acids from ultrasonically ruptured white spot syndrome virus (WSSV) can infect crayfish and cause death as effectively as intact WSSV virions. In this study, ultrasound was used to rupture the virus and the resulting suspension was filtered through a 50 nm membrane. Analysis by PCR and SDS-PAGE showed that both viral genes (VP19, VP26, VP28 and DNA polymerase) and proteins (VP15, VP19, VP26 and VP28) were present in the filtered solution. Electron microscopy showed that there were no intact virions in the filtered solution. When crayfish were injected with the filtered solution or with intact WSSV, the mortality in each group was 100 %. The same result was seen when crayfish were challenged orally with the filtered solution and intact WSSV. The filtered solution of ultrasonically ruptured virus, which contains viral proteins and residual DNA genome, can thus infect the host as effectively as intact virions. When the solution of viral proteins and residual DNA genome was digested with DNase I and then injected into crayfish, the survival rate was 100 %. We also found that, although viral proteins (except VP15) in the solution of ruptured virus were destroyed by treatment with DNase I, DNase I did not destroy the structural proteins of intact virions. A remaining viral protein in the DNase I-treated solution protects the DNA genome from degradation and we concluded that this protein is VP15, which is a DNA-binding protein. Our study highlights the extreme danger in producing vaccines from proteins obtained by ultrasonic rupture of viruses sincethe viral DNA genome is difficult to degrade and, if present, will lead to viral infection.

Assessment of the origin of white spot syndrome virus DNA sequences in farmed Penaeus monodon in Australia

Until 2016, the Australian shrimp industry was considered free of White Spot Syndrome Virus (WSSV), a destructive pathogen responsible for the huge economic losses in the shrimp culture industry in most shrimp producing countries worldwide. In December 2016, there was an outbreak of WSSV in commercial Penaeus monodon shrimp farms located on the Logan River in Queensland, northeast Australia which resulted in the loss of all shrimp production on the Logan river either directly from the virus itself or indirectly through destruction of the shrimp production as part of quarantine measures.The first objective of this study was to consider the possible origins of the outbreak, whether the WSSV outbreak arose of an exotic introduction or a local endemic strain. The Australian WSSV DNA sequences, from nine farmed samples, amplified using the 146F2R2 primers spanning relatively conserved regions, were identical to each other and had very close identity to overseas WSSV strains which did not refute the working hypothesis that the outbreak was caused by an invasive species from overseas rather than from entry into aquaculture of a long term Australian endemic strain.The second objective was to consider the possible source of the outbreak (i.e. country or region). To do this, highly variable genomic WSSV regions were identified and novel primers designed for this study to amplify variable genomic regions so it would be possible to conduct DNA sequence comparisons, theoretically, from a hundreds of samples in a relatively short period of time and low cost, something not readily possible with full genomic sequencing. In practice we generated DNA sequences using up to five of these novel primers (and the published ORF 125 variable primers) from three Australian farmed shrimp samples, from two farmed Vietnamese samples (from the north and south of the country), one retail product from Vietnam and one retail product from China sold in Australia and compared these sequences to 10 published overseas WSSV genomes. Available data indicated the Australian sequences were most similar to published sequences from China, and less closely related to sequences from other regions, for example sequences we derived from Vietnamese farmed samples.The third objective was to assess possible physical routes of infection into Australia by testing for the presence of WSSV DNA in aquaculture feed samples used at the time of the outbreak and in imported shrimp retail items. No PCR positives were detected from two samples of shrimp feeds used at the time of the outbreak. Nearly every Australian retail item of imported shrimp product (12 out of 13) tested positive for WSSV and DNA sequences could be obtained from the amplified PCR products indicating that at some point in their history, nearly all the imported samples were in contact with WSSV.In conclusion, our data, considered together, are consistent with the following hypotheses: an exotic shrimp virus, WSSV, which is of considerable economic importance, has breached Australia's biosecurity; the Australian WSSV strain is mostly closely related to one reported from Asia; a plausible physical route of entry may have been through the importation of WSSV infected shrimp for retail sale.

Molecular Characterization of Viral Responsive Protein 15 and Its Possible Role in Nuclear Export of Virus in Black Tiger Shrimp Penaeus monodon

A viral responsive protein 15 from Penaeus monodon (PmVRP15) has been reported to be important for white spot syndrome virus (WSSV) infection in vivo. This work aims to characterize PmVRP15 and investigate its possible role in nuclear import/export of the virus. Circular dichroism spectra showed that PmVRP15 contains high helical contents (82%). Analytical ultracentrifugation suggested that PmVRP15 could possibly form oligomers in solution. A subcellular fractionation study showed that PmVRP15 was found in heavy and light membrane fractions, indicating that PmVRP15 may be associated with endoplasmic reticulum. Double-stranded RNAi-mediated knockdown of PmVRP15 gene expression in vitro showed no effect on WSSV copy number in whole hemocyte cells. However, PmVRP15 silencing resulted in an accumulation of WSSV DNA in the nucleus of PmVRP15-silenced hemocytes. Immunofluorescence confocal microscopy showed that PmVRP15 knockdown hemocytes had a much lower level of VP28 (WSSV envelope protein), in comparison to that in the control. It is likely that PmVRP15 may play a role in viral nuclear egress.

Highly Sensitive Detection of Low-Abundance White Spot Syndrome Virus by a Pre-Amplification PCR Method.

White spot syndrome virus (WSSV) is a major threat to the shrimp farming industry and so far there is no effective therapy for it, and thus early diagnostic of WSSV is of great importance. However, at the early stage of infection, the extremely low-abundance of WSSV DNA challenges the detection sensitivity and accuracy of PCR. To effectively detect low-abundance WSSV, here we developed a pre-amplification PCR (pre-amp PCR) method to amplify trace amounts of WSSV DNA from massive background genomic DNA. Combining with normal specific PCR, 10 copies of target WSSV genes were detected from ~1010 magnitude of backgrounds. In particular, multiple target genes were able to be balanced amplified with similar efficiency due to the usage of the universal primer. The efficiency of the pre-amp PCR was validated by nested-PCR and quantitative PCR, and pre-amp PCR showed higher efficiency than nested-PCR when multiple targets were detected. The developed method is particularly suitable for the super early diagnosis of WSSV, and has potential to be applied in other low-abundance sample detection cases.

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei (Boone), and the outcome for virus and cell.

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)-killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV-killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV-killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV-killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60min post-inoculation) and that afterwards the capsid started to become disintegrated (from 360min post-inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV-killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular-sensing molecules.

Development of a rapid method for identifying carryover contamination of positive control DNA, using a chimeric positive control and restriction enzyme for the diagnosis of white spot syndrome virus by nested PCR.

Chimeric positive plasmids have been developed to minimize false-positive reactions caused by polymerase chain reaction (PCR) contamination. Here, we developed a rapid method for identifying false-positive results while detecting white spot syndrome virus (WSSV) by nested PCR, using chimeric positive plasmids. The results of PCRs using WSSV diagnostic primer sets showed PCR products of a similar size (WSSV 1st PCR product, 1,447bp; WSSV 2nd PCR product, 941bp) using WSSV chimeric plasmids or DNA from shrimp infected with WSSV. The PCR products were digested with DraI for 1h at 37°C. The digested chimeric DNA separated into two DNA bands; however, the WSSV-infected shrimp DNA did not separate. Thus, chimeric plasmid DNA may be used as positive control DNA instead of DNA from WSSV-infected shrimp, in order to prevent PCR contamination. Thus, the use of restriction enzyme digestion allowed us to rapidly distinguish between WSSV DNA and WSSV chimeric plasmid DNA.

Crystal structure of the shrimp proliferating cell nuclear antigen: structural complementarity with WSSV DNA polymerase PIP-box.

DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV) that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA). This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein). The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.

Viability of white spot syndrome virus (WSSV) in sediment during sun-drying (drainable pond) and under non-drainable pond conditions indicated by infectivity to shrimp

White spot syndrome virus (WSSV), is a highly virulent rapidly replicating large, enveloped, double-stranded DNA (dsDNA) virus, causing an estimated losses of over US$6billion since its emergence in 1992. As part of the better management practices (BMPs), ploughing, tilling and sun-drying of shrimp culture ponds are advocated for prevention and control of this disease. Despite this, instances of outbreaks of white spot disease (WSD) recur, causing loss to shrimp farmers. Some studies have indicated that WSSV DNA is reported to persist for over 20months in sediments as detected by PCR. Since mere detection of virus using PCR methods does not indicate its viable nature and ability to infect, information on its viability outside the host in water and pond sediment is necessary. Hence, in this study, the viability of WSSV in seawater and shrimp pond sediments under experimentally simulated drainable and non-drainable pond conditions was examined by shrimp infectivity experiments. WSSV with an initial viral load of 1000virionsmL−1 was found to be viable for a period of 12days in seawater of 27ppt salinity, pH of 7.5 at 29–33°C as revealed by its ability to infect juvenile shrimp, whereas, in shrimp pond sediment (with initial viral load of 211,500copiesg−1), the virus was viable and infective up to 19days despite sun-drying. In the case of non-drainable conditions, WSSV (753,600copiesg−1) remained infective for a period of 35days. Although the sediment samples tested nested PCR positive after 19days of sun-drying and 40days under water-logged conditions, shrimps did not develop WSD, suggesting that WSSV was not viable. Over a period of time after 21days under sun-drying and 40days under non-drainable experimental conditions, due to reduction in viral load, sediments were positive only by nested PCR, and by this time, viability of WSSV was almost lost as revealed by shrimp infectivity. Hence, PCR testing of shrimp farm sediment before starting culture as one of the BMPs may help in ensuring biosecurity from WSSV. The information generated here would help in the improvement of better management practices (BMPs) with regard to pond preparation protocols for shrimp aquaculture.

Molecular modeling and expression of the Litopenaeus vannamei proliferating cell nuclear antigen (PCNA) after white spot syndrome virus shrimp infection

Proliferating cell nuclear antigen (PCNA) is the eukaryotic sliding clamp that tethers DNA polymerase to DNA during replication. The full-length cDNA of the Pacific white shrimp Litopenaeus vannamei PCNA (LvPCNA) was cloned and encoded a protein of 260 amino acids that is highly similar to other Crustacean PCNAs. The theoretical shrimp PCNA structure has all the domains that are necessary for its interaction with template DNA and DNA polymerase. RT-PCR analysis showed that LvPCNA is expressed mainly in muscle and hemocytes and much less in hepatopancreas and gills. LvPCNA mRNA levels are not statistically different in muscle from healthy and challenged shrimp with the white spot syndrome virus (WSSV). In contrast, the mRNA levels of the viral DNA polymerase show a biphasic pattern with expression at 6h post-infection and later at 24 and 48h. These results suggest that in shrimp muscle LvPCNA levels are steadily kept to allow viral replication and that WSSV DNA polymerase (WSSV-DNApol) is more responsive towards later stages of infection. More knowledge of the DNA replication machinery would result in a better understanding of the mechanism and components of viral replication, since the WSSV genome does not have all the components required for assembly of a fully functional replisome.

Effect of processing treatments on the white spot syndrome virus DNA in farmed shrimps (Penaeus monodon)

To investigate the effect of processing treatments on the destruction of white spot syndrome virus (WSSV) DNA in WSSV-infected farmed shrimps (Penaeus monodon). The presence of WSSV was tested by single step and nested polymerase chain reaction (PCR). The primers 1s5 & 1a16 and IK1 & IK2 were used for the single step PCR and primers IK1 & IK2-IK3 & IK4 were used for the nested PCR. Various processing treatments such as icing, freezing, cooking, cooking followed by slow freezing, cooking followed by quick freezing, canning, and cold storage were employed to destroy the WSSV DNA. Of the processing treatments given, cooking followed by quick freezing was efficient in destroying WSSV DNA in WSSV-infected shrimp products. Canning, and cooking followed by slow freezing process had some destructive effect on the WSSV DNA, as WSSV DNA in such processed shrimp products was detected only by nested PCR. Icing, slow freezing, quick freezing, and cooking processes had no effect on the destruction of WSSV DNA. A gradual increase in the destruction of WSSV DNA was observed as the cold storage period increased. The results indicated that cooking followed by quick freezing process destroy the WSSV DNA. WSSV can be destroyed by cooking followed by quick freezing and this combined process can reduce the disease transmission risks from commodity shrimps to native shrimps.

Shrimp Pm-fortilin inhibits the expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model

Fortilin plays an important role in anti-apoptotic mechanisms and cell proliferation in many eukaryotic organisms. This work confirmed previous reports that Sf9 can support the replication of white spot syndrome virus (WSSV) genomic material by using immunohistochemistry with a specific antibody to detect the immediate early gene 1 ( ie1) and by amplification of WSSV DNA and mRNA products. Using this insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the expression of WSSV early genes and late genes ( WSSV- DNA polymerase, VP15 and VP28) but not an immediate early gene ie1. This is the first time that an insect cell line has been used to demonstrate interaction between a shrimp gene and genes of a shrimp virus.

Infection of WSSV-negative Shrimp, Litopenaeus vannamei, Cultivated under Fluctuating Temperature Conditions

To test whether effluents released from white spot syndrome virus (WSSV)-infected farm ponds are a pathway for spreading WSSV, WSSV-negative Pacific whiteleg shrimp, Litopenaeus vannamei, were exposed to WSSV-containing water under conditions of fluctuating water temperatures. White spot disease outbreaks occurred at the shrimp ponds before and during the experiment. Two cages were placed inside each test pond, and one was placed at the outlet canal. Each cage was stocked with 30 shrimp. Hemolymph from stocked shrimp was collected at intervals of 24, 48, 72, 120, 168, and 360 h after exposure and analyzed for presence of WSSV DNA by nested polymerase chain reaction. At diurnal variation of water temperature from 28.0 to 33.4 C, WSSV was detected as early as 120 h (ca. 11% of shrimp hemolymph pools) and 168 h (ca. 18% of shrimp hemolymph pools). WSSV was detected by 360 h (ca. 33% of shrimp hemolymph pools) in all cages, when water temperature varied from 24.9 to 28.5 C during a 48-h period. Cumulative mortality in cages inside ponds was ≤50.0 and 86.7% at the outlet canal. These data show that grow-out operations during the summer–autumn transition are at risk of WSSV outbreaks. The experiment demonstrated that WSSV can be spread by shrimp farm water drainage.

Characterization of WSSV resistance in selected families of Litopenaeus vannamei

Disease outbreaks caused by the white spot syndrome virus (WSSV) have resulted in high economic losses in the shrimp-culture industry worldwide. After three generations of selective breeding for WSSV resistance, 29 families of Litopenaeus vannamei (G3) were selected and divided into three groups: relatively high-resistance, moderate-resistance, and susceptible families, which had average survival rates of 22.7±12.30, 9.08±1.46, and 0.78±1.70%, respectively, after WSSV infection. Appearance of the mortality peak in the relatively high-resistance families was postponed by 2–3days compared to the same in susceptible families and unselected shrimps. Using real-time quantitative polymerase chain reaction, the copy number of WSSV DNA in the muscles of surviving shrimps from the relatively high-resistance family 14 was significantly lower than that of the moderate-resistance family 17, the susceptible family 4, and the unselected family of shrimps at 36hourspostinfection (hpi) and thereafter; for example, at 48hpi, the DNA copy number was (23.87±2.56)×106copiesg−1 in the high-resistance families against (40.03±2.54)×106, (65.70±3.44)×106, and (47.87±3.95)×106copiesg−1 body weight, respectively, in the other groups. The WSSV copy number in the muscles of dead shrimps from the relatively high-resistance family 14 was significantly higher than that of the moderate-resistance family 17, the susceptible family 4, and the unselected family of shrimps at 48hpi and thereafter; for example, at 72hpi, the DNA copy number was (54.1±3.9)×106copiesg−1 compared to values of (31.5±2.6)×106, (11.6±2.9)×106, and (25.4±2.7)×106copiesg−1 body weight in the other groups, respectively. The results indicated that shrimps from the relatively high-resistance families could inhibit the replication of WSSV and therefore had apparent WSSV resistance. These results provide a direction for the future selective breeding of shrimps for WSSV resistance.

Detection of white spot syndrome virus (WSSV) in the Pacific oyster Crassostrea gigas

Oysters Crassostrea gigas were placed at water supply canals of three shrimp farms in Guasave, Mexico where WSSV outbreaks occur. Animals were sampled through April–August and September–December to detect WSSV DNA. By using three different PCR protocols, only oysters from a farm undergoing a WSSV outbreak were found WSSV-positive in gills and digestive gland. Two WSSV amplicons were sequenced and they corresponded over 99% to WSSV genome segments. Results showed that oysters can capture WSSV particles suspended in water. Susceptibility of oysters to WSSV infection and their role as a carrier remain to be determined.