White Spot Syndrome Virus Particles Research Articles

Vertical transmission and prevalence of white spot syndrome virus (WSSV) in the wild spawning population of the Indian white shrimp, Penaeus indicus

White spot disease, caused by white spot syndrome virus (WSSV), has historically been the most devastating disease in shrimp aquaculture industry across the world. The mode of virus transmission is the most crucial stage in the dynamics and management of virus infection. This study explored the mechanism of vertical transmission of WSSV in Indian white shrimp, Penaeus indicus, potential native species for domestication and genetic improvement, using quantitative real time PCR (q RT PCR), light and electron microscopy, and in situ hybridization. Wild brooders of P. indicus (n = 2576) were sampled along the South east coast of India, during 2016 to 2021. Of these ∼ 58 % of the brooders were positive for WSSV, and almost 50 % of infected wild brooders were at the various stages of reproductive maturation. WSSV-PCR positive brooders (n = 200) were analysed for vertical WSSV transmission. The q RT PCR studies of reproductive tissues revealed that 61 % (n = 13) of spermatophore, 54 % (n = 28) of immature ovaries and 48 % (n = 27) of ripe ovaries were infected with WSSV. The lowest level of infection was recorded in females with ripe ovaries (6.84 × 101 ± 9.79 × 100 ng genomic DNA) followed by fertilized eggs (1.59 × 102 ± 3.69 × 101 ng genomic DNA), and larvae (nauplius and zoea). The histology of gravid females with high WSSV copies showed pyknotic and karyorrhectic germinal vesicle with degenerated cortical rods. Conversely, the gravid females with low WSSV copies showed fully developed ovary without characteristic signs of WSSV infection. Transmission electron microscopic studies clearly established the presence of WSSV particles in both ovaries and spermatophores. When subjected to in situ hybridization, WSSV-specific signals were observed in connective tissues of spermatophore, although gravid ovary and fertilized eggs were failed to produce WSSV specific signals. The present study provides the first molecular and histological evidence for trans-ovarian vertical transmission of WSSV. Development of disease-free base population being the cornerstone and first step in establishing the breeding program, the present findings could be a basis for development of such programs.

The first description of the blue swimming crab (Portunus pelagicus) transcriptome and immunological defense mechanism in response to white spot syndrome virus (WSSV)

In the global shellfish farming industry, white spot syndrome virus (WSSV) is a major cause of mortality and a significant factor in economic losses. However, information on molecular immune responses to WSSV in blue swimming crabs (Portunus pelagicus) has never been reported. First, viral loads were measured in the gills, hepatopancreas, intestines, subcuticular epithelium and hemocytes of blue swimming crabs (50 ± 10 g) (n = 4) after WSSV induction at 0, 24, 48 and 96 h post injection (hpi). A significant increase in WSSV particles was observed in gills at 48 and 96 hpi, as supported by histopathology. To further investigate the acute immune response to WSSV, total RNA from the same gill tissues at 0, 24, and 96 hpi was used to construct 16 high-quality RNA-seq cDNA libraries. In summary, 162,740 unigenes were discovered in these transcriptomic libraries analyzed with the GO, KO, KOG, NR, NT, PFAM and SwissProt databases. Intensive sequence analysis against control crabs using three major categories of gene oncology (GO) of DEGs, biological processes (BPs), molecular functions (MFs), and cellular components (CCs), indicated that induction of WSSV in blue swimming crabs strongly affected the immune responses of the target animals significantly during the early stages of infection from 24 to 96 hpi. Furthermore, KEGG identified approximately twenty biological pathways of gene expression that were both downregulated and upregulated. Interestingly, at 24 and 96 hpi, several immune-related genes involved in virus defense in the blue swimming crab, particularly crustin 2, chitinase, anti-lipopolysaccharide, proteinase inhibitor, and lysozyme, were highly expressed during the WSSV early infection stages. At the same time, viral mRNA transcripts, including WSV289, WSV343, WSV306, deoxyuridine 5’ triphosphate nucleohydrolase, RING finger containing E3 ubiquitin-protein ligase WSV403 and WSV404, were recorded in the top twenty upregulated genes. Moreover, some immune-responsive genes related to growth development, such as chitinase, tubulin alpha and beta chains, trypsin, and the cathepsin family, were also differentially expressed during these periods. Expression validation of 20 upregulated and 11 downregulated immune-related genes using qRT‒PCR showed similar patterns with transcriptome information. Overall, the data showed that during WSSV infection, a number of immune-, metabolism-, and growth-related pathways were activated, and several of the pathways involved differed depending on the stage of virus invasion. These findings could effectively help us better understand the impact of WSSV on the physiology of blue swimming crabs and serve as a valuable reference for future research on the immune system and disease control in this target species.

First Discovery of Beta-Sitosterol as a Novel Antiviral Agent against White Spot Syndrome Virus.

The outbreak of white spot syndrome (WSS) is a looming challenge, due to dramatic losses to the crustacean aquaculture industry. However, at present, there are no prophylactic or therapeutic means to control this infectious viral disease. Here, we screened fifteen medicinal plants for their inhibitory activity on the white spot syndrome virus (WSSV), using red swamp crayfish (Procambarus clarkii) as a model species. The results showed that the crude extracts of Pinellia ternata (Thunb.) Breit. had the highest inhibitory effect (91.59%, 100 mg/kg) on WSSV proliferation, and its main component, beta-sitosterol, showed a much higher activity (95.79%, 50 mg/kg). Further, beta-sitosterol potently reduced (p < 0.01) viral loads and viral gene transcription levels in a concentration-dependent fashion, and significantly promoted the survival rate of WSSV-challenged crayfish (57.14%, 50 mg/kg). The co-incubation assay indicated that beta-sitosterol did not influence the infectivity of WSSV particles. Both pre- and post-treatment of beta-sitosterol exerted a significant inhibitory effect (p < 0.01) on the viral load in vivo. Mechanistically, beta-sitosterol not only interfered with the expression of viral genes (immediate early gene 1, ie1; DNA polymerase, DNApol) that are important in initiating WSSV transcription, but it also attenuated the hijacking of innate immune signaling pathways (Toll, IMD, and JAK/STAT pathways) by viral genes to block WSSV replication. Moreover, the expression of several antiviral immune, antioxidant, pro-inflammatory, and apoptosis-related genes changed significantly in beta-sitosterol-treated crayfish. Beta-sitosterol is a potent WSSV inhibitor and has the potential to be developed as an effective anti-WSSV agent against a WSS outbreak in crustacean aquaculture.

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.

SpgC1qR interacts with WSSV VP28 exhibiting antiviral activity

Although human gC1qR is a multi-ligand binding protein with diverse biological functions, the functions of invertebrate gC1qR homologues remain largely unknown. In the present study, we characterized a novel gC1qR homologue, namely SpgC1qR, from mud crab Scylla paramamosain. SpgC1qR shared high identity and similar three-dimensional structure with human gC1qR. After challenge with White spot syndrome virus (WSSV), the transcripts of SpgC1qR were significantly increased, suggesting that SpgC1qR may be involved in antiviral immune response. To reveal the likely antiviral activity of SpgC1qR, the proliferation profile of WSSV in SpgC1qR-silenced crabs was examined. The result showed that knockdown of SpgC1qR by RNAi facilitated viral proliferation in vivo. This result was further confirmed by a SpgC1qR pre-incubation assay, in which pre-incubating WSSV particles with rSpgC1qR dramatically suppressed viral replication. Moreover, a GST pull-down assay revealed that SpgC1qR specifically bound to the viral envelope protein VP28. These findings clearly demonstrated that SpgC1qR specifically interacted with viral envelope protein VP28 and restricted WSSV replication, suggesting that it played a crucial role in anti-WSSV immune response of mud crab. This study provided new insights into the antiviral mechanism mediated by SpgC1qR and the biological functions of invertebrate gC1qR homologues.

The dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus recognizes white spot syndrome virus and restricts its replication

Dyslexia-associated protein KIAA0319, with immunoglobulin-like polycystic kidney disease (PKD) domains, is a dyslexia susceptibility protein. KIAA0319 participates in neuronal migration and is involved in the interaction between neurons and glial fibers. Recent studies found that KIAA0319 acts as a viral receptor via an interaction between its PKD domains with viruses. KIAA0319 mediates viral internalization via a clathrin-dependent pathway involving interaction with adaptor protein 2 (AP2). In the present study, we identified a cDNA encoding a dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus (designated as MjKIAA0319L). MjKIAA0319L was upregulated in shrimp challenged by white spot syndrome virus (WSSV). After knockdown of MjKIAA0319L using RNA interference, followed by WSSV infection in shrimp, WSSV replication increased and the shrimp survival rate decreased significantly. Further study found that the extracellular domain of MjKIAA0319L expressed in Escherichia coli bound WSSV particles by interacting with the WSSV envelope protein VP28. Fluorescent immunocytochemical analysis showed that MjKIAA0319L was internalized into the cytoplasm of hemocytes during WSSV infection. The intracellular domain of MjKIAA0319L, comprising a YXXΦ motif, interacted with AP2μ, suggesting that MjKIAA0319L binds to WSSV and induces endocytosis in a clathrin-dependent manner. Therefore, MjKIAA0319L recognizes WSSV via its extracellular domain and induces endocytosis of WSSV through its intracellular domain in shrimp. Thus, the MjKIAA0319L-AP2-clathrin pathway is involved in the phagocytosis of WSSV to restrict viral infection in shrimp.

SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein.

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

Antiviral activity of esculin against white spot syndrome virus: A new starting point for prevention and control of white spot disease outbreaks in shrimp seedling culture.

White spot syndrome virus (WSSV) is a pathogenic and threatening virus in shrimp culture for which there is no effective control strategy. Finding antiviral lead compounds for the development of anti-WSSV drugs is urgent and necessary; in this study, esculin from 12 monomeric compounds exhibited an excellent anti-WSSV activity. The results showed that esculin increased the survival rate of WSSV-infected shrimps by 59% and reduced the virus copy number in vivo over 90% at 100μM. In the pre-treatment and post-treatment experiments, esculin could prevent and treat WSSV infection. Compared with the control group, the virus copy number decreased by 30% after 6h of esculin pre-incubation with WSSV particles and inhibited horizontal transmission of WSSV to a certain extent. Considering that the antiviral activity of esculin was stable in the aquacultural water for 2days, we evaluated the dosing pattern of continuous medication changes. Obviously, the survival rate of WSSV-infected shrimps was 0% at 108h when no esculin exchange was made, while at 120h the survival rate was over 40% at continuous medicine changes. In addition, esculin significantly increased the expression of antimicrobial peptides and thus improved the ability of shrimp to resist WSSV. Overall, our findings suggest that esculin has the potential to be developed into an anti-WSSV medicine.

Evaluation on the antiviral activity of a nitrile phenylpropanoid against white spot syndrome virus infection in Litopenaeus vannamei

The white spot syndrome virus (WSSV) is an efficiently destructive pathogen in shrimp farming, which has contributed to enormous economic losses in the global shrimp sector. After antiviral activity screening in our small molecule drug library, we found a medicine, 2-amino-4-(4-hydroxy-3-methoxyphenyl)-5-oxo-4H,5H-pyrano [3,2-c] chromene-3‑carbonitrile (serial number: Coumarin 3004), significantly inhibited WSSV replication in Litopenaeus vannamei and reduced the mortality of WSSV-infected L. vannamei. The data indicated that the replication inhibition of WSSV exceeded 60% after WSSV particles pre-incubation with 10 mg/L Coumarin 3004 for 4 h. Moreover, Coumarin 3004 showed a stable efficacy for two days in aquacultural water, which provided a potential for its aquaculture application. The approach of consecutive Coumarin 3004 changes further decreased the mortality of WSSV-infected L. vannamei, which allows novel possibilities for treating WSSV infection. Meanwhile, up-regulation of four antimicrobial peptides (AMPs) related gene expressions revealed that Coumarin 3004 may promote AMPs-related expressions and inhibit WSSV replication. Although Coumarin 3004 blocked the horizontal transmission of WSSV in a cohabitation model, it did not entirely block the transmission. Taken together, Coumarin 3004 is a potentially excellent therapeutic agent against WSSV propagation in shrimp farming.

Role of Clathrin Assembly Protein-2 Beta Subunit during White Spot Syndrome Virus Infection in Black Tiger Shrimp Penaeus monodon

White spot syndrome virus (WSSV) is one of the most lethal viruses severely affecting shrimp industry. This disease can cause 100% mortality of farmed shrimp within a week. This work aims to characterize clathrin assembly proteins in Penaeus monodon and investigate their roles in WSSV entry. In general, clathrin assembly proteins form complexes with specific receptors and clathrins, leading to clathrin-mediated endocytosis. Adaptor protein 2 (AP-2), which is responsible for endocytosis at plasma membrane, consists of four subunits including α, β2, μ2 and σ2. Knockdown of clathrin coat AP17, or σ subunit of AP-2 dramatically reduced WSSV infectivity. Similar results were observed, when shrimp were pre-treated with chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis. The complete open reading frames of AP-2β and μ subunits of P. monodon are reported. PmAP-2 β was up-regulated about 4-fold at 6 and 36 h post-WSSV infection. Knockdown of PmAP-2β delayed shrimp mortality during WSSV infection, of which WSSV intermediate early 1 gene expression was also down-regulated. Immunogold-labelling and transmission electron microscopy revealed that PmAP-2β co-localized with WSSV particles at plasma membrane. In addition, PmAP-2β-silencing significantly affected the expression levels of PmSTAT, PmDOME, PmDorsal and ALFPm3 during WSSV infection. It is possible that PmAP-2β is associated with the JAK/STAT and the Toll pathway.

Effect of white spot syndrome virus on the activity of immune-related enzymes in the red claw crayfish (Cherax quadricarinatus)

In this study, we explored the pathogenic effects of white spot syndrome virus (WSSV) and effects of yeast cell wall in the red claw crayfish, Cherax quadricarinatus, by investigating the activity of enzymes related to innate immune function following artificial infection of immunized and non-immunized crayfish. Our results reveal that the activity of four enzymes, phenoloxidase (PO), peroxidase (POD), superoxide dismutase (SOD) and lysozyme (LSZ), increased in the hepatopancreas and antennal gland of C. quadricarinatus 6 – 24 hours after exposure to WSSV. After, activity levels of these enzymes decreased rapidly, such that 72 h post-exposure the activity of the enzymes in WSSV-infected crayfish were significantly lower than those of the control individuals (except for LSZ). Interestingly, we found that immunization with yeast cell wall before challenge with WSSV conferred an immune protection rate of 51.86% at 7 days post-infection, demonstrating that yeast cell wall could improve immune-related enzyme activity in the crayfish and enhance the anti-viral defenses of this species. Morphological examination by transmission electron microscopy revealed significant damage in the hepatopancreas and antennal glands of infected crayfish. WSSV infection caused damage to the epithelium of the hepatopancreas and antennas and reduced the number and size of microvilli. In addition, mitochondria morphology changed following infection, with parts of the cristae diminished leaving large vacuoles. Moreover, the dictyosome morphology changed, the lysosome membranes ruptured, and heterochromatinized nuclei could be seen in cells with ruptured nuclear membranes. Despite this no WSSV particles appeared in the hepatopancreas or in the antennal gland.

FmLC6: An ultimate dual-CRD C-type lectin from Fenneropenaeus merguiensis mediated its roles in shrimp defense immunity towards bacteria and virus

C-type lectins are a member of pattern recognition receptors (PRRs) that can interact with pathogen-associated molecular patterns of invading microorganisms by using their conserved motifs in carbohydrate recognition domain (CRD). The binding can trigger various immune responses in both direct and indirect mechanisms. Hereby, an ultimate C-type lectin with dual CRDs each of which containing a different motif was identified from hepatopancreas of Fenneropenaeus merguiensis (mentioned as FmLC6). The full-length cDNA of FmLC6 consisted of 1148 bp comprising one 1005 bp open reading frame (ORF) encoding a signal peptide and a mature protein of 317 residues. FmLC6 was composed of two CRDs with a highly conserved QPD (Gln-Pro-Asp) motif and one variant EPQ (Glu-Pro-Gln) motif for illustrating the carbohydrate binding affinity. The transcription of FmLC6 was detected only in hepatopancreas of normal shrimp. After injection with pathogens or immunostimulants, the expression of FmLC6 was significantly up-regulated and reached the highest level at 12 h post-injection except with lipoteichoic acid challenge. The FmLC6 expression was severely suppressed by knockdown based-silencing. This gene silencing with co-injection by Vibrio parahaemolyticus caused increasing in cumulative mortality and reduction of the median lethal time. Purified recombinant proteins of an entire ORF and two individual CRDs of FmLC6 produced in Escherichia coli could induce a broad spectrum of microbial agglutination with calcium dependence. The agglutination induced by rFmLC6, rCRD1 and rCRD2 was suppressed by galactose plus mannose, galactose and mannose, respectively which this event was confirmed by the inhibition of hemagglutination. All three recombinant proteins possessed ability to inhibit the bacterial growth with a dose-response. Purified rFmLC6 could bind directly to white spot syndrome virus particles and also its recombinant proteins including VP15, VP39A and VP28 with different affinity. Altogether, these results indicate that FmLC6 acts as a PRR to recognize invading microorganisms and leads to mediating the immune response to cooperation in pathogenic elimination via the binding, agglutination and antimicrobial activity.

Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).

BackgroundWhite spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses.Methods/Principal FindingsHere, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional “gold standard test”, viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen’s kappa coefficient of 0.983 suggested "very good agreement” between the developed LFIA and the conventional one-step PCR.ConclusionThe LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.

Effect of white spot syndrome virus (WSSV) infection on immune enzyme activity and ultrastructure in the haemolymph tissue of Cherax quadricarinatus (Decapoda, Astacidea)

To explore the pathogenic mechanism of white spot syndrome virus (WSSV) in crayfish (Cherax quadricarinatus), we analysed activities of the three immune-related enzymes PO, SOD and LSZ in haemolymph tissue ofC. quadricarinatusbefore and after infection, and simultaneously studied the ultrastructural pathology. The results show that WSSV infection affects activities of the three enzymes. After 6-24 h of WSSV infection, the activities of PO, SOD and LSZ increased, but decreased significantly during longer infection times. The enzyme activities in WSSV-infected crayfish were significantly lower than those in controls at 72 h, except for LSZ (). Interestingly, the activities of PO, SOD and LSZ in the group treated with immune-polysaccharides before challenge with WSSV were higher than in the directly infected group, and the immune protection rate reached 51.9%, suggesting that the polysaccharides could improve enzyme activities and enhance antiviral defences of the organism. Ultrastructural pathological changes showed damaged haemolymph tissue, deformed golgiosomes, fuzzy damage in the mitochondrial structures, and nuclear membrane deformation and fracture. High levels of heterochromatin appeared in the nucleus; organoid and chromatin dissolved in dying blood cells, cytoplasm appeared oedematous and cells dissolved. WSSV particles were visible in blood cell nuclei of infected crayfish.

Effects of total ammonia, temperature and salinity on the mortality and viral replication of WSSV-infected Chinese shrimp (Fenneropenaeus chinensis)

In this study, we evaluated the effects of three factors, total ammonia, temperature and salinity, on the mortality of and viral proliferation in white spot syndrome virus (WSSV)-infected Chinese shrimp. Shrimp maintained in 30‰ seawater at 25°C with 0.34 mg L−1 total ammonia (control group) were injected with approximately 20 WSSV particles per shrimp and subsequently subjected to the following conditions: 30‰ seawater at 25°C, with 6 mg L−1 (N1 group) or 14 mg L−1 (N2 group) total ammonia; 30‰ seawater at 18°C (T1 group) or 30°C (T2 group), with 0.34 mg L−1 total ammonia and 20‰ (S1 group) or 40‰ (S2 group) seawater at 25°C, with 0.34 mg L−1 total ammonia. An anova analysis revealed that the cumulative mortality of WSSV-infected Chinese shrimp was significantly lower when reared in the T1 group compared to that of the T2 and control group. Similarly, the mortality of the shrimp in the S1 group was also significantly lower than that of the S2 and control group. No significant differences were detected among the N1, N2 and control groups. Accordingly, the WSSV level in the T1 and S1 groups was significantly lower than those in the control, T2 or S2 groups respectively. No significant differences in viral loads were detected among the control, N1 and N2 groups. The transfer of Chinese shrimp to lower temperature and lower salinity enhanced their resistance to WSSV infection, whereas a change in the concentration of total ammonia had no significant effect on the mortalities and viral loads of WSSV-infected shrimp.

Effects of white spot syndrome virus infection on immuno-enzyme activities and ultrastructure in gills of Cherax quadricarinatus

In this study, we explored the pathogenic mechanism of white spot syndrome virus (WSSV) in crayfish, Cherax quadricarinatus, by investigating activities of enzymes related to innate immune function during infection. After 6–12 h of exposure to WSSV, the activities of four enzymes, phenoloxidase (PO), peroxidase (POD), superoxide dismutase (SOD) and lysozyme (LSZ), increased in the gills of C. quadricarinatus but then sharply decreased during longer infection times. Except for PO, the activities of other enzymes in the WSSV-infected crayfish (Group II) were significantly lower than those of the controls at 72 h post-exposure (P < 0.01). Interestingly, the enzyme activities in the group treated with polysaccharides before challenge with WSSV (Group III) were higher than those in Group II. This phenomenon demonstrated that the polysaccharides could improve the immuno-enzyme activities and enhance the organism’s antiviral defenses. Morphological examination by transmission electron microscopy revealed abundant WSSV particles and significant damage in the gills of infected crayfish. WSSV infection caused parts of the gill epithelium and microvilli to be reduced in number and size or damaged; meanwhile, the mitochondria morphology changed, with parts of the cristae diminished leaving large vacuoles. Moreover, electron dense deposits appeared and heterochromatinized nuclei could be seen in blood cells with ruptured nuclear membranes and outflow of nucleoplasm. The findings of this study furthers our understanding of the biochemical alterations induced by viral infections, including changes in the antioxidant status, oxidative stress and lysozyme activity, which could help to advance strategies for control of WSSV in crayfish.

Protection of Fenneropenaeus chinensis (Osbeck, 1765) against the white spot syndrome virus using specific chicken egg yolk immunoglobulins by oral delivery

Two kinds of specific chicken egg yolk immunoglobulins (IgYs), IgY-WSSV and IgY-VP28, were, respectively, raised against the 2 mM binary ethylenimine (BEI)-inactivated white spot syndrome virus (WSSV) and a principal envelope protein VP28. The activity of purified specific IgYs was stable under the conditions of 20–70 °C, pH 3.0–10.0 and 0–700 g L−1 sucrose solution. In the neutralization assay, these high-affinity IgY antibodies can specifically bind with the virus particles to protect shrimp (Fenneropenaeus chinensis) against WSSV infection. After oral delivery for 20 days, the IgY-WSSV exerted a higher protection effect (RPS: 71.5%) than IgY-VP28 (RPS: 63.7%). Moreover, an increase in RPS (79.2%) was found on addition of IgY-WSSV:VP28 (0.1% IgY-VP28 plus 0.2% IgY-WSSV). This may indicate that neutralization of WSSV refers to the multiple-hit model. By time-course study of the levels of the specific IgYs in vivo, the data showed that the titre was enhanced to a relatively high level (P/N=8.35±0.45) at 3 days post administration, declined slightly (P/N=7.13±1.01) at 7 days post administration and then remained stable for further investigation. The stable antibody level potentially contributes towards blocking a large number of WSSV particles from entering and infecting on the major tissues at the early and late stages after challenge in shrimp.

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti-rVP28 antiserum to detect the presence of the virus in WSSV-infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR-positive WSSV-infected shrimp samples. Immunodot blot was found to be equivalent to first-step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 105 viral DNA copies.

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.

Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus infecting shrimp and other crustaceans. The potentiality of binary ethylenimine (BEI)-inactivated WSSV against WSSV in crayfish, Procambarus clarkii, was investigated in this study. Efficacy of BEI-inactivated WSSV was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish injected with BEI-inactivated WSSV showed a better survival ( P < 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared to the control. Calculated relative percent survival (RPS) values were 77% and 60% on the 7th and 21st dpv for 2 mM BEI-inactivated WSSV, and 63%, 30% on 7th and 21st dpv for 3 mM BEI-inactivated WSSV. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to 3 mM BEI and heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect P. clarkii from WSSV.