White Spot Syndrome Virus VP28 Research Articles

Use of brome mosaic virus-like particles in feed, to deliver dsRNA targeting the white spot syndrome virus vp28 gene, reduces Penaeus vannamei mortality.

Numerous strategies have been investigated to combat viral infections in shrimp, specifically targeting the white spot syndrome virus (WSSV) that has caused outbreaks worldwide since the 1990s. One effective treatment involves intramuscular application of dsRNA-mediated interference against the viral capsid protein VP28. However, this approach presents challenges in terms of individual shrimp management, limiting its application on a large scale. To address this, our study aimed to evaluate the efficacy of oral delivery of protected dsRNA using chitosan nanoparticles or virus-like particles (VLPs) synthesized in brome mosaic virus (BMV). These delivery systems were administered before, during, and after WSSV infection to assess their therapeutic potential. Our findings indicate that BMV-derived VLPs demonstrated superior efficiency as nanocontainers for dsRNA delivery. Notably, the treatment involving vp28 dsRNA mixed in the feed and administered simultaneously to shrimp already infected with WSSV exhibited the highest survival rate (48%), while the infected group had a survival rate of zero, suggesting the potential efficacy of this prophylactic approach in commercial shrimp farms.

Interaction between a Peptide and White Spot Syndrome Virus VP28 Envelope Protein

Interaction between a Peptide and White Spot Syndrome Virus VP28 Envelope Protein

Variability of White Spot Syndrome Virus (WSSV) Envelope Protein VP28 from Diseased Shrimp (Litopenaeus vannamei) in Indonesia

White Spot Disease (WSD) is a viral disease affecting crustaceans. Caused by the White Spot Syndrome Virus (WSSV), this disease has caused significant mortality in commercially cultivated marine shrimp species with severe impacts on the shrimp farming industry and may be a threat to wild shrimp stocks. Thorough studies on the molecular biology of this pathogen are urgently needed to improve understanding of the virus at a molecular level, including variation in key viral protein (VP) components of the WSSV virion. This study aimed to isolate and characterize WSSV VP28 gene encoding envelope proteins from Indonesian Pacific white shrimp (Litopenaeus vannamei) isolates. Infected juvenile shrimp were collected from Pangkep, Barru, and Pinrang Districts in South Sulawesi, Indonesia. Genomic DNA was isolated from infected shrimp muscle tissue using a DTAB-CTAB (dodecyle trimethyl ammonium bromide-hexadecyl trimethyl ammonium bromide) DNA extraction procedure. The WSSV VP28 DNA sequences from Pangkep, Barru, and Pinrang isolates were 640-680 bp in length. Homology of Pangkep isolates with isolates from Barru and Pinrang was 97-99%. BLAST-N (Basic Local Alignment Search Tool-Nucleotide) analysis showed isolates from all three sites clustered with WSSV VP28 accessions from China, Indonesia, Japan, South Carolina and Vietnam. These results increase the geographic spread and host taxon coverage of WSSV VP28 sequence data for Indonesia.

Monoclonal antibodies (mAbs) and single chain variable fragment (scFv) antibodies targeting envelope protein VP28 of white spot syndrome virus provide protection against viral infection

White spot syndrome virus (WSSV) is extremely pathogenic and causes huge economic losses in the shrimp farming industry. Neutralizing antibodies against WSSV is expected to be an effective means of preventing infection with the virus. In the present study, eight monoclonal antibodies (mAbs) against VP28 were developed by immunizing BALB/c mice with WSSV-VP28 recombinant protein. Among them, three mAbs named 3B7, 2G3 and 5D2 were determined to be able to delay the mortality of WSSV-infected shrimp in vivo neutralization assay, suggesting their neutralizing ability against WSSV infection. Immunoblotting results showed that the three mAbs reacted specifically with native VP28 of WSSV, and could also recognize the virions in the gills of WSSV-infected shrimp by IFA. Furthermore, the single chain variable fragment (scFv) genes specific for WSSV-VP28 were cloned from the three hybridoma cells and expressed in Escherichia coli. After purification and refolding, three biologically active scFv recombinant proteins were all capable of recognizing the native VP28 of WSSV and delayed the mortality of WSSV-infected shrimp, indicating their neutralizing capacity against WSSV. Subsequently, the eukaryotic expression plasmids of three scFv genes were constructed and the transcriptional properties of expression vectors in shrimp were analyzed. Animal experiments also proved that the scFv eukaryotic expression plasmids were able to partially neutralize WSSV infection. Thus, the production of neutralizing mAb and recombinant scFv antibodies against WSSV has a promising therapeutic potential in prevention and treatment of white spot disease of shrimp.

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.

A barrier-to-autointegration factor promotes white spot syndrome virus infection in a crustacean Cherax quadricarinatus

Barrier-to-autointegration factor (BAF) is a highly conserved DNA binding protein that participates in a variety of biological processes such as transcription, epigenetic regulation and antiviral immunity in vertebrates. However, the function of BAF is poorly understood in crustaceans. In this study, we identified a barrier-to-autointegration factor (CqBAF) from red claw crayfish Cherax quadricarinatus, which was responsive to white spot syndrome virus (WSSV) infection. The full-length cDNA sequence of CqBAF was 544 bp, including an open reading frame of 273 bp encoding 90 amino acids, a 107 bp of 5′-Untranslated Regions (5′-UTR) and a 164 bp of 3′-UTR. Gene expression analysis showed that CqBAF was distributed in all tissues examined with the highest expression in the crayfish haematopietic tissue (Hpt), which protein expression was also significantly up-regulated by WSSV infection in Hpt cells. Furthermore, the transcripts of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV were clearly reduced in Hpt cells after gene silencing of CqBAF. Importantly, the promoter activity of two immediate early genes of WSSV, including WSV051 and IE1, was strongly enhanced by the increased phosphorylation of CqBAF, which also facilitated the accumulation of CqBAF protein in the cytoplasm of Sf9 cells. Taken together, these data suggest that CqBAF is likely to increase the replication of WSSV by promoting the transcription of viral immediate early genes, probably regulated by phosphorylation of CqBAF, which sheds new light on the molecular mechanism of WSSV infection.

An oral delivery system for controlling white spot syndrome virus infection in shrimp using transgenic microalgae

White spot disease (WSD) is a longstanding and serious viral disease of various shrimp species that has caused high mortality rates for many decades. Currently, there is no practical method to control this disease. Therefore, we have explored the development of a novel vaccine-based method to control this disease using transgenic algae. During infection by white spot syndrome virus (WSSV), the interaction between viral envelope proteins and cell surface protein receptors on target cells is the key step of viral entry and replication. Hence, transgenic lines of the green microalga Chlamydomonas reinhardtii harbouring a WSSV VP28 viral envelope protein were created as an oral delivery system for vaccinating shrimp. Two separate transplastomic lines containing wild-type and codon optimized gene sequences for VP28 were evaluated for recombinant protein levels. Only the codon optimized line gave rise to detectable VP28 in western blot analysis, which demonstrated that optimization for chloroplast codon bias improved the efficiency of expression and that the gene design produced a favourable RNA secondary structure with suitable free energy for translation. In addition, bile salt and acid tolerance tests demonstrated that this transgenic Chlamydomonas can tolerate mildly acidic (pH 5.0) conditions and 0.30% bile salts. These features indicated that algal cells are suitable for delivering viral antigens through a shrimp's digestive system. In WSSV infection experiments, the highest survival rate (87%) was recorded in shrimps fed with the codon optimized VP28 line mixed into their feed, indicating that this line could be employed in the control of WSSV spread in shrimp populations. This algal strategy offers a new, efficient, fast and less labour-intensive method for the control of other diseases in aquatic animals through oral delivery.

Penaeidins restrict white spot syndrome virus infection by antagonizing the envelope proteins to block viral entry

ABSTRACT Emerging studies have indicated that some penaeidins restrict virus infection; however, the mechanism(s) involved are poorly understood. In the present study, we uncovered that penaeidins are a novel family of antiviral effectors against white spot syndrome virus (WSSV), which antagonize the envelope proteins to block viral entry. We found that the expression levels of four identified penaeidins from Litopenaeus vannamei, including BigPEN, PEN2, PEN3, and PEN4, were significantly induced in hemocytes during the early stage of WSSV infection. Knockdown of each penaeidin in vivo via RNA interference resulted in elevated viral loads and rendered shrimp more susceptible to WSSV, while the survival rate was rescued via the injection of recombinant penaeidins. All penaeidins, except PEN4, were shown to interact with several envelope proteins of WSSV, and all four penaeidins were observed to be located on the outer surface of the WSSV virion. Co-incubation of each recombinant penaeidin with WSSV inhibited virion internalization into hemocytes. More importantly, we found that PEN2 competitively bound to the envelope protein VP24 to release it from polymeric immunoglobulin receptor (pIgR), the cellular receptor required for WSSV infection. Moreover, we also demonstrated that BigPEN was able to bind to VP28 of WSSV, which disrupted the interaction between VP28 and Rab7 – the Rab GTPase that contributes to viral entry by binding with VP28. Taken together, our results demonstrated that penaeidins interact with the envelope proteins of WSSV to block multiple viral infection processes, thereby protecting the host against WSSV.

A novel RING finger protein CqRNF152-like with self-ubiquitination activity inhibits white spot syndrome virus infection in a crustacean Cherax quadricarinatus

Really Interesting New Gene (RING) finger proteins are highly conserved molecules that participate in a variety of biological processes such as regulation of development, apoptosis and antiviral immunity in vertebrates. However, the functions of RING finger proteins are still poorly understood in crustaceans. Previously, we found that the transcript of a homolog of RING finger protein 152 (CqRNF152-like) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, which is one of the most devastating viral diseases for crustaceans like shrimp and crayfish. The full-length cDNA sequence of CqRNF152-like was then identified with 975 bp, including an ORF of 685 bp that encoded a 195 amino acids protein, a 5′- UTR of 180 bp, and a 3′-UTR with a poly (A) tail of 207 bp. The conserved domain prediction showed that CqRNF152-like contained a conserved RING-finger domain. Gene expression analysis showed that CqRNF152-like was distributed in all tissues examined and the transcript is significantly up-regulated after WSSV challenge both in vivo in Hpt tissue and in vitro in cultured Hpt cells. Furthermore, the transcripts of both an immediate early gene ie1 and a late envelope protein gene vp28 of WSSV were clearly increased in the Hpt tissues, hemocytes and cultured Hpt cells after gene silencing of CqRNF152-like, which were further proved to be significantly decreased after overloading of recombinant CqRNF152-like protein in Hpt cell cultures. Meanwhile, CqRNF152-like was found to bind with WSSV envelope protein VP28 by proteins pull-down assay. Similar to most of RNF proteins, CqRNF152-like protein sequence contained a conserved RING-finger domain and showed self-ubiquitination activity in a RING finger domain dependent manner. Taken together, CqRNF152-like is likely to function as an antiviral molecular against WSSV infection through interaction with the envelope protein VP28 in a crustacean C. quadricarinatus. This is the first report that a RING finger protein with directly antiviral functions via interaction with viral protein and self-ubiquitination activity in crustacean, which sheds new light on the molecular mechanism of WSSV infection and the control of white spot disease.

Computational identification of self-inhibitory peptides from white spot syndrome virus envelope protein VP28

Since effective chemotherapeutics or preventive measures are still unavailable, finding feasible approaches against white spot syndrome virus (WSSV) has always been the vital subject in shrimp farming field. Envelope proteins are the ideal targets for antiviral strategies development due to their indispensable roles in virus entry, and inhibitory peptides targeting them have been proved to be promising in blocking virus infection. In this study, the Wimley-White interfacial hydrophobicity scale (WWIHS) in combination with known structural data was applied to identify potential inhibitory peptides that targeted the envelope protein VP28 of WSSV. Results showed that two potential inhibitory peptides were identified, one of which exhibited not only obvious antiviral activity, but also broad-spectrum antimicrobial activity. The inhibitory peptide identified here can serve as a lead compound for anti-WSSV strategies development.

Interaction of the small GTP-binding protein (Rab7) with β-actin in Litopenaeus vannamei and its role in white spot syndrome virus infection

In this study, GST Pull-down and mass spectrometry was applied to the precipitation and identification of the small GTP-binding protein (Rab7) interacting protein in hemocyte of Litopenaeus vannamei. According to the search in GenBank with the peptide mass fingerprint, the 45 kDa protein which was pulled down with the GST-tagged Rab7 (GST-Rab7, GTP bound form) was identified to be β-actin with 28% coverage of amino acid sequences. The interaction of Rab7 with β-actin was verified by both GST Pull-down and ELISA in vitro. Meanwhile, confocal microscopic observation showed that Rab7 could be co-localized with β-actin in hemocytes at 12 h post white spot syndrome virus (WSSV) infection (hpi). GST Pull-down and western blotting were used to analyze the cross-interaction between WSSV VP28, Rab7 and β-actin. The results showed that the GST-VP28, His-tagged Rab7 (His-Rab7) and His-β-actin formed a tripartite complex. At 12 hpi, confocal microscopic observation showed that WSSV could be co-localized with Rab7 and β-actin in hemocytes respectively. Furthermore, based on the in vivo neutralization assay, recombinant His-β-actin accelerated the infection of WSSV, conversely, recombinant His-Rab7 delayed WSSV infection in shrimp. These results suggested the interaction of Rab7 with β-actin and this interaction was involved in WSSV infection.

Identification of a CqCaspase gene with antiviral activity from red claw crayfish Cherax quadricarinatus

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.

Litopenaeus vannamei attenuates white spot syndrome virus replication by specific antiviral peptides generated from hemocyanin

Recent studies have shown that hemocyanin plays immune-related functions apart from its canonical respiratory function. While shrimp hemocyanin is found to generate antimicrobial peptides, antiviral related peptides have not been reported. In the present study, the serum of white spot syndrome virus (WSSV) infected Litopenaeus vannamei analyzed by two-dimensional gel electrophoresis, revealed 45 consistently down-regulated protein spots and 10 up-regulated protein spots. Five of the significantly up-regulated spots were identified as hemocyanin derived peptides. One of the five peptides, designated LvHcL48, was further characterized by analyzing its primary sequence via Edman N-terminal sequencing, C-terminal sequencing and amino acid sequence alignment. LvHcL48 was found to be a 79 amino acid fragment (aa584-662) from the C-terminal domain of L. vannamei hemocyanin protein (ADZ15149). Both in vivo and in vitro functional studies revealed that LvHcL48 has immunological activities, as recombinant LvHcL48 protein (rLvHcL48) significantly inhibited the transcription of the WSSV genes wsv069 and wsv421 coupled with a significant reduction in WSSV copy numbers. Further analysis showed that LvHcL48 could interact with the WSSV envelope protein 28 (VP28). Our present data therefore reveals the generation of an antiviral hemocyanin derived peptide LvHcL48 from WSSV infected shrimp, which binds to the envelope protein VP28 of WSSV.

Marine derived compounds as binders of the White spot syndrome virus VP28 envelope protein: In silico insights from molecular dynamics and binding free energy calculations

White spot syndrome virus (WSSV) remains as one of the most dreadful pathogen of the shrimp aquaculture industry owing to its high virulence. The cumulative mortality reaches up to 100% within in 2–10days in a shrimp farm. Currently, no chemotherapeutics are available to control WSSV. The viral envelope protein, VP28, located on the surface of the virus particle acts as a vital virulence factor in the initial phases of inherent WSSV infection in shrimp. Hence, inhibition of envelope protein VP28 could be a novel way to deal with infection by inhibiting its interaction in the endocytic pathway. In this direction, a timely attempt was made to recognize a potential drug candidate of marine origin against WSSV using VP28 as a target by employing in silico docking and molecular dynamic simulations. A virtual library of 388 marine bioactive compounds was extracted from reports published in Marine Drugs. The top ranking compounds from docking studies were chosen from the flexible docking based on the binding affinities (ΔGb). In addition, the MD simulation and binding free energy analysis were implemented to validate and capture intermolecular interactions. The results suggested that the two compounds obtained a negative binding free energy with −40.453kJ/mol and −31.031kJ/mol for compounds with IDs 30797199 and 144162 respectively. The RMSD curve indicated that 30797199 moves into the hydrophobic core, while the position of 144162 atoms changes abruptly during simulation and is mostly stabilized by water bridges. The shift in RMSD values of VP28 corresponding to ligand RMSD gives an insight into the ligand induced conformational changes in the protein. This study is first of its kind to elucidate the explicit binding of chemical inhibitor to WSSV major structural protein VP28.

A sensitive and specific hyperbranched rolling circle amplification assay and test strip for white spot syndrome virus.

White spot syndrome virus (WSSV) is a global threat to the prawn industry, and there is no simple method for field-based testing of this virus. We designed a padlock probe and primers to the capsid protein gene VP28 of WSSV, and established a hyperbranched rolling circle amplification (HRCA) assay and a corresponding strip-based test. The assay and the test strip both had similar high accuracy and specificity, and their sensitivity was about 10 copies/μL, which is 100 times higher than conventional PCR. In this study, 68 batches of prawns were tested for WSSV with the HRCA assay and test strip, and the results were compared with the PCR assay. The results indicated that both the assay and test strip had accuracy similar to each other and to the PCR results. However, the assay and strip were more sensitive and user-friendly than PCR. Establishment of this method will provide a rapid detection of WSSV and also a basis for field-based detection of animal disease.

The Endemic Copepod Calanus pacificus californicus as a Potential Vector of White Spot Syndrome Virus

The susceptibility of the endemic copepod Calanus pacificus californicus to white spot syndrome virus (WSSV) was established by the temporal analysis of WSSV VP28 transcripts by quantitative real-time PCR (qRT-PCR). The copepods were collected from a shrimp pond located in Bahia de Kino Sonora, Mexico, and challenged per os with WSSV by a virus-phytoplankton adhesion route. Samples were collected at 0, 24, 48 and 84 h postinoculation (hpi). The VP28 transcripts were not detected at early stages (0 and 24 hpi); however, some transcript accumulation was observed at 48 hpi and gradually increased until 84 hpi. Thus, these results clearly show that the copepod C. pacificus californicus is susceptible to WSSV infection and that it may be a potential vector for the dispersal of WSSV. However, further studies are still needed to correlate the epidemiological outbreaks of WSSV with the presence of copepods in shrimp ponds.

Characterization of Four Novel Caspases from Litopenaeus vannamei (Lvcaspase2-5) and Their Role in WSSV Infection through dsRNA-Mediated Gene Silencing

Apoptosis plays an important role in white spot syndrome virus (WSSV) pathogenesis, and caspases are central players in apoptosis. Here, we cloned four novel caspases (Lvcaspase2-5) from the Pacific white shrimp Litopenaeus vannamei, and investigated their potential roles in WSSV replication using dsRNA-mediated gene silencing. Lvcaspase2-5 have the typical domain structure of caspase family proteins, with the conserved consensus motifs p20 and p10. Lvcaspase2 and Lvcaspase5 were highly expressed in muscle, while Lvcaspase3 was highly expressed in hemocytes and Lvcaspase4 was mainly expressed in intestine. Lvcaspase2-5 could also be upregulated by WSSV infection, and they showed different patterns in various tissues. When overexpressed in Drosophila S2 cells, Lvcaspase2-5 showed different cellular localizations. Using dsRNA-medicated gene silencing, the expression of Lvcaspase2, Lvcaspase3, and Lvcaspase5 were effectively knocked down. In Lvcaspase2-, Lvcaspase3- or Lvcaspase5-silenced L. vannamei, expression of WSSV VP28 gene was significantly enhanced, suggesting protective roles for Lvcaspase2, Lvcaspase3 and Lvcaspase5 in the host defense against WSSV infection.

Preparation of transgenic Dunaliella salina for immunization against white spot syndrome virus in crayfish

Although a white spot syndrome virus (WSSV) subunit vaccine could significantly enhance the immune response and benefit the shrimp host, its practical application is currently not feasible because of drawbacks in existing expression systems. We generated a transgenic Dunaliella salina (D. salina) strain by introducing the WSSV VP28 gene to produce a novel oral WSSV subunit vaccine. Following transformation of D. salina, VP28 gene expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) assays, enzyme-linked immunosorbent assays (ELISAs), and western blot analysis. The RT-PCR results indicated that the VP28 gene was successfully expressed in D. salina cells. The presence of recombinant VP28 proteins with natural bioactivity was confirmed by western blot analysis and ELISA. Animal vaccination experiments indicated that transgenic D. salina can induce protection against WSSV by oral delivery in crayfish. Our findings indicate that the VP28 gene can be successfully expressed in transgenic D. salina and can be applied as an oral vaccine to protect crayfish against WSSV. We have demonstrated that it is feasible to produce an oral vaccine using D. salina, and thereby provide a new method for controlling other viral diseases in crustaceans.

Analysis of Expression, Cellular Localization, and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)

Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei . LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrio alginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V . alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibrio penaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.

Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans

BackgroundThe White Spot Syndrome Virus (WSSV), the sole member of the family Whispoviridae, is the etiological agent that causes severe mortality events in wild and farmed shrimp globally. Given its adverse effects, the WSSV has been included in the list of notifiable diseases of the Office of International Epizootic (OIE) since 1997. To date there are no known therapeutic treatments available against this lethal virus, and a surveillance program in brood-stock and larvae, based on appropriate diagnostic tests, has been strongly recommended. However, some currently used procedures intended for diagnosis of WSSV may be particularly susceptible to generate spurious results harmfully impacting the shrimp farming industry.MethodsIn this study, a sensitive one-step SYBR green-based real-time PCR (qPCR) for the detection and quantitation of WSSV was developed. The method was tested against several WSSV infected crustacean species and on samples that were previously diagnosed as being positive for WSSV from different geographical locations.ResultsA universal primer set for targeting the WSSV VP28 gene was designed. This method demonstrated its specificity and sensitivity for detection of WSSV, with detection limits of 12 copies per sample, comparable with the results obtained by other protocols. Furthermore, the primers designed in the present study were shown to exclusively amplify the targeted WSSV VP28 fragment, and successfully detected the virus in different samples regardless of their geographical origin. In addition, the presence of WSSV in several species of crustaceans, including both naturally and experimentally infected, were successfully detected by this method.ConclusionThe designed qPCR assay here is highly specific and displayed high sensitivity. Furthermore, this assay is universal as it allows the detection of WSSV from different geographic locations and in several crustacean species that may serve as potential vectors. Clearly, in many low-income import-dependent nations, where the growth of shrimp farming industries has been impressive, there is a demand for cost-effective diagnostic tools. This study may become an alternative molecular tool for a less expensive, rapid and efficient detection of WSSV.