White Spot Syndrome Virus Infection In Shrimp Research Articles

White spot syndrome virus IE1 protein blocks the integrin-FAK signaling to enhance viral infection in shrimp

DNA viruses commonly utilize immediate-early proteins to manipulate cellular signaling pathways in order to facilitate their infection. Our previous research has suggested that IE1, an immediate-early protein encoded by the white spot syndrome virus (WSSV), may modulate the shrimp integrin-FAK signaling pathway. However, the specific molecular mechanism and role of IE1 in regulating this signaling pathway remain unclear. In this study, we demonstrated that IE1 competes for binding to the cytoplasmic tail of Penaeus vannamei integrin-α5, resulting in the inhibition of the integrin-α5-FAK interaction, thereby suppressing FAK activation and cell adhesion. Furthermore, we observed a significant increase in the expression of P. vannamei integrin-α5 and FAK following WSSV infection. Additionally, knockdown of integrin-α5 or FAK through RNA interference has been shown to reduce cell adhesion and enhance WSSV infection. In conclusion, our findings reveal that IE1 disrupts integrin-FAK signaling to inhibit cell adhesion, ultimately promoting WSSV infection in shrimp.

Genome editing of WSSV CRISPR/Cas9 and immune activation extends the survival of infected Penaeus vannamei

White spot syndrome virus (WSSV) is an exceptionally harmful virus that generally causes high levels of mortality in cultured shrimp. Attempts at viral suppression have been made to control the disease and have achieved limited efficiency. Recent advances in genome editing technology using CRISPR/Cas9 have led to potential innovations to prevent or treat many viral diseases. In this study, a CRISPR/Cas9 system was applied to WSSV genome cleavage to suppress WSSV infection in shrimp. The U6 promoter sequence was identified. A chimeric DNA vector consisting of the shrimp U6 promoter with gRNA expression sequences specific to two sites of the WSSV genome and the WSSV ribonucleotide reductase promoter with the Cas9 DNA sequence in pAC-sgRNA-Cas9 was constructed. The expression of gRNAs specific to the WSSV genome and Cas9 was determined in primary cultured hemocyte cells and in shrimp tissue via RT‒PCR. The efficacy of CRISPR/Cas9-WSSV for WSSV genome cleavage was determined in vitro and against WSSV-infected Penaeus vannamei. The reaction of synthetic gRNAs and recombinant Cas9 was able to cleave WSSV DNA amplicons, and shrimp that received CRISPR/Cas9-WSSV presented significantly lower WSSV DNA. In addition to interfering with viral DNA propagation, CRISPR/Cas9-WSSV encapsulated with IHHNV-VLP also stimulated an immune-related gene response. Treatment with CRISPR/Cas9-WSSV against WSSV challenge resulted in a significantly longer survival period. This finding has led to the development and application of a CRISPR/Cas9 system for WSSV infectious disease control, which could be used for managing shrimp aquaculture in the future.

A computational approach to identifying peptide inhibitors againstWhite Spot Syndrome Virus: Targeting the virus envelope protein

The white spot syndrome virus (WSSV), a rapidly replicating and highly lethal pathogen that targets Penaeid shrimp, has emerged as one of the most widespread viruses globally due to its high virulence. With effective chemotherapeutics still unavailable, the pursuit of novel and viable strategies against WSSV remains a crucial focus in the field of shrimp farming. The envelope proteins of WSSV are essential for virus entry, serving as excellent targets for the development of antiviral therapeutics. Novel strategies in the design of inhibitory peptides, especially those targeting envelope protein (VP28) located on the surface of the virus particle, play a critical role as a significant virulence factor during the early stages of inherent WSSV infection in shrimp. In this direction, the current computational study focused on identifying self-inhibitory peptides from the hydrophobic membrane regions of the VP28 protein, employing peptide docking and molecular dynamics simulation (MDS) approaches. Such inhibitory peptides could be useful building blocks for the rational engineering of inhibitory therapeutics since they imitate the mechanism of binding to homologous partners used by their origin domain to interact with other molecules. The N-terminal sequence of VP28 has been reported as the potential site for membrane interactions during the virus entry. Moreover, drug delivery systems mediated by chitosan and gold nanoparticles are being developed to enhance the therapeutic efficacy of anti-viral peptides. These systems can increase the solubility, stability, and selectivity of peptides, possessing better qualities than conventional delivery methods. This computational study on self-inhibitory peptides could be a valuable resource for further in vitro and in vivo studies on anti-viral therapeutics in the aquaculture industry.

DnaJC16, the molecular chaperone, is implicated in hemocyte apoptosis and facilitates of WSSV infection in shrimp.

Chaperone proteins, including heat shock proteins (HSPs) and DnaJ proteins, are highly conserved and well known for their quick responses to environmental stresses and pathogen infections, especially viruses. However, how DnaJ, an HSP family member, in Penaeus vannamei responds to viral invasion has not been reported. In this research, the novel DnaJ homolog subfamily C member 16-like, or DnaJC16, was characterized in P. vannamei. It contains the DnaJ and thioredoxin domains. Phylogenetic tree analysis demonstrated the conservation of DnaJC16 among penaeid shrimp, where PvDnaJC16 was found to be closely related to DnaJC16 from Fenneropenaeus chinensis and Marsupenaeus japonicus. The transcripts of PvDnaJC16 were expressed in all the tissues tested, and the highest expression was in the lymphoid organs. As hemocytes are major immune tissue, we found significant upregulation of PvDnaJC16 in shrimp hemocytes after white spot syndrome virus (WSSV) infection. Furthermore, the suppression of PvDnaJC16 expression by RNA interference in WSSV-infected shrimp showed a decrease in replication and WSSV copy number. Interestingly, a dramatically high cumulative survival rate following the WSSV challenge (over 60%) was observed in PvDnaJC16-silenced shrimp. Meanwhile, the total hemocyte number was significantly increased in PvDnaJC16 knockdown. In addition, the expression of caspase-3 was reduced, as was the caspase-3/7 activity in PvDnaJC16 silencing. Additionally, the percentage of late apoptotic hemocytes diminished after PvDnaJC16 reduction, whereas the percentage of hemocyte viability increased. Our data reflect the fact that the upregulation of PvDnaJC16 expression upon WSSV infection enhances hemocyte apoptosis, which can accelerate viral spreading in shrimp.

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.

Natural ingredient paeoniflorin could be a lead compound against white spot syndrome virus infection in Litopenaeus vannamei.

White spot syndrome virus (WSSV) is an important pathogen causing high mortality in the shrimp industry in aquaculture, yet there is no treatment available to date. In order to find a treatment against WSSV infection, this study examined the anti-WSSV activity of eight natural compounds using shrimp larvae as a model. Among the eight compounds, paeoniflorin showed the most obvious anti-WSSV effect, with a maximum protection efficiency of WSSV-infected shrimp >60% at 100μM. Furthermore, pretreatment and post-treatment experiments revealed that paeoniflorin could prevent and treat WSSV infection in shrimp. The antiviral activity of paeoniflorin in aquaculture water decreased rapidly with time, and the results showed that the stable anti-WSSV activity of paeoniflorin could only remain in water for 1day. Thus, the dosing pattern of continuous medication changes was evaluated. Obviously, in the model of continuous change of paeoniflorin, WSSV copy numbers in the virus-treated shrimp group still progressively increased, while the virus content in WSSVpaeoniflorin -treated group continued to decrease. Interestingly, paeoniflorin inhibited horizontal transmission of WSSV to a certain extent. Notably, paeoniflorin significantly increased the expression of antimicrobial peptides of shrimp to resist WSSV. In conclusion, paeoniflorin has the potential to protect shrimp against WSSV.

Ftz-F1H promotes white spot syndrome virus infection in shrimp by suppressing the Dorsal pathway

White spot syndrome virus (WSSV) is currently a major viral pathogen of cultured shrimp. The NF-κB signaling is central to the regulation of immune responses in both invertebrates and vertebrates. Dorsal is a member of the NF-κB family in arthropods, playing important role in shrimp antiviral response. The fushi tarazu (FTZ) transcription factor (Ftz-F1) homologue (Ftz-F1H) is a novel identified orphan nuclear receptor in Pacific white shrimp Penaeus vannamei with an inhibitory effect on anti-Vibrio parahaemolyticus response. The current study investigated the role of Ftz-F1H in the immune response against WSSV infection. We showed that Ftz-F1H directly promoted the expression of Cactus, the cytoplasmic inhibitor of the Dorsal pathway in shrimp, and inhibited the nuclear translocation of Dorsal. Silencing of Ftz-F1H in vivo suppressed the WSSV infection in shrimp. These suggested that Ftz-F1H could promote WSSV infection by inhibiting the activation of the Dorsal pathway. This study, together with the previous finding, indicated that inhibition of Ftz-F1H in shrimp could enhance the defense against both bacterial and viral infections.

A novel C-type lectin LvCTL 4.2 has antibacterial activity but facilitates WSSV infection in shrimp (L. vannamei)

Glycan-binding protein C-type lectin (CTL), one of the pattern recognition receptors (PRRs), binds to carbohydrates on the surface of pathogens and elicits antimicrobial responses in shrimp innate immunity. The objective was to identify and characterize a novel C-type lectin LvCTL 4.2 in Litopenaeus vannamei. The LvCTL 4.2 protein consisted of a signal peptide at the N terminal and a carbohydrate-recognition domain (CRD) with a mutated mannose-binding (Glu-Pro-Ala; EPA) motif at the C terminal, and thereby has a putative secreted mannose-binding C-type lectin architecture. LvCTL 4.2 was highly expressed in nervous tissue and stomach. Infection with white spot syndrome virus (WSSV) induced expression of LvCTL 4.2 in shrimp stomach at 12 h post infection. Conversely, there was no obvious upregulation in expression of LvCTL 4.2 in stomach or hepatopancreas of shrimp with AHPND (acute hepatopancreas necrosis disease). Pathogen binding assays confirmed recombinant LvCTL 4.2 protein (rLvCTL 4.2) had significant binding ability with the WSSV virion, Gram-negative, and Gram-positive bacteria. Moreover, rLvCTL 4.2 had strong growth inhibition of Vibrio parahaemolyticus. Silencing LvCTL 4.2 suppressed WSSV replication, whereas pretreatment of WSSV with rLvCTL 4.2 facilitated viral replication in vivo. In conclusion, LvCTL 4.2 acted as a PRR that inhibited AHPND-causing bacteria, but facilitated WSSV pathogenesis.

Different expression pattern of thrombospondin gene in the presence and absence of β-glucan fed Penaeus monodon challenged with white spot syndrome virus

Thrombospondins (TSPs) are extracellular, calcium-binding glycoproteins that play an essential role in cell homeostasis and development, wound-healing, angiogenesis, connective tissue organization, immune response etc. and it conserves from sea sponges to mammals. However, their role in shrimp immunity is poorly understood. In the present study, the differential expression profiling of TSP transcripts in Penaeus monodon tissues such as gills, lymphoid organs, hepatopancreas, and hemolymph challenged with white spot syndrome virus (WSSV), were studied by quantitative real-time PCR. Further, shrimps fed with the immunostimulant (β-glucan) when challenged with WSSV showed significant upregulation of TSP expression in gills, hepatopancreas, and lymphoid organ at the early phase of WSSV infection. The results suggest that TSP may be an inducible acute phase response protein to WSSV infection. The possibility of differences in mRNA expression pattern seen in immunostimmulated shrimp after the viral challenge, possibility due to altered immune mechanisms getting triggered during immunostimulant administration and virus infections in the host.

Toll receptor 2 (Toll2) positively regulates antibacterial immunity but promotes white spot syndrome virus (WSSV) infection in shrimp

The Toll family of receptors are a group of conserved pattern recognition receptors (PRRs) essentially controlling the initiation of innate immune responses. The white spot syndrome virus (WSSV) and Vibrio parahaemolyticus are major pathogens of aquaculture shrimp. Previous study has suggested that expression of the Toll2 receptor in Pacific white shrimp Penaeus vannamei was up-regulated by white spot syndrome virus (WSSV) infection but did not significantly changed upon infection with the bacterial pathogen Vibrio parahaemolyticus. The current study intends to investigate the role of P. vannamei Toll2 in antibacterial and antiviral immunity. We demonstrated that compared with the control, the Toll2-silenced shrimp was more susceptible to V. parahaemolyticus infection, suggesting that Toll2 may play a positive role in antibacterial immunity. However, silencing of Toll2 significantly enhanced survivorship of shrimp infected with WSSV and reduced the viral load in shrimp tissues. The expression of WSSV structural protein VP28 was also inhibited in Toll2-silenced shrimp. Histologic pathology analysis further showed that the WSSV infection was attenuated in stomach tissues from Toll2-silenced shrimp. These suggested that Toll2 could promote WSSV infection in shrimp. In Toll2-silenced shrimp, expression of antimicrobial peptides ALFs and PENs was significantly changed, which may contribute to the role of Toll2 in antibacterial immunity and WSSV infection.

The Two NF-κB Pathways Regulating Bacterial and WSSV Infection of Shrimp.

The outbreak of diseases ordinarily results from the disruption of the balance and harmony between hosts and pathogens. Devoid of adaptive immunity, shrimp rely largely on the innate immune system to protect themselves from pathogenic infection. Two nuclear factor-κB (NF-κB) pathways, the Toll and immune deficiency (IMD) pathways, are generally regarded as the major regulators of the immune response in shrimp, which have been extensively studied over the years. Bacterial infection can be recognized by Toll and IMD pathways, which activate two NF-κB transcription factors, Dorsal and Relish, respectively, to eventually lead to boosting the expression of various antimicrobial peptides (AMPs). In response to white-spot-syndrome-virus (WSSV) infection, these two pathways appear to be subverted and hijacked to favor viral survival. In this review, the recent progress in elucidating microbial recognition, signal transduction, and effector regulation within both shrimp Toll and IMD pathways will be discussed. We will also highlight and discuss the similarities and differences between shrimps and their Drosophila or mammalian counterparts. Understanding the interplay between pathogens and shrimp NF-κB pathways may provide new opportunities for disease-prevention strategies in the future.

Role of DCP1-DCP2 complex regulated by viral and host microRNAs in DNA virus infection

The DCP1-DCP2 complex can regulate the antiviral immunity of animals by the decapping of retrovirus RNAs and the suppression of RNAi during RNA virus infection. However, the influence of DCP1-DCP2 complex on DNA virus infection and the regulation of DCP1-DCP2 complex by microRNAs (miRNAs) remain unclear. In this study, the role of miRNA-regulated DCP1-DCP2 complex in DNA virus infection was characterized. Our results showed that the DCP1-DCP2 complex played a positive role in the infection of white spot syndrome virus (WSSV), a DNA virus of shrimp. In the DCP1-DCP2 complex, the N-terminal regulatory domain of DCP2 was interacted with the EVH1 domain of DCP1. Furthermore, shrimp miRNA miR-87 inhibited WSSV infection by targeting the host DCP2 gene and viral miRNA WSSV-miR-N46 took a negative effect on WSSV replication by targeting the host DCP1 gene. Therefore, our study provided novel insights into the underlying mechanism of DCP1-DCP2 complex and its regulation by miRNAs in virus-host interactions. ImportanceDuring RNA virus infection, the DCP1-DCP2 complex can play important roles in the animal antiviral immunity by decapping retrovirus RNAs and suppressing RNAi. In the present study, the findings indicated that the silencing of DCP1 and DCP2 inhibited the infection of WSSV, a DNA virus of shrimp, suggesting that the DCP1-DCP2 complex facilitated DNA virus infection. Due to the suppressive role of the DCP1-DCP2 complex in shrimp RNAi against WSSV infection, the DCP1-DCP2 complex could promote WSSV infection in shrimp. The results showed that WSSV-miR-N46 and shrimp miR-87 could respectively suppress the expressions of DCP1 and DCP2 to affect virus infection. Therefore, our study contributed novel aspects of the DCP1-DCP2 complex and its regulation by miRNAs in virus-host interactions.

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.

The polymeric immunoglobulin receptor-like protein from Marsupenaeus japonicus is a receptor for white spot syndrome virus infection.

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

An effective white spot syndrome virus challenge test for cultured shrimp using different biomass of the infected papilla

White spot syndrome virus (WSSV) is one of the most virulent pathogens of cultured penaeid shrimp. Several control strategies are used commonly to mitigate the economic losses caused by the pathogen, such as application of antiviral products at farm level. One of the most practical method for the screening of potential anti-WSSV products is through challenge tests. Therefore, it is essential to develop simple, reproducible and effective bioassays able to simulate specific mortality levels. The purpose of this study was to develop a simple and reproducible bioassay that simulate different mortality levels by varying the proportion of WSSV-infected and noninfected shrimp tissues administered to susceptible shrimp during a per os challenge test. This method mimics one of the natural transmission routes of WSSV infection in shrimp and could be applied to identify potential antiviral products to different cultured shrimp species susceptible to WSSV.Here we report:•A simple and economic method to evaluate therapeutic antiviral products against WSSV through a challenge test, that uses different biomass amounts of WSSV-infected papilla.•Allows to simulate a wide and reproducible range of mortalities observed in shrimp farms.•A challenge test that simulates one mode of natural WSSV infection in shrimp.

Analysis and identification of tyrosine phosphorylated proteins in hemocytes of Litopenaeus vannamei infected with WSSV

Previous studies have demonstrated that protein tyrosine phosphorylation plays an important role in WSSV infection. In the present work, in order to further elucidate the potential role of protein tyrosine phosphorylation in white spot syndrome virus (WSSV) infection. The expression variation of tyrosine phosphorylated proteins in hemocytes of shrimp (Litopenaeus vannamei) after WSSV infection were examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA), and results showed that the level of protein tyrosine phosphorylation in hemocytes fluctuated significantly after WSSV infection and exhibited two peaks at 6 and 24 h post infection (hpi). Meanwhile, tyrosine phosphorylated proteins in hemocytes after WSSV infection were also detected by cell immunofluorescence, and results showed that the fluorescence intensity in hemocytes was altered with the course of WSSV infection and showed stronger fluorescent signals at 6 and 24 hpi compared to other time points. Furthermore, two dimensional gel electrophoresis (2-DE) and 2-DE western blotting were applied to identify the differentially expressed tyrosine phosphorylated proteins in hemocytes before and after WSSV infection. The result of 2-DE western blotting showed that there were nine tyrosine phosphorylated proteins in the hemocytes of healthy shrimp, whereas twenty-one tyrosine phosphorylated proteins were detected in the hemocytes of shrimp at 6hpi. Then, the differential tyrosine phosphorylated proteins were analyzed by Mass Spectrometry (MS), and eight of them were identified to be sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, actin-D, phosphopyruvate hydratase, beta-actin, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase. Moreover, the expression levels of sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, phosphopyruvate hydratase, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase were examined to be up-regulated post WSSV infection by quantitative real-time RT-PCR. Taken together, these results demonstrated that protein tyrosine phosphorylation was involved in the process of WSSV infection, which might play an important role in the immune response to WSSV infection in shrimp.

Increased nucleoside diphosphate kinase activity induces white spot syndrome virus infection in Litopenaeus vannamei.

Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP’s) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp.

White spot syndrome virus VP28 specific double-stranded RNA provides protection through a highly focused siRNA population

Several studies have demonstrated that injection of double-stranded RNAs (dsRNA) homologous to mRNA for the white spot syndrome virus (WSSV) viral protein 28 (VP28) can induce protection in shrimp against WSSV through RNA interference (RNAi). In comparison to shrimp injected with either PBS or a green fluorescent protein (GFP) nonspecific dsRNA, we obtained nearly complete protection against WSSV infection in shrimp injected with VP28 dsRNA. Upregulation of host genes associated with small RNA silencing was measured 48 hours post treatment in groups injected with dsRNA, and although the VP28-treated group remained moderately upregulated after challenge with WSSV, many-fold higher induction was observed in both control groups reflecting the ongoing viral infection. RNA sequencing of VP28-treated shrimp demonstrated a siRNA population dominated by high levels of 22 nt long molecules narrowly targeting the VP28 mRNA both before and after challenge with WSSV. Conversely, while no siRNAs targeting WSSV were detected before challenge, a broad response of 22 nt siRNAs mapping across the entire WSSV genome were found in both control groups after challenge. These results give detailed insight to how dsRNA targeting VP28 function to induce protection against WSSV, by generating a highly focused population of 22 nt long siRNA molecules.

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.

Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection

White spot syndrome virus (WSSV) is a major causative agent in shrimp farming. Consequently, RNAi technology is an effective strategy to prevent WSSV infection in shrimp especially dsRNA targeting to rr2 of WSSV. In an effort to develop dsRNA expression in shrimp for control of WSSV infection, we developed a recombinant baculovirus expressing recombinant VP28 as the gene delivery system to carry a gene encoding dsRNA specific to rr2 for triggering the RNAi process in shrimp. The results showed that the recombinant baculovirus harboring VP28 was able to express VP28 indicated by Western blot with polyclonal antibody specific to VP28. VP28 transcript was detected in shrimp hemocytes after co-culture hemocytes with the recombinant baculovirus displaying VP28. In addition, we found that shrimp injected with the recombinant baculovirus displaying VP28 and encoding dsRNA synthetic gene specific to rr2 (Bac-VP28-dsrr2) showed the lowest cumulative mortality (33%) at 14days post infection (dpi) when compared to shrimp injected with baculovirus displaying VP28 (Bac-VP28) (64% cumulative mortality) (p<0.05). According to the results, shrimp injected with Bac-VP28-dsrr2 also showed significantly lower WSSV copies than shrimp injected with Bac-VP28 (p<0.05) along with the down-regulation of rr2 expression at 1, 3 and 7dpi. In conclusion, the Bac-VP28-dsrr2 was effective in prevention of WSSV infection. Therefore, the results obtained here can be applied to the prevention of WSSV infection by mixing the recombinant baculovirus with shrimp feed in the future.