Molecular docking analyses of Avicennia marinaderived phytochemicals against white spot syndrome virus (WSSV) envelope protein-VP28

White spot syndrome (WSS) is one of the most common and most disastrous diseases of shrimp worldwide. It causes up to 100% mortality within 3 to 4 days in commercial shrimp farms, resulting in large economic losses to the shrimp farming industry. VP28 envelope protein of WSSV is reported to play a key role in the systemic infection in shrimps. Considering the most sombre issue of viral disease in cultivated shrimp, the present study was undertaken to substantiate the inhibition potential of Avicennia marinaderived phytochemicals against the WSSV envelope protein VP28. Seven A. marina-derived phytochemicals namely stigmasterol, triterpenoid, betulin, lupeol, avicenol-A, betulinic acid and quercetin were docked against the WSSV protein VP28 by using Argus lab molecular docking software. The chemical structures of the phytochemicals were retrieved from Pubchem database and generated from SMILES notation. Similarly the protein structure of the envelope protein was obtained from protein data bank (PDB-ID: 2ED6). Binding sites were predicted by using ligand explorer software. Among the phytochemicals screened, stigmasterol, lupeol and betulin showed the best binding exhibiting the potential to block VP28 envelope protein of WSSV, which could possibly inhibit the attachment of WSSV to the host species. Further experimental studies will provide a clear understanding on the mode of action of these phytochemicals individually or synergistically against WSSV envelope protein and can be used as an inhibitory drug to reduce white spot related severe complications in crustaceans.     

Envelope Proteins of White Spot Syndrome Virus (WSSV) Interact with Litopenaeus vannamei Peritrophin-Like Protein (LvPT)

White spot syndrome virus (WSSV) is a major pathogen in shrimp cultures. The interactions between viral proteins and their receptors on the surface of cells in a frontier target tissue are crucial for triggering an infection. In this study, a yeast two-hybrid (Y2H) library was constructed using cDNA obtained from the stomach and gut of Litopenaeus vannamei, to ascertain the role of envelope proteins in WSSV infection. For this purpose, VP37 was used as the bait in the Y2H library screening. Forty positive clones were detected after screening. The positive clones were analyzed and discriminated, and two clones belonging to the peritrophin family were subsequently confirmed as genuine positive clones. Sequence analysis revealed that both clones could be considered as the same gene, LV-peritrophin (LvPT). Co-immunoprecipitation confirmed the interaction between LvPT and VP37. Further studies in the Y2H system revealed that LvPT could also interact with other WSSV envelope proteins such as VP32, VP38A, VP39B, and VP41A. The distribution of LvPT in tissues revealed that LvPT was mainly expressed in the stomach than in other tissues. In addition, LvPT was found to be a secretory protein, and its chitin-binding ability was also confirmed.     

Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells

White spot disease (WSD) is caused by the white spot syndrome virus (WSSV), which results in devastating losses to the shrimp farming industry around the world. However, the mechanism of virus entry and spread into the shrimp cells is unknown. A binding assay in vitro demonstrated VP28-EGFP (envelope protein VP28 fused with enhanced green fluorescence protein) binding to shrimp cells. This provides direct evidence that VP28-EGFP can bind to shrimp cells at pH 6.0 within 0.5 h. However, the protein was observed to enter the cytoplasm 3 h post-adsorption. Meanwhile, the plaque inhibition test showed that the polyclonal antibody against VP28 (a major envelope protein of WSSV) could neutralize the WSSV and block an infection with the virus. The result of competition ELISA further confirmed that the envelope protein VP28 could compete with WSSV to bind to shrimp cells. Overall, VP28 of the WSSV can bind to shrimp cells as an attachment protein, and can help the virus enter the cytoplasm.     

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.     

Multiple proteins of White spot syndrome virus involved in recognition of β-integrin

The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that β-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of β-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with β-integrin. The β-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of β-integrin. Identification of proteins in WSSV that are associated with β-integrin will assist development of new agents for effective control of the white spot syndrome.     

Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship

White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 A, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt beta-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core beta-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.     

A putative cell surface receptor for white spot syndrome virus is a member of a transporter superfamily

White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral disease in shrimp and has a wide host range among crustaceans. In this study, we identified a surface protein, named glucose transporter 1 (Glut1), which could also interact with WSSV envelope protein, VP53A. Sequence analysis revealed that Glut1 is a member of a large superfamily of transporters and that it is most closely related to evolutionary branches of this superfamily, branches that function to transport this sugar. Tissue tropism analysis showed that Glut1 was constitutive and highly expressed in almost all organs. Glut1's localization in shrimp cells was further verified and so was its interaction with Penaeus monodon chitin-binding protein (PmCBP), which was itself identified to interact with an envelope protein complex formed by 11 WSSV envelope proteins. In vitro and in vivo neutralization experiments using synthetic peptide contained WSSV binding domain (WBD) showed that the WBD peptide could inhibit WSSV infection in primary cultured hemocytes and delay the mortality in shrimps challenged with WSSV. These findings have important implications for our understanding of WSSV entry.     

Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine

Although invertebrates lack a true adaptive immune response, the potential to vaccinate Penaeus monodon shrimp against white spot syndrome virus (WSSV) using the WSSV envelope proteins VP19 and VP28 was evaluated. Both structural WSSV proteins were N-terminally fused to the maltose binding protein (MBP) and purified after expression in bacteria. Shrimp were vaccinated by intramuscular injection of the purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection. As controls, purified MBP- and mock-vaccinated shrimp were included. VP19-vaccinated shrimp showed a significantly better survival (p<0.05) as compared to the MBP-vaccinated control shrimp with a relative percent survival (RPS) of 33% and 57% at 2 and 25 days after vaccination, respectively. Also, the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival when challenged two days after vaccination (RPS of 44% and 33%, respectively), but not after 25 days. These results show that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine. This suggests that the shrimp immune system is able to specifically recognize and react to proteins. This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, opening the way to new strategies to control viral diseases in shrimp and other crustaceans.     

Prohibitin Interacts with Envelope Proteins of White Spot Syndrome Virus and Prevents Infection in the Red Swamp Crayfish,Procambarus clarkii

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.     

Involvement of interaction between viral VP466 and host tropomyosin proteins in virus infection in shrimp

The accumulating evidence indicates that the viral structural proteins play critical roles in virus infection. However, the interaction between the viral structural protein and host cytoskeleton protein in virus infection remains to be addressed. In this study, the viral VP466 protein, one of the major structural proteins of shrimp white spot syndrome virus (WSSV), was characterized. The results showed that the suppression of VP466 gene expression led to the inhibition of WSSV infection in shrimp, indicating that the VP466 protein was required in virus invasion. It was found that the VP466 protein was interacted with the host cytoskeleton protein tropomyosin. As documented, the VP466–tropomyosin interaction facilitated the WSSV infection. Therefore our findings revealed a novel molecular mechanism in the virus invasion to its host, which would be helpful to better understand the molecular events in virus infection in invertebrate.     

Protection of Penaeus monodon against white spot syndrome virus by oral vaccination

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. No adequate treatments against this virus are available. It is generally accepted that invertebrates such as shrimp do not have an adaptive immune response system such as that present in vertebrates. As it has been demonstrated that shrimp surviving a WSSV infection have higher survival rates upon subsequent rechallenge, we investigated the potential of oral vaccination of shrimp with subunit vaccines consisting of WSSV virion envelope proteins. Penaeus monodon shrimp were fed food pellets coated with inactivated bacteria overexpressing two WSSV envelope proteins, VP19 and VP28. Vaccination with VP28 showed a significant lower cumulative mortality compared to vaccination with bacteria expressing the empty vectors after challenge via immersion (relative survival, 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection, challenges were subsequently performed 3, 7, and 21 days after vaccination. A significantly higher survival was observed both 3 and 7 days postvaccination (relative survival, 64% and 77%, respectively), but the protection was reduced 21 days after the vaccination (relative survival, 29%). This suggests that contrary to current assumptions that invertebrates do not have a true adaptive immune system, a specific immune response and protection can be induced in P. monodon. These experiments open up new ways to benefit the WSSV-hampered shrimp farming industry.     

Protection of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV) challenge by double-stranded RNA

To determine whether Penaeus chinensis can be protected against white spot syndrome virus (WSSV) infection by intramuscular injection with long double-stranded RNAs (dsRNAs) as in other shrimp species and whether the protection degree by WSSV-specific dsRNAs is correlated with the roles of viral genes, P. chinensis juveniles were intramuscularly injected with long dsRNAs corresponding to VP28, VP281, protein kinase genes of WSSV, and an unrelated long dsRNA corresponding to a green fluorescence protein (GFP) gene. All shrimp injected with long dsRNAs including GFP dsRNA showed higher survival rates against WSSV infection than shrimp injected with PBS alone. Furthermore, shrimp injected with dsRNAs corresponding to VP28 and protein kinase showed higher survival rates than those injected with dsRNAs corresponding to VP281 and GFP. These results indicate that the introduction of long dsRNAs corresponding to viral proteins, which are essential for WSSV infection, is quite effective in blocking WSSV infection in P. chinensis, and suggest that dsRNA-mediated protection is a common feature across shrimp species.     

Conservation of the DNA sequences encoding the major structural viral proteins of WSSV

A cDNA library was constructed from white spot syndrome virus (WSSV)-infected penaeid shrimp tissue. cDNA clones with WSSV inserts were isolated and sequenced. By comparison with DNA sequences in GenBank, cDNA clones containing sequence identical to those of the WSSV envelope protein VP28 and nucleoprotein VP15 were identified. Poly(A) sites in the mRNAs of VP28 and VP15 were identified. Genes encoding the major viral structural proteins VP28, VP26, VP24, VP19 and VP15 of 5 WSSV isolates collected from different shrimp species and/or geographical areas were sequenced and compared with those of 4 other WSSV isolate sequences in GenBank. For each of the viral structural protein genes compared, the nucleotide sequences were 100 to 99% identical among the 9 isolates. Gene probes or PCR primers based on the gene sequences of the WSSV structural proteins can be used for diagnoses and/or detection of WSSV infection.     

Penaeus monodon chitin-binding protein (PmCBP) is involved in white spot syndrome virus (WSSV) infection

White spot syndrome virus (WSSV) can cause the most serious viral disease of shrimp and has a wide host range among crustaceans. Although researches show a lot about its genome and structure, information concerning the mechanism of how WSSV infects' cells is lacking. In this study, some experiments were applied to confirm the biological meaning of the protein–protein interaction between WSSV envelope protein, VP53A, and Penaeus monodon chitin-binding protein (PmCBP). Immunofluorescent study indicated that PmCBP is located on the cell surface of host cells. PmCBP amounts of about 34 kDa can be detected in both P. monodon and Litopenaeus vannamei tissues by Western blotting. In the in vivo neutralization experiment, both rVP53A and rPmCBP that were produced by Esherichia coli can promote resp. a 40% and 20% survival rate of the shrimp which were challenged by WSSV. Furthermore, a yeast-two-hybrid result revealed that PmCBP could interact with at least 11 WSSV envelope proteins. Those findings suggest that PmCBP may be involved in WSSV infection.     

Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV

Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests.     

VP37 of white spot syndrome virus interact with shrimp cells

Aims:  To investigate VP37 [WSV 254 of White spot syndrome virus (WSSV) genome] interacting with shrimp cells and protecting shrimp against WSSV infection.
Methods and Results:  VP37 was expressed in Escherichia coli and was confirmed by Western blotting. Virus overlay protein binding assay (VOPBA) technique was used to analyse the rVP37 interaction with shrimp and the results showed that rVP37 interacted with shrimp cell membrane. Binding assay of recombinant VP37 with shrimp cell membrane by ELISA confirmed that purified rVP37 had a high-binding activity with shrimp cell membrane. Binding of rVP37 to shrimp cell membrane was a dose-dependent. Competition ELISA result showed that the envelope protein VP37 could compete with WSSV to bind to shrimp cells. In vivo inhibition experiment showed that rVP37 provided 40% protection. Inhibition of virus infection by rVP37 in primary cell culture revealed that rVP37 counterparted virus infection within the experiment period.
Conclusions:  VP37 has been successfully expressed in E . coli . VP37 interacted with shrimp cells.
Significance and Impact of the Study:  The results suggest that rVP37 has a potential application in prevention of virus infection.     

Protection of Shrimp Penaeus monodon from WSSV Infection Using Antisense Constructs

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3–VP24, pPn–VP24), VP28 (pH3–VP28, pPn–VP28), TS (pH3–TS, pPn–TS), and RR2 (pH3–RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.     

White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP)

White spot syndrome virus (WSSV) is the causative agent of a severe disease of cultivated shrimp. Using purified WSSV virions, VP53A encoded by open reading frame wssv067 was identified as a structural protein by SDS-PAGE and proteomics. Immunoelectron microscopy with a gold-labeled secondary antibody revealed that VP53A was distributed on the viral envelope. In order to further explore the link between WSSV067 and host proteins, we performed a yeast 2-hybrid screening of a Penaeus monodon cDNA library, using WSSV067C as bait. One of the molecules that specifically interacted with WSSV067C was the P. monodon chitin-binding protein (PmCBP). An in vitro binding assay showed that c-myc-WSSV067C was capable of co-precipitating HA-PmCBP-C. Furthermore, PmCBP was expressed in almost all organs but appeared to be up-regulated at the late stage of WSSV infection.