Identification of two major virion protein genes of white spot syndrome virus of shrimp

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 x 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.     

Identification of a collagen-like protein gene from white spot syndrome virus

Summary. The most unique feature of white spot syndrome virus (WSSV) is the presence of a collagen-like protein (termed as WSSV-CLP). In this report, the N-terminal fragment of WSSV-CLP (CLPn) was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified. Specific antibody was then raised against the purified fusion protein (GST-CLPn). Temporal analysis showed that the WSSV collagen gene was an early viral gene. Immunogold localization using specific antibody revealed that the gold particles, under a transmission electron microscope, were presented along the outer envelope of WSSV virions. This experiment suggested that the collagen gene encoded an envelope protein of WSSV. Using immuno-affinity chromatography, the WSSV-CLP was purified from crudely purified WSSV virions. The WSSV-CLP was N-glycosylated, as indicated by the increased migration in SDS-PAGE after treatment with N-linked glycosidase F.     

Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31

Based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 kDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag, and then specific antibody was raised. Western blot analysis and the immunoelectron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity.     

Inactivation of White Spot Syndrome Virus (WSSV) by normal rabbit serum: implications for the role of the envelope protein VP28 in WSSV infection of shrimp

White Spot Syndrome Virus (WSSV) is a highly pathogenic and prevalent virus affecting crustacea. A number of WSSV envelope proteins, including vp28, have been proposed to be involved in viral infectivity based on the ability of specific antibodies to attenuate WSSV-induced mortality in vivo. In the present study, a series of monoclonal and polyclonal antibodies targeting vp28 were tested for their ability to neutralize WSSV infectivity, with the purpose of identifying epitopes potentially involved in vp28-mediated infection of shrimp. Surprisingly, when used as protein A-purified immunoglobulin, none of the antibodies tested were capable of inhibiting WSSV infectivity. This included one polyclonal preparation that has been previously shown to inactivate WSSV, when used as whole rabbit serum. Moreover, strong inactivation of WSSV by some rabbit sera was observed, in a manner independent of anti-vp28 antibodies. These results underscore the problems associated with using heterogeneous reagents (e.g. whole rabbit antiserum) in viral neutralization experiments aimed at defining proteins involved in infection by WSSV. In light of this, the potential of anti-vp28 antibodies to specifically neutralize WSSV should be reconsidered.     

The C-terminal region of envelope protein VP38 from white spot syndrome virus is indispensable for interaction with VP24

White spot syndrome virus (WSSV) is a large, rod-shaped, enveloped double-stranded DNA virus. In this study, VP38, a viral envelope protein, was expressed as a glutathione S-transferase (GST) fusion protein, and a polyclonal antibody against VP38 was obtained. Far-Western blotting and GST pull-down showed that VP38 interacted directly with VP24, a major WSSV envelope protein. In addition, to delineate the interaction region of VP38 with VP24, GST-VP38n (aa 1–142) and GST-VP38c (aa 143–309) were expressed. The GST pull-down assay revealed that VP38 binds via its C-terminal region to VP24. The result implies that VP38 may participate in the formation of the WSSV envelope.     

Development of a polyclonal antibody specific to VP19 envelope protein of white spot syndrome virus (WSSV) using a recombinant protein preparation

The VP19 gene encoding a structural envelope protein of white spot syndrome virus was cloned into an expression vector and introduced into E. coli. The objective was to produce a recombinant VP19 structural protein. After induction, the recombinant VP19 protein (rVP19) was produced, purified by SDS-PAGE and used for immunization of Swiss mice for polyclonal antibody production. The mouse anti rVP19 antiserum had specific immunoreactivity to the viral antigen in WSSV infected Penaeus monodon as verified by immunohistochemistry and Western blot. The production of monoclonal antibodies against this rVP19 may be useful in order to combine with anti-VP28 monoclonal antibodies for enhancing the sensitivity of various WSSV serological assays.     

Identification of white spot syndrome virus (WSSV) envelope proteins involved in shrimp infection

White spot syndrome virus (WSSV) is a major shrimp pathogen causing large economic losses. In an attempt to identify the envelope proteins involved in virus infection, antisera against six WSSV envelope proteins were used in neutralization assays conducted in vivo. The results showed that the virus infection could be significantly delayed or neutralized by antibodies against three WSSV envelope proteins (VP68, VP281 and VP466). This neutralization was further confirmed by quantitative PCR. It could be concluded that the viral envelope proteins VP68, VP281 and VP466 played roles in WSSV infection to shrimp.     

Tetramerization of white spot syndrome virus envelope protein VP33 and its interaction with VP24

VP33, also termed VP281, VP37 or VP36B, is a minor envelope protein of white spot syndrome virus (WSSV). Because of its low abundance and lack of a transmembrane domain, we hypothesized that VP33 is likely to be attached to the viral envelope by interaction with other envelope proteins. In this study, we employed far-western blotting and pull-down assays to demonstrate that VP33 interacts with itself, as well as with VP24, which is one of the four major viral envelope proteins. Moreover, a gel-filtration analysis was performed to show that this self-interaction led to the formation of stable VP33 tetramers. These results implied that VP33 tetramers were anchored to the viral envelope through interaction with VP24, suggesting that VP33 may participate in the formation of the WSSV envelope.     

A vector that expresses VP28 of WSSV can protect red swamp crayfish from white spot disease

White spot disease caused by white spot syndrome virus (WSSV) leads to devastating losses in shrimp farming. The WSSV envelope protein VP28, can be used as subunit vaccines that can efficiently protect shrimp against WSSV disease. However, the function of the envelope protein VP19 was not confirmed, some researches found that VP19 could protect shrimp against WSSV, and other reports found it no any protection. To detect the functions of VP28 and VP19 and find a method to prevent this disease in red swamp crayfish Procambarus clarkii, we constructed the plasmid vectors pIevp28 and pIevp19, which contains the ie1 promoter and coding region of vp28 or vp19 of WSSV, respectively. The results of quantitative real-time PCR and western blot showed that the injected vectors could transcribe corresponding mRNAs and translate to the protein VP28 or VP19 in the crayfish. The vp28 or vp19 signal was detected on the third day post injection, and maintained its expression for 30 days. The mortality of the crayfish with pIevp28 showed obvious decline compared with the controls (pIe and PBS injection). However, pIevp19 seems did not affect the mortality of the crayfish compared with the controls. Furthermore, only VP28 was found tightly bound to the host haemocytes under immunocytochemistry. The results suggest that the VP28 protein might protect shrimp from the virus through competitive inhibition. We also found that oral administration of Escherichia coli with pIevp28 could protect crayfish from white spot disease, but the E. coli with pIevp19 was not. Therefore, we think that oral administration of bacteria with pIevp28 is a potentially easy therapeutic way against white spot disease in aquaculture.     

Multiple envelope proteins are involved in white spot syndrome virus (WSSV) infection in crayfish

Summary. White spot syndrome virus (WSSV) is a devastating viral pathogen of cultured shrimp worldwide. Previous studies have shown that the intact virion consists of at least 39 structural proteins and, among them, six were identified as envelope proteins involved in the virus infection. In this paper, the structural proteins VP36A, VP36B and VP31 (J Virol 2004; 78: 11360–11370), containing the RGD motif, were expressed in Escherichia coli and used to produce specific antibodies. Western blot confirmed that VP36A is a newly reported envelope protein. A neutralization assay with these three antibodies demonstrated that VP36A, VP36B and VP31 could significantly delay the initial infection of crayfish, but mortality still reached 100% at day 11 post-injection. However, a neutralization assay with the combination of antibodies against different envelope proteins showed that a combination of VP36B and VP31 antibodies could strongly inhibit WSSV infection in crayfish. These results revealed that multiple envelope proteins are involved in WSSV infection in crayfish and that VP36B and VP31 play a key role during this process.     

Interaction of white spot syndrome virus VP26 protein with actin

VP26 protein, the product of the WSV311 gene of white spot syndrome virus (WSSV), is one of major structural proteins of virus. In this study, when purified virions were treated with Triton X-100 detergent, VP26 protein was present in both the envelope and the nucleocapsid fraction. We have rationalized this finding by suggesting that VP26 protein might be located in the space between the envelope and the nucleocapsid. By using a fluorescent probe method, we have investigated the interaction between VP26 protein and some proteins of host cells. Three major VP26-binding proteins were purified from crayfish hemocytes by affinity-chromatography, in which the protein with an apparent molecular mass of 42 kDa was identified as actin by mass spectrometry (MS). Moreover, the association of VP26 protein with actin microfilaments was confirmed by coimmunoprecipitation.     

Reassortment and interspecies transmission of North American H6N2 influenza viruses

To identify the protein encoded by a 687-bp open reading frame (ORF) of a salI genomic DNA fragment of shrimp white spot syndrome virus (WSSV), we expressed the ORF in a baculovirus/insect cell expression system. The apparent molecular mass of the recombinant protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 35 kDa in insect cells. Antibody raised against bacterially synthesized protein of the ORF identified a nucleocapsid protein (VP35) in the extracts of both the purified WSSV virions and the nucleocapsids which comigrated with the 35-kDa baculovirus-expressed recombinant protein on SDS-PAGE. We also show by transient expression in insect cells (Sf9) that VP35 targets the nucleus. Two potential nuclear localization signals (NLSs) were characterized, but only one of them was important for targeting VP35 to the nuclei of transfected insect cells. Replacement of a cluster of four positively charged residues ((24)KRKR(27)) at the N terminus of the protein with AAAA resulted in mutant proteins that were distributed only in the cytoplasm, thus confirming that this sequence is a critical part of the functionally active NLS of VP35.     

Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry

White spot syndrome virus (WSSV) is a large enveloped virus, but the organization of its envelope proteins remains largely unknown. In the present study, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and SDS-PAGE in combination with mass spectrometry to analyze the envelope protein complexome of WSSV. Our results show that the viral envelope consists of multi-protein complexes (MPCs). Within them, the envelope protein VP19 exists as a homotrimer, while another major envelope protein, VP28, mainly exists as a homotetramer. The most notable feature is that the majority of MPCs include VP26 and VP24, suggesting that these two proteins might serve as hub proteins to recruit low-abundance proteins to MPCs and play crucial roles in the process of protein complex formation. Furthermore, we found significant evidence for interactions between several low-abundance proteins, such as VP52B/VP38/VP33 and VP12/VP150. The result of this study may promote the further research on WSSV envelope assembly.     

Molecular cloning and prokaryotic expression of vp5 gene of grass carp reovirus strain GCRV096

VP5 is an outer capsid protein of grass carp reovirus (GCRV). It is predicted to involve in helping GCRV enter the host cells. In this study, the full-length vp5 gene (accession number in GenBank: JN206664.1) was cloned from GCRV strain GCRV096, which was isolated from diseased grass carp (Ctenopharyngodon idella) in southern China by RT-PCR technique using the primers designed from the known vp5 gene sequences of other strains of GCRV published in GenBank. The ORF sequence of vp5 is composed of 1,947 nucleotides encoding a 648-residues protein with a calculated molecular mass of 68.6 kDa and an estimated isoelectric point of 6.1. Sequence analysis results showed that VP5 might serve as a penetration protein and play an important role in GCRV penetration into the host cells. A full length of vp5 gene was subcloned into the prokaryotic expression vector pET-28a (+) and the recombinant plasmid (pET/GCRV-VP5) was then transduced into Escherichia coli BL21 (DE3) cells to express VP5 in vitro. SDS-PAGE and western blotting analysis indicated that the protein expressed successfully. Results also showed that the fusion protein expressed in the form of inclusion body, and it expressed in the highest level when induced with 0.2-mM IPTG at 28 °C for 4 h. These results are important for the future study on the molecular structure, function, and immunogenicity of GCRV capsid protein.     

Identification and stability of a 30-kDa nonstructural protein encoded by mRNA 2 of mouse hepatitis virus in infected cells

A bacterial expression vector encoding a fusion protein containing almost the entire first open reading frame (ORF1) of mRNA 2 of MHV-A59 has been constructed. The purified fusion protein was used to raise antibodies to the protein encoded by mRNA 2 ORF1. Specificity of the antibodies was verified by immunoprecipitation of the in vitro translation product of ORF1, which was reconstructed downstream of a T7 promoter. In vivo the antiserum reacted specifically with a 30-kDa protein synthesized in MHV-A59- and MHV-JHM-infected cells. This 30-kDa protein could not be identified in purified virions and is therefore a nonstructural viral protein. The expression pattern of this 30-kDa nonstructural viral protein in infected cells was shown to be identical to that of the viral structural proteins. However, in comparison to the nucleocapsid protein pulse-chase studies revealed a relative short half life for this 30-kDa protein in vivo.     

Beta-integrin mediates WSSV infection

White Spot Syndrome Virus (WSSV) is a virulent and widespread dsDNA virus with a wide range of hosts. Although remarkable progress has been made on virus characterization, however, its mechanism of infection is poorly understood. In this study, by analyzing the phage display library of the WSSV genome, a WSSV envelope protein VP187 (wsv209) was found to interact with shrimp integrin. VP187 possesses the RGD motif. The interaction between integrin and VP187 was confirmed with coimmunoprecipitation. These results demonstrate for the first time an interaction between the WSSV envelope protein and a cell surface molecule. Soluble integrin, integrin-specific antibody and an RGD-containing peptide were found to block the WSSV infection in vivo and in vitro. Gene silencing using a sequence-specific dsRNA targeting beta-integrin effectively inhibited the virus infection. These findings suggest that beta-integrin may function as a cellular receptor for WSSV infection.     

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS–PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.