Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii

Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28ΔN), C-terminal (rVP28ΔC) and middle (rVP28ΔM). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1–27) and middle region (residues 35–95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV.     

一种抗对虾白斑综合症病毒(WSSV)线性抗原表位单链抗体的筛选

对虾白斑综合症病毒(WSSV)是全世界对虾养殖业最主要的病原体之一, 虽然对该病毒的研究已较为深入, 但目前仍无有效的防治方法。本研究应用噬菌体展示技术, 构建了抗变性WSSV的单链抗体噬菌体展示文库, 分别以WSSV病毒粒子和原核表达的囊膜蛋白VP28为靶分子对该文库进行淘选。经过数轮淘选后, 得到5个能特异识别WSSV的单链抗体, 且首次获得了能特异识别WSSV线性抗原表位的单链抗体P75E8。并通过免疫胶体金电镜分析, 对5种单链抗体对应在病毒粒子上的表位进行了定位。为获取识别多种WSSV抗原的抗体提供了新的方法路线, 也为获取特异性识别线性表位的单链抗体提供了一种新的淘选技巧。     

Cloning and characterization of three novel WSSV recognizing lectins from shrimp Marsupenaeus japonicus

C-type lectins (CTLs) acting as pattern recognition receptors play essential roles in shrimp innate immune responses. Using WSSV envelope proteins (VP26, VP28, and VP281) to screen a phage display library of Marsupenaeus japonicus, three lectins (termed as MjLecA, MjLecB, and MjLecC) were found to interact with WSSV. Sequence analysis revealed that these MjLecs shared low similarities with each other. Phylogenetic analysis indicated MjLecA and MjLecB are likely to belong to the same lectin sub-family, while MjLecC belongs to another sub-family. These MjLecs showed broad, unique carbohydrate binding spectra. Also, the three MjLecs could interact with several envelope proteins of WSSV and could recognize a wide range of microorganisms. Moreover, binding of MjLecA or MjLecB to WSSV reduced the viral infection rate in vitro. These results suggest that various kinds of CTLs with structural and functional diversities may constitute a recognizing network against invading pathogens such as bacteria and virus, and play essential roles in the defence system of shrimp.     

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.     

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.     

Proteomic analysis of the major envelope and nucleocapsid proteins of white spot syndrome virus

White spot syndrome virus (WSSV) virions were purified from the tissues of infected Procambarus clarkii (crayfish) isolates. Pure WSSV preparations were subjected to Triton X-100 treatment to separate into the envelope and nucleocapsid fractions, which were subsequently separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major envelope and nucleocapsid proteins were identified by either matrix-assisted laser desorption ionization-time of flight mass spectrometry or defined antibody. A total of 30 structural proteins of WSSV were identified in this study; 22 of these were detected in the envelope fraction, 7 in the nucleocapsid fraction, and 1 in both the envelope and the nucleocapsid fractions. With the aid of specific antibodies, the localizations of eight proteins were further studied. The analysis of posttranslational modifications revealed that none of the WSSV structural proteins was glycosylated and that VP28 and VP19 were threonine phosphorylated. In addition, far-Western and coimmunoprecipitation experiments showed that VP28 interacted with both VP26 and VP24. In summary, the data obtained in this study should provide an important reference for future molecular studies of WSSV morphogenesis.     

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.     

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.     

Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: An uptake and processing study in the midgut of Penaeus monodon

Several oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.     

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.     

Characterization and diagnostic use of a monoclonal antibody for VP28 envelope protein of white spot syndrome virus

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21. After induction, the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production. It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection. Competitive PCR showed that the viral level was approximately 10⁴ copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV 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.     

Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa)

The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.     

Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Co...     

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immun-ological diagnosis methods for WSSV infection.     

Production and characterization of monoclonal antibodies of shrimp White spot syndrome virus envelope protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.     

Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion

The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.