Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV)

This study investigates white spot syndrome virus (WSSV) gene expression levels in the cells of 2 hosts (Penaeus monodon and Litopenaeus vannamei). Microarray and expressed sequence tag (EST) analysis of the mRNA profiles in WSSV-infected P. monodon cells were used to identify WSSV genes that were very highly expressed. Results showed that the mRNA of the WSSV icp11 gene consistently had the highest copy number of all (3x higher than the major envelope protein, VP28). At the protein level in WSSV-infected L. vannamei, 2-dimensional gel analysis and liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) protein identification also showed that this WSSV non-structural protein has the highest expression levels reported to date. ICP11 is capable of self-multimerization, and it becomes located in both the cytoplasm and nucleus of the host cell. These data suggest that ICP11 plays an important, but presently unknown, role during viral infection, and that expression of the WSSV icp11 gene/WSSV ICP11 protein is potentially a good and diagnostically useful indicator of WSSV infection.     

Bacterial Lipid Modification of ICP11 and a New ELISA System Applicable for WSSV Infection Detection

In ELISA, a popular analytical diagnostic tool, the stable non-covalent immobilization (coating) of hydrophilic proteins/peptides on to hydrophobic polystyrene surface has remained a major common challenge. Recombinant bacterial lipid modification of proteins in Escherichia coli system has been shown in this study to solve this problem owing to the hydrophobic anchorage provided by three fatty acyl groups in N-acyl-S-diacylglyceryl Cys at the N-terminus. Exploiting this first post-translational protein engineering, the most abundantly expressed white spot syndrome viral protein ICP11 was lipid-modified and tested as a new target in a new ELISA method useful to shrimp farming. The lipid served as a potent adjuvant to enhance the titer (16 times) of higher affinity antibodies where amino terminal lipoamino acid N-acyl-S-diacylglyceryl cysteine of bacterial lipoproteins induce inflammatory responses through TLR and stimulate humoral immune responses without additional adjuvant and also aided in the immobilization of even a few nanograms of ICP11. Competition between the immobilized and the free antigen from the sample provided a sensitive measure of antigen in the infected shrimp tissues. The detection limit for ICP11 protein using competitive ELISA was 250 pg and the linear range of the assay was 15–240 ng.     

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.     

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 g l⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT₅₀/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC₅₀. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.     

Development of TaqMan Probe-Based Insulated Isothermal PCR (iiPCR) for Sensitive and Specific On-Site Pathogen Detection

Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensity_after/fluorescent intensity_before) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 10¹ copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.     

The VP37-binding protein F1ATP synthase β subunit involved in WSSV infection in shrimp Litopenaeus vannamei

To investigate the interaction between white spot syndrome virus (WSSV)-VP37 and gill membrane proteins (GMPs) of Pacific white shrimp (Litopenaeus vannamei), the VP37 protein was expressed and purified, and a distinct 53 kDa VP37-binding protein band was identified in GMPs by virus overlay protein binding assay and GST pull-down assay. By electroelution, the VP37 binding protein was purified and identified as F₁ATP synthase β (F₁ATPase β) subunit by Mass Spectrometry. The purified F₁ATPase β subunit was used to immunize BALB/C mice to produce monoclonal antibodies (Mabs). After cell fusion, sixteen hybridomas secreting Mabs against F₁ATPase β subunit of L. vannamei were screened by enzyme-linked immunosorbant assay (ELISA), three of which designated as 1D5, 1E8 and 2H4 were cloned by limiting dilution and further characterized by indirect immunofluorescence assay (IIFA) and western blotting. The results of IIFA showed that specific fluorescence signals located at the peripheral zone of the gills of L. vannamei. Western blotting demonstrated that three Mabs reacted specifically with the 53 kDa protein band in GMPs of L. vannamei. By IIFA, the Mabs could also cross-react with the gill cells of three other WSSV-susceptible shrimps Fenneropenaeus chinensis, Penaeus monodon and Marsupenaeus japonicus. Furthermore, the three anti-F₁ATPase β subunit Mabs could partially block the binding of WSSV to GMPs by ELISA in vitro, and also exhibited direct anti-WSSV activity in shrimp by neutralization assay in vivo. These findings suggested that F₁ATPase β subunit involved in WSSV infection in L. vannamei.     

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.     

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.     

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.     

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.     

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.     

Effects of dominant microalgae species and bacterial quantity on shrimp production in the final culture season

The dominant microalgal species, quantity of heterotrophic bacteria and Vibrio in the intestines and gills of Litopenaeus vannamei (Pacific white shrimp), positive detection rate of white spot syndrome virus (WSSV), and water quality indices were investigated at the final culture stage (88th day in culture season). Correlation of microalgal community, bacteria quantity, and shrimp production were analyzed by statistical analysis methods. Every 10 days, probiotics were used in groups A, B, and C, consisting of Bacillus, photosynthetic bacteria (PSB), and equal parts Bacillus and PSB, respectively. The results showed that production (25597.33?±?928.46 kg ha^?1) and survival rate (77.06?±?9.00 %) was the highest in group C, but positive detection rate of WSSV was the lowest. The microalgal community of group C was significantly dominated by Chlorella pyrenoidosa, with an average density and dominance of (289.52?±?142.10)?×?10⁷ cells L^?1 and 0.878?±?0.161, respectively. The correlation analysis indicated a significant negative correlation between Cyanophyta dominance and shrimp production (P?<?0.05), while the relationship between production and Vibrio quantity was not significantly correlated (P?>?0.05). Accordingly, microalgal dominant species should be controlled as a key factor in the shrimp culture season; in particular, the dominance of Cyanophyta should be restricted to a low level. Meanwhile, the combined use of Bacillus and PSB probiotics was considered an effective solution to optimize microalgal communities and controlling the cell density of Cyanophyta.     

Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp.

Monoclonal antibodies (MAbs) were produced against white spot syndrome virus (WSSV) of penaeid shrimp. The virus isolate used for immunization was obtained from China in 1994 and was passaged in Penaeus vannamei. The 4 hybridomas selected for characterization all produced MAbs that reacted with the 28 kD structural protein by Western blot analysis. The MAbs tested in dot-immunoblot assays were capable of detecting the virus in hemolymph samples collected from moribund shrimp during an experimentally induced WSSV infection. Two of the MAbs were chosen for development of serological detection methods for WSSV. The 2 MAbs detected WSSV infections in fresh tissue impression smears using a fluorescent antibody for final detection. A rapid immunohistochemical method using the MAbs on Davidson's fixed tissue sections identified WSSV-infected cells and tissues in a pattern similar to that seen with digoxigenin-labeled WSSV-specific gene probes. A whole mount assay of pieces of fixed tissue without paraffin embedding and sectioning was also successfully used for detecting the virus. None of the MAbs reacted with hemolymph from specific pathogen-free shrimp or from shrimp infected with infectious hypodermal and hematopoietic necrosis virus, yellow head virus or Taura syndrome virus. In Western blot analysis, the 2 MAbs did not detect any serological differences among WSSV isolates from China, Thailand, India, Texas, South Carolina or Panama. Additionally, the MAbs did not detect a serological difference between WSSV isolated from penaeid shrimp and WSSV isolated from freshwater crayfish.     

Enzyme E2 from Chinese white shrimp inhibits replication of white spot syndrome virus and ubiquitinates its RING domain proteins

Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.     

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.