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.     

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.     

Vaccination of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV)

Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.     

Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV)

White spot disease is an important viral disease caused by white spot syndrome virus (WSSV) and is responsible for huge economic losses in the shrimp culture industry worldwide. The VP28 gene encoding the most dominant envelope protein of WSSV was used to construct a DNA vaccine. The VP28 gene was cloned in the eukaryotic expression vector pcDNA3.1 and the construct was named as pVP28. The protective efficiency of pVP28 against WSSV was evaluated in Penaeus monodon by intramuscular challenge. In vitro expression of VP28 gene was confirmed in sea bass kidney cell line (SISK) by fluorescence microscopy before administering to shrimp. The distribution of injected pVP28 in different tissues of shrimp was studied and the results revealed the presence of pVP28 in gill, head soft tissue, abdominal muscle, hemolymph, pleopods, hepatopancreas and gut. RT-PCR and fluorescence microscopy analyses showed the expression of pVP28 in all these tissues examined. The results of vaccination trials showed a significantly higher survival rate in shrimp vaccinated with pVP28 (56.6-90%) when compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups revealed that the vaccinated shrimp showed significantly high level of prophenoloxidase and superoxide dismutase (SOD) when compared to the control groups. The high levels of prophenoloxidase and superoxide dismutase (SOD) might be responsible for developing resistance against WSSV in DNA vaccinated shrimp.     

Oral vaccination of baculovirus-expressed VP28 displays enhanced protection against White Spot Syndrome Virus in Penaeus monodon

White Spot Syndrome Virus (WSSV) is an infectious pathogen of shrimp and other crustaceans, and neither effective vaccines nor adequate treatments are currently available. WSSV is an enveloped dsDNA virus, and one of its major envelope proteins, VP28, plays a pivotal role in WSSV infection. In an attempt to develop a vaccine against WSSV, we inserted the VP28 gene into a baculovirus vector tailored to express VP28 on the baculovirus surface under the WSSV ie1 promoter (Bac-VP28). The Bac-VP28 incorporated abundant quantity (65.3 μg/ml) of VP28. Shrimp were treated by oral and immersion vaccination with either Bac-VP28 or wild-type baculovirus (Bac-wt). The treatment was followed by challenge with WSSV after 3 and 15 days. Bac-VP28 vaccinated shrimp showed significantly higher survival rates (oral: 81.7% and 76.7%; immersion: 75% and 68.4%) than Bac-wt or non-treated shrimp (100% mortality). To verify the protective effects of Bac-VP28, we examined in vivo expression of VP28 by immunohistochemistry and quantified the WSSV copy number by qPCR. In addition to that, we quantified the expression levels shrimp genes LGBP and STAT by real-time RT-PCR from the samples obtained from Bac-VP28 vaccinated shrimp at different duration of vaccine regime. Our findings indicate that oral vaccination of shrimp with Bac-VP28 is an attractive preventative measure against WSSV infection that can be used in the field.     

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.     

Cloning and sequencing of envelope proteins (VP19, VP28) and nucleocapsid proteins (VP15, VP35) of a white spot syndrome virus isolate from Korean shrimp

Since our first report in 1998, white spot syndrome virus (WSSV) has become wide-spread on the southern and western coasts of Korea. Almost all shrimp in ponds die within 3 to 4 d after the first dead shrimp are observed with gross lesions ranging from abnormal red body discoloration to white spots in the cuticle. From one isolate, we cloned and sequenced WSSV genomic DNA coding for VP19 and VP28 envelope proteins and VP15 and VP35 nucleocapsid proteins. Putative protein sequences were submitted to GenBank and assigned accession numbers AY316119 (VP19), AY324881 (VP28), AY374120 (VP15) and AY325896 (VP35). At the nucleotide level, VP19, VP28 and VP15 sequences were, respectively, 99, 100 and 100% identical to those of China, Indonesia, Japan and the United States and the VP35 sequence was 100% identical to that of a Taiwanese isolate. The deduced amino-acid sequences were 99 to 100% identical to those from other countries. In VP19, C and T in the foreign isolates were replaced by T and A in the Korean isolate at Positions 57 and 218 nt, respectively, downstream of A (+) of the VP19 start codon. The change at Position 218 nt resulted in valine in the foreign isolates being replaced by aspartate in the Korean isolate.     

Shrimp vaccination trials with the VP292 protein of white spot syndrome virus

AIMS: Construction of a recombinant vector that expresses VP292 protein of white spot syndrome virus (WSSV) and to exploit the possibility of obtaining the vaccine conferring protection against WSSV infection in shrimps. METHODS AND RESULTS: VP292 protein of WSSV was amplified from WSSV genomic DNA by PCR. The target 814 bp amplified product specific for VP292 protein was inserted in to pQE30 expression vector. The recombinant plasmid of VP292 protein was transformed and expressed in Escherichia coli under induction of isopropyl-1-1-thio-beta-D-galactoside (IPTG) and the immunoreactivity of the fusion protein was detected by Western blot. Shrimp were vaccinated by intramuscular injection of the purified protein VP292 of WSSV and challenged for 0-30 days. Vaccination trial experiments show that two injections with recombinant VP292 (rVP292) protein induced a higher resistance, with 52% relative percentage survival value, in the shrimp at the 30th day postvaccination. CONCLUSIONS: The expression system of protein VP292 of WSSV with a high efficiency has been successfully constructed. Vaccination trials show significant resistance in the shrimp vaccinated twice with recombinant VP292. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study prosper the development of WSSV protein vaccine against WSSV infection in shrimps.     

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.     

Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis

White spot syndrome virus (WSSV) disease is a major threat to shrimp culture worldwide. Here, we assessed the efficacy of the oral administration of purified recombinant VP28, an envelope protein of WSSV, expressed in a Gram-positive bacterium, Brevibacillus brevis, in providing protection in shrimp, Penaeus japonicus, upon challenge with WSSV. Juvenile shrimp (2-3g in body weight) fed with pellets containing purified recombinant VP28 (50mug/shrimp) for 2weeks showed significantly higher survival rates than control groups when challenged with the virus at 3days after the last day of feeding. However, when shrimp were challenged 2weeks after the last day of feeding, survival rates decreased (33.4% and 24.93%, respectively). Survival rate was dose-dependent, increasing from 60.7 to 80.3% as the dose increased from 1 to 50mug/shrimp. At a dose of 50mug/shrimp, the recombinant protein provided protection as soon as 1day after feeding (72.5% survival). Similar results were obtained with larger-sized shrimp. These results show that recombinant VP28 expressed in a Gram-positive bacterium is a potential oral vaccine against WSSV.     

Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV‐vaccines and live white spot syndrome virus

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate ‘vaccines’, WSSV envelope protein VP28 and formalin‐inactivated WSSV, can provide short‐lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live‐WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV‐intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune‐related, intracellular organelle part, intracellular calcium‐binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV‐intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.     

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.     

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.     

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.     

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。     

Highly conserved sequences of three major virion proteins of a Korean isolate of white spot syndrome virus (WSSV)

In Korea, mass mortality occurred among cultured shrimp with visible macroscopic white spots in 2000, and we confirmed the presence of white spot syndrome virus (WSSV) in the tissues of moribund shrimp by electron microscopy. In order to identify the characteristics of this Korean isolate of WSSV, we cloned and characterized its genomic DNA coding for VP24, VP26, and VP28. On the nucleotide level, VP24, VP26, and VP28 of the Korean isolate were found to be 100%, 100%, and 99% identical to those of Taiwan, Thailand and Chinese isolates, respectively. On the deduced amino-acid level, all 3 virion proteins showed 100% identity to those of the foreign isolates. The extent of sequence identity suggests that the Korean isolate originated from the same ancestor as the Taiwanese, Thai and Chinese isolates.     

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.