Clearance of white spot syndrome virus (WSSV) and immunological changes in experimentally WSSV-injected Macrobrachium rosenbergii

A time course experimental challenge of WSSV was carried out to examine the clearance of WSSV in Macrobrachium rosenbergii and the consequent immunological changes. The experimental animals were injected with WSSV and the samples of gills, pleopods, head soft tissue and hemolymph were collected at different intervals of 1, 3, 5, 10, 25, 50, 75 and 100days post infection (p.i.). WSSV infection and clearing were confirmed by single step PCR, nested PCR and bioassay. At 3days p.i., M. rosenbergii became lethargic and stopped feeding in contrast to the control prawns that behaved and fed normally. However, the WSSV-injected prawns suffered no mortality during the experimental period and recovered without any further gross signs of disease or any mortality over a period of 100days p.i. The single step PCR analysis showed positive at 1, 3 and 5days p.i. in gills, head soft tissue, pleopods and hemolymph, and all the organs showed negative at 10days p.i. onwards. The nested PCR results showed that all organs were positive for WSSV from 3days p.i. and extended up to 25days p.i. At 50days p.i, head soft tissue sample alone showed WSSV-positive while all other organs were negative by nested PCR. All the organs at 75 and 100days p.i. showed nested PCR negative for WSSV as observed in the control prawn. The hemolymph collected from experimentally infected M. rosenbergii at 1, 3 and 5days p.i. caused 100% mortality at 40h p.i., 55h p.i. and 72h p.i, respectively in Penaeus monodon whereas hemolymph collected at 10, 25, 50, 75 and 100days p.i. failed to cause mortality in shrimp. The moribund shrimp showed WSSV-positive and surviving shrimp showed negative by PCR. Immunological parameters such as proPO, O(2)(-) and clotting time in WSSV-injected M. rosenbergii were found to be significantly higher than those of the control groups, whereas THC and superoxide dismutase were significantly lower when compared to control groups.     

Appendage deformity syndrome--a nutritional disease of Macrobrachium rosenbergii

Culture of the freshwater prawn Macrobrachium rosenbergii as an alternative to penaeid shrimp has recently increased in coastal areas of southern India in order to avoid numerous problems, particularly with white spot syndrome virus (WSSV). However, M. rosenbergii culture is now threatened by a new disease, appendage deformity syndrome (ADS), that also results in high mortality. Analysis of ADS prawns for viruses such as WSSV, monodon baculovirus (MBV) and infectious hypodermal and hematopoeitic necrosis virus (IHHNV) gave negative results. ADS prawns were also negative for bacterial pathogens and affected animals did not respond to antibiotic therapy. A study of potential nutritional deficiency revealed that carotenoid supplementation in the diet led to a significant decrease in ADS prawns.     

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.     

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.     

Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28

It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture.     

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.     

Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus

This study focused on apoptosis in various tissues of the black tiger shrimp Penaeus monodon following white spot syndrome virus (WSSV) injection. The study included: (1) light microscopy (LM) and transmission electron microscopy (TEM) of various tissues; (2) fluorescent LM of nuclear DNA by staining with 4, 6-diamidine-2-phenyl indole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labelling (TUNEL) techniques; and (3) determination of caspase-3 activity. Juvenile P. monodon were injected with WSSV, and several tissues of ectodermal and mesodermal origin were studied at different intervals after injection. The total haemocyte count had decreased to one-tenth of its original level 60 h after WSSV injection. By LM, extensive destruction by WSSV was observed in the stomach epithelium, gills, hematopoietic tissue, hemocytes and the heart, but the most severely affected tissue was the subcuticular epithelium. TEM revealed that at 6 h post-injection (p.i.) the chromatin of infected nuclei was marginated, and by 24 h p.i. the nuclei were filled with enveloped and non-enveloped WSSV virions. At later stages of the infection, the nucleus extruded WSSV particles. Chromatin margination and nuclear condensation and fragmentation (i.e. signs of apoptosis) were observed as early as 6 h p.i. in all affected tissues, but occurred in cells without WSSV virions rather than in cells with virions. The occurrence of apoptosis was supported by data obtained using TUNEL and by DAPI-staining and progressed from 6 to 60 h p.i. In addition, caspase-3 activity in WSSV-infected shrimp was about 6-fold higher than that in uninfected shrimp. The data strongly suggests that apoptosis occurs following WSSV infection in P. monodon, but the extent to which it contributes to shrimp mortality requires further investigation.     

Prevalence of white spot syndrome virus (WSSV) in wild shrimp Penaeus monodon in the Philippines

Prevalence of white spot syndrome virus (WSSV) was determined using polymerase chain reaction (PCR) methodology on DNA extracted from the gills of wild black tiger shrimp Penaeus monodon collected from 7 sampling sites in the Philippines. These 7 sampling sites are the primary sources of spawners and broodstock for hatchery use. During the dry season, WSSV was detected in shrimp from all sites except Bohol, but during the wet season it was not detected in any site except Palawan. None of the WSSV-PCR positive shrimp showed signs of white spots in the cuticle. Prevalence of WSSV showed seasonal variations, i.e. prevalence in dry season (April to May) was higher than in the wet season (August to October). These results suggest that WSSV has already become established in the local marine environment and in wild populations of P. monodon. Thus, broodstock collected during the dry season could serve as the main source of WSSV contamination in shrimp farms due to vertical transmission of the virus in hatcheries.     

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.     

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.     

对虾白斑综合征杆状病毒体内增殖模型的建立

应用对虾白斑综合征杆状病毒（WSSV）,对淡水克氏螯虾、罗氏沼虾、日本沼虾和两种淡水蟹（中华绒螯蟹、长江华溪蟹）进行人工感染实验。结果除淡水克氏螯虾之外,其它受试的虾蟹均不能感染WSSV。克氏螯虾3个不同剂量级感染至12d平均死亡率为94％。从发病或死亡个体采集血淋巴,经电镜负染色可观察到完整的病毒粒子,其形态大小、靶细胞组织病理均与从中国对虾中分离的WSSV相似或相同。同时,通过原位杂交技术进一步证明该实验的可靠性。克氏螯虾重复感染效果良好,有可能成为研究WSSV的一种理想的病毒体内增殖模型。     

Haemocyte reactions in WSSV immersion infected Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8(+) haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut.     

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.     

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.     

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.     

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.     

Silencing VP28 Gene of White Spot Syndrome Virus of Shrimp by Bacterially Expressed dsRNA

An in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria to provide a practical control of white spot syndrome virus (WSSV) in shrimp was developed. The bacterially synthesized dsRNA specific to VP28 gene of WSSV promoted gene-specific interference with the WSSV infection in shrimp. Virus infectivity was significantly reduced in WSSV-challenged shrimp injected with VP28-dsRNA and 100% survival was recorded. The inhibition of the expression of WSSV VP28 gene in experimentally challenged animals by VP28-dsRNA was confirmed by RT-PCR and Western blot analyses. Furthermore, we have demonstrated the efficacy of bacterially expressed VP28-dsRNA to silence VP28 gene expression in SISK cell line transfected with eukaryotic expression vector (pcDNA3.1) inserted with VP28 gene of WSSV. The expression level of VP28 gene in SISK cells was determined by fluorescent microscopy and ELISA. The results showed that the expression was significantly reduced in cells transfected with VP28dsRNA, whereas the cells transected with pcDNA-VP28 alone showed higher expression. The in vivo production of dsRNA using prokaryotic expression system could be an alternative to in vitro method for large-scale production of dsRNA corresponding to VP28 gene of WSSV for practical application to control the WSSV in shrimp farming.     

Performance of WSSV-infected and WSSV-negative Penaeus monodon postlarvae in culture ponds.

In a survey of 27 Penaeus monodon culture ponds stocked with postlarvae (approximately PL10) at medium density (approximately 40 shrimp m(-2)), single-step nested white spot syndrome virus (WSSV) PCR was used to measure the WSSV infection rates in the shrimp populations within 1 mo after stocking. Seven ponds were initially WSSV-free, and the shrimp in 5 of these were harvested successfully. In the ponds (n = 6) where detection rates were higher than 50%, mass mortality occurred during the growth period, and none of these ponds was harvested successfully. In a subsequent study, P. monodon brooders were classified into 3 groups according to their WSSV infection status before and after spawning: brooders that were WSSV-positive before spawning were assigned to group A; spawners that became WSSV-positive only after spawning were assigned to group B; and group C consisted of brooders that were still WSSV-negative after spawning. WSSV screening showed that 75, 44 and 14%, respectively, of group A, B and C brooders produced nauplii that were WSSV-positive. Most (57%; 16/28) of the brooders in group A produced nauplii in which the WSSV prevalence was high (>50%).When a pond was stocked with high-prevalence nauplii from 1 of these group A brooders, an outbreak of white spot syndrome occurred within 3 wk and only approximately 20% of the initial population survived through to harvest (after 174 d). By contrast, 2 other ponds stocked with low-prevalence and WSSV-negative nauplii (derived respectively from 2 brooders in group B), both had much higher survival rates (70 to 80%) and yielded much larger (approximately 3x by weight) total harvests. We conclude that testing the nauplii is an effective and practical screening strategy for commercially cultured P. monodon.     

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.