Proteomic analyses of the shrimp white spot syndrome virus

White spot syndrome virus (WSSV), a unique member within the virus family Nimaviridae, is the most notorious aquatic virus infecting shrimp and other crustaceans and has caused enormous economic losses in the shrimp farming industry worldwide. Therefore, a comprehensive understanding of WSSV morphogenesis, structural proteins, and replication is essential for developing prevention measures of this serious parasite. The viral genome is approximately 300kb and contains more than 180 open reading frames (ORF). However, most of proteins encoded by these ORF have not been characterized. Due to the importance of WSSV structural proteins in the composition of the virion structure, infection process and interaction with host cells, knowledge of structural proteins is essential to understanding WSSV entry and infection as well as for exploring effective prevention measures. This review article summarizes mainly current investigations on WSSV structural proteins including the relative quantities, localization, function and protein-protein interactions. Traditional proteomic studies of 1D or 2D gel electrophoresis separations and mass spectrometry (MS) followed by database searches have identified a total of 39 structural proteins. Shotgun proteomics and iTRAQ were initiated to identify more structural proteins. To date, it is estimated that WSSV is assembled by at least 59 structural proteins, among them 35 are defined as the envelope fraction (including tegument proteins) and 9 as nucleocapsid proteins. Furthermore, the interaction within several major structural proteins has also been investigated. This identitification and characterization of WSSV protein components should help in the understanding of the viral assembly process and elucidate the roles of several major structural proteins.     

VP37 of white spot syndrome virus interact with shrimp cells

Aims:  To investigate VP37 [WSV 254 of White spot syndrome virus (WSSV) genome] interacting with shrimp cells and protecting shrimp against WSSV infection.
Methods and Results:  VP37 was expressed in Escherichia coli and was confirmed by Western blotting. Virus overlay protein binding assay (VOPBA) technique was used to analyse the rVP37 interaction with shrimp and the results showed that rVP37 interacted with shrimp cell membrane. Binding assay of recombinant VP37 with shrimp cell membrane by ELISA confirmed that purified rVP37 had a high-binding activity with shrimp cell membrane. Binding of rVP37 to shrimp cell membrane was a dose-dependent. Competition ELISA result showed that the envelope protein VP37 could compete with WSSV to bind to shrimp cells. In vivo inhibition experiment showed that rVP37 provided 40% protection. Inhibition of virus infection by rVP37 in primary cell culture revealed that rVP37 counterparted virus infection within the experiment period.
Conclusions:  VP37 has been successfully expressed in E . coli . VP37 interacted with shrimp cells.
Significance and Impact of the Study:  The results suggest that rVP37 has a potential application in prevention of virus infection.     

Genotyping of white spot syndrome virus prevalent in shrimp farms of India

DNA extracts from white spot syndrome virus (WSSV) that had infected post-larvae and juveniles of cultured shrimp, wild shrimp and crabs, which had been collected from different hatcheries and farms located along both the east and west coasts of India, revealed considerable variation in several previously identified WSSV DNA repeat regions. These include the 54 bp repeat in ORF 94, the 69 bp repeat in ORF 125 and the compound 45 and 57 bp repeat region in ORF 75. In ORF 94, 13 genotypes were observed with the number of repeats ranging from 2 to 16 units. While 7 repeat units were commonly observed (11.3%), no samples with 11 or 15 repeat units were found. In ORF 125, 11 types were found, with repeats ranging from 2 to 14 units. The most prevalent genotype displayed 4 repeat units (47.1%); no samples with 6 or 13 repeats were observed. The compound repeat region of ORF 75 displayed 6 different patterns of repeats. Samples with the same repeat pattern in one ORF did not always show identical repeat patterns in one or both of the other repeat regions. These data suggest that combined analysis of all 3 variable loci could be used to differentiate and characterize specific WSSV strains. For general epidemiological studies, the best marker with maximum variation is ORF 94, followed by ORF 125 and ORF 75. The 3 repeat regions above were used to compare WSSV genotypes from disease outbreaks on 3 sets of farms from different locations in the state of Andhra Pradesh. The genotypes within each farm set were almost identical, but differed between farm sets, suggesting that WSSV transmission occurred directly through virus carriers or water exchange between adjacent farms at each location. These findings show that genotyping can be a useful epidemiological tool for tracing the movement of WSSV within infected populations.     

A model for apoptotic interaction between white spot syndrome virus and shrimp

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.     

白斑综合症病毒与对虾血淋巴细胞的体外结合实验

通过差速离心和蔗糖密度梯度离心,从感染了白斑综合症病毒(WSSV)的病虾头胸部分离了WSSV,利用地高辛对病毒蛋白进行了标记(DIG-WSSV),以体外培养的对虾血淋巴细胞为吸附基底,观察和分析了病毒与细胞间的结合现象及特性。以NBT/BCIP为酶反应显色底物观察到在细胞周围形成许多暗紫色颗粒,证实病毒与细胞间存在着稳定的结合。以OPD为酶反应显色底物分析了结合反应的特性:当DIG-WSSV维持恒定值时,随着血淋巴细胞数量的增加结合显色增强,细胞数量达到1.2104cells/孔,492nm处的吸光值达到饱和;当血淋巴细胞数量维持恒定值时,随着DIG-WSSV蛋白含量的增加显色增强,且在DIG-WSSV的蛋白浓度达到4g/孔时,492nm处的吸光值达到饱和;未标记WSSV可竞争抑制血淋巴细胞与DIG-WSSV间的结合作用。进一步的研究得出:4℃下,随着结合时间的延长显色增强,但继续延长结合时间显色反而减弱;缓冲液的渗透压对结合结果影响甚微,而酸性条件利于病毒与细胞间的结合。37℃孵育对病毒结合活性影响不大,55℃和70℃孵育可显著影响病毒的结合活性;短时间超声波处理病毒可增加病毒结合能力,长时间超声波处理可破坏病毒结合能力;有机溶剂处理同样可破坏病毒结合能力,其中尤以氯仿/甲醇的处理更为激烈;不同的去垢剂对病毒结合活性的影响结果不同:SDS和脱氧胆酸钠可以降低病毒的结合活性,而Triton X-100和NP-40可以提高病毒的结合活性。       

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.     

Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus

White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 + 6) WSSV structural protein genes. Only 61.5% of the WSSV structural genes have a polyadenylation signal, and preliminary analysis by 3' rapid amplification of cDNA ends suggested that some structural protein genes produced mRNA without a poly(A) tail. Microarray analysis showed that gene expression started at 2, 6, 8, 12, 18, 24, and 36 hpi for 7, 1, 4, 12, 9, 5, and 1 of the genes, respectively. Based on similarities in their time course expression patterns, a clustering algorithm was used to group the WSSV structural genes into four clusters. Genes that putatively had common or similar roles in the viral infection cycle tended to appear in the same cluster.     

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.     

Absence of surface-associated microorganisms in adult oysters (Crassostrea gigas).

Healthy, actively feeding intertidal oysters were removed from an estuarine environment (Pipeclay Lagoon, Tasmania). The epithelial surfaces of various organs of the mantle cavity and alimentary tract were explored by scanning and transmission electron microscopy. All epithelial tissues examined were ciliated, and nearly all were partly covered with secreted mucus. However, microorganisms were seen rarely in the adhesive mucus and never attached to the epithelium. Electron microscopy also failed to demonstrate a surface microflora in emersed oysters which had been incubated at 5 to 25 degrees C for 6 or 24 h. The absence of an internal surface microflora did not vary on a seasonal basis. In laboratory experiments, oysters were allowed to filter feed from seawater containing diverse types of marine bacteria at concentrations of 10(3) to 10(7)/mL. However, no surface microflora could be found within actively feeding oysters or in emersed animals incubated at 20 degrees C for 6 or 24 h. In contrast, surface-associated microorganisms were detected readily by scanning electron microscopy on the external shell of healthy oysters and on various internal tissues in spoiled oysters. It is suggested that the major mechanisms restricting microbial growth within oysters are ciliary movement and mucus secretion.     

Sampling and evaluation of white spot syndrome virus in commercially important Atlantic penaeid shrimp stocks

In 1997, white spot syndrome virus (WSSV) was discovered in shrimp culture facilities in South Carolina, USA. This disease was known to cause devastating mortalities in cultured populations in Southeast Asia and prompted concern for the health of wild populations in the USA. Our study surveyed wild shrimp populations for the presence of WSSV by utilizing molecular diagnostics and bioassay techniques. A total of 1150 individuals (586 Litopenaeus setiferus, 477 Farfantepenaeus aztecus and 87 F. dourarum) were examined for the presence of WSSV DNA by PCR. A total of 32 individuals tested positive and were used in a bioassay to examine the transmission of disease to healthy individuals of the culture species L. vannamei. DNA sequencing of PCR products from a positive individual confirmed that the positive individuals carried WSSV DNA. Significant mortalities were seen in test shrimp injected with tissue extracts from heavily infected wild shrimp. These data confirm the existence of WSSV in wild shrimp stocks along the Atlantic Coast and that the virus can cause mortalities in cultured stocks.     

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.     

Complete genome sequence of the shrimp white spot bacilliform virus.

We report the first complete genome sequence of a marine invertebrate virus. White spot bacilliform virus (WSBV; or white spot syndrome virus) is a major shrimp pathogen with a high mortality rate and a wide host range. Its double-stranded circular DNA genome of 305,107 bp contains 181 open reading frames (ORFs). Nine homologous regions containing 47 repeated minifragments that include direct repeats, atypical inverted repeat sequences, and imperfect palindromes were identified. This is the largest animal virus that has been completely sequenced. Although WSBV is morphologically similar to insect baculovirus, the two viruses are not detectably related at the amino acid level. Rather, some WSBV genes are more homologous to eukaryotic genes than viral genes. In fact, sequence analysis indicates that WSBV differs from all known viruses, although a few genes display a weak homology to herpesvirus genes. Most of the ORFs encode proteins that bear no homology to any known proteins, either suggesting that WSBV represents a novel class of viruses or perhaps implying a significant evolutionary distance between marine and terrestrial viruses. The most unique feature of WSBV is the presence of an intact collagen gene, a gene encoding an extracellular matrix protein of animal cells that has never been found in any viruses. Determination of the genome of WSBV will facilitate a better understanding of the molecular mechanism underlying the pathogenesis of the WSBV virus and will also provide useful information concerning the evolution and divergence of marine and terrestrial animal viruses at the molecular level.     

The role of Pm-fortilin in protecting shrimp from white spot syndrome virus (WSSV) infection

Crustacean fortilin or the product of the translationally controlled tumor protein (TCTP) gene isolated from Penaeus monodon, is well conserved and has a Ca(++) binding domain. Pm-fortilin has anti-apoptotic properties and is present at high levels during the onset of viral infections in P. monodon. The possibility of using rFortilin to protect against white spot syndrome virus (WSSV) infection was tested. Injection of shrimp with rFortilin, after infection with WSSV, resulted in 80-100% survival and detection of very low levels of WSSV by PCR, whereas in moribund samples WSSV levels were very high. This result implies that injection of recombinant rFortilin decreases viral infection by an unknown mechanism, but probably by inhibiting viral replication. Using a yeast two-hybrid screen for cellular protein partners to rFortilin we identified an unknown protein that bound to fortilin. This is a novel polypeptide of 93 amino acids with a number of XPPX signature sequences that are often reported to have a function in antiviral peptides.     

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.     

安徽地区克氏原螯虾白斑综合征的诊断及朔源

[目的]诊断安徽地区养殖的克氏原螯虾发生疫情的病原,追踪感染源,为疫病的防控提供依据.[方法]采集病料,参照OIE推荐的套式PCR法检测白斑综合征病毒(WSSV).将病螯虾鳃丝组织研磨后离心,取上清液接种健康克氏原螯虾.透射电镜观察病原特征.综合流行病源学及病原检测,分析养殖地区的可能感染源.[结果]发病螯虾感染了WSSV,动物实验可见和发病螯虾相同的临床症状,证实该病原引起养殖螯虾发病.电镜观察可见典型的白斑综合症病毒颗粒.流行病学调查显示,水产颗粒膨化饲料和冷冻饲料甲壳类检测阳性.[结论]该地区养殖的克氏原螯虾近年爆发的疫病病原为WSSV,推断地区性克氏原螯虾WSSV的感染并流行与污染的饲料有关.     

Immune response of white shrimp, Litopenaeus vannamei, after a concurrent infection with white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus

In the present study, we investigated immunological changes in viral-infected white shrimp, Litopenaeus vannamei. White shrimp were infected with white spot syndrome virus (WSSV) or co-infected with WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV) as detected by polymerase chain reaction (PCR). The complete (100%) mortality rate of shrimp was caused by viral infection due to immune parameters being suppressed including decreases in phenoloxidase activity, total hemocyte counts, differential hemocyte counts, and the gene expressions of prophenoloxidase and peroxinectin. In addition, increases in lipopolysaccharide and beta-1,3-glucan-binding protein of hemocytes and the hepatopancreas, and respiratory bursts per cell, and a decrease in superoxide dismutase were found in viral-infected shrimp, which may have been related to the defense against viral infection.