对虾白斑杆状病毒DNA聚合酶调控序列的克隆

为了揭示对虾白斑杆状病毒的致病机理,将该病毒基因组中推测的DNA聚合酶上游调控序列克隆进荧光素酶报告基因载体中,以便寻找一个能表达该病毒基因的细胞系统.     

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.     

Complete genome sequence of the European sheatfish virus

Viral diseases are an increasing threat to the thriving aquaculture industry worldwide. An emerging group of fish pathogens is formed by several ranaviruses, which have been isolated at different locations from freshwater and seawater fish species since 1985. We report the complete genome sequence of European sheatfish ranavirus (ESV), the first ranavirus isolated in Europe, which causes high mortality rates in infected sheatfish (Silurus glanis) and in other species. Analysis of the genome sequence shows that ESV belongs to the amphibian-like ranaviruses and is closely related to the epizootic hematopoietic necrosis virus (EHNV), a disease agent geographically confined to the Australian continent and notifiable to the World Organization for Animal Health.     

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.     

Indel-II region deletion sizes in the white spot syndrome virus genome correlate with shrimp disease outbreaks in southern Vietnam

Sequence comparisons of the genomes of white spot syndrome virus (WSSV) strains have identified regions containing variable-length insertions/deletions (i.e. indels). Indel-I and Indel-II, positioned between open reading frames (ORFs) 14/15 and 23/24, respectively, are the largest and the most variable. Here we examined the nature of these 2 indel regions in 313 WSSV-infected Penaeus monodon shrimp collected between 2006 and 2009 from 76 aquaculture ponds in the Mekong Delta region of Vietnam. In the Indel-I region, 2 WSSV genotypes with deletions of either 5950 or 6031 bp in length compared with that of a reference strain from Thailand (WSSV-TH-96-II) were detected. In the Indel-II region, 4 WSSV genotypes with deletions of 8539, 10970, 11049 or 11866 bp in length compared with that of a reference strain from Taiwan (WSSV-TW) were detected, and the 8539 and 10970 bp genotypes predominated. Indel-II variants with longer deletions were found to correlate statistically with WSSV-diseased shrimp originating from more intensive farming systems. Like Indel-I lengths, Indel-II lengths also varied based on the Mekong Delta province from which farmed shrimp were collected.     

Complete nucleotide sequence of the Eastern equine encephalomyelitis virus genome

The complete nucleotide sequence of the genomic 42S RNA of Eastern equine encephalitis virus has been defined for the first time. The strategy of this viral genome occurred analogous to the ones of the other alfa viruses. The comparison of amino acid sequences of E1 and E2 proteins from the two strains of the virus has revealed a number of differences. Partially, they are localized in the hydrophilic regions of the protein molecules and evidently participate in organization of the specific antigenic structures. The amino acid sequences of all viral proteins have been comparatively analysed with the sequences of the analogous proteins of other known alfa viruses.     

Complete nucleotide sequence of the mycoplasma virus P1 genome

Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma. The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins. The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties. The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified. The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP. Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known. One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins.     

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.     

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.     

Complete genome sequence of a street rabies virus from Mexico

A canine rabies virus (RABV) has been used as a street rabies virus in laboratory investigations. Its entire genome was sequenced and found to be closely related to that of canine RABV circulating in Mexico. Sequence comparison indicates that the virus is closely related to those in the "cosmopolitan" group, with high homology (89 to 93%) to clade I of rabies viruses. The virus is now termed dog rabies virus-Mexico (DRV-Mexico).     

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.     

Complete genome sequence of a double-stranded RNA virus from avocado

A number of avocado (Persea americana) cultivars are known to contain high-molecular-weight double-stranded RNA (dsRNA) molecules for which a viral nature has been suggested, although sequence data are not available. Here we report the cloning and complete sequencing of a 13.5-kbp dsRNA virus isolated from avocado and show that it corresponds to the genome of a new species of the genus Endornavirus (family Endornaviridae), tentatively named Persea americana endornavirus (PaEV).     

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.     

Complete genome sequence of a porcine epidemic diarrhea virus variant

In 2011, outbreaks of viral diarrhea were observed on most swine-breeding farms in most of the provinces of China. The disease is characterized by vomiting, severe diarrhea, and a high mortality rate (82.3%) in newborn piglets. The clinical appearance was similar to that of porcine epidemic diarrhea virus (PEDV) infection. PEDVs were detected in samples (feces or small intestines) from most farms. In order to investigate whether there is a PEDV variant circulating in China, we sequenced and analyzed the complete genome of the recently identified field strain, CH/FJND-3/2011. The sequence data indicate that this PEDV variant prevails in China.     

Crassostrea gigas oysters as a shrimp farm bioindicator of white spot syndrome virus

This study explored whether Crassostrea gigas oysters can be used as a bioindicator of white spot syndrome virus (WSSV) in shrimp farm water canals. Bioassays showed that C. gigas can accumulate WSSV in their gills and digestive glands but do not become infected, either by exposure to seawater containing WSSV or by cohabitation with infected shrimp. The use of a WSSV nested PCR to screen oysters placed in water canals at the entry of a shrimp farm allowed WSSV to be detected 16 d prior to the disease occurring. The finding that C. gigas can concentrate small amounts of WSSV present in seawater without being harmed makes it an ideal sentinel species at shrimp farms.     

对虾白斑综合症病毒结构蛋白60B的原核表达和其基本氨基酸序列的初步分析

VP60B是对虾白斑综合症病毒(WSSV)中含量很少的一个结构蛋白。VP60B的一段序列与腺病毒纤维蛋白(Adenovirus type 5 fiber protein)的knob domain的一段序列具有同源性。本试验将VP60B基因克隆到原核表达系统中,在低温条件下,诱导了VP60B蛋白的表达。结果显示VP60B在该系统主要是以包含体的形式存在。原核表达的VP60B不能被鼠抗WSSV多克隆血清所识别,预示该蛋白的免疫原性较弱。通过对VP60B氨基酸序列的分析,发现有一个跨膜区,这预示着该蛋白可能位于WSSV病毒的囊膜上。     

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。