Prevalence of three shrimp viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV), Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang (93.3%), liuao (66.7%), Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.     

Molecular Epidemiological Investigation of Infectious Hypodermal and Hematopoietic Necrosis Virus and Taura Syndrome Virus in Penaeus Vannamei Cultured in China

The Infectious hypodermal and hematopoietic necrosis virus(IHHNV) and Taura syndrome virus(TSV) are two important shrimp viruses in cultured shrimp in America.These two viruses were transmitted to China at the beginning of the 21st century.In this study,214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection.The results showed that there were a high prevalence of IHHNV(65.42%) and low prevalence of TSV(3.27%) in the tested samples.Several samples were found to be co-infected with these two viruses.A 3 kb fragment of 7 positive IHHNV samples and a structure protein region(ORF2) of three TSV positive samples were amplified and sequenced.The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii.Phylogenetic analysis showed that TSV isolates were clustered into two groups,Asia and America group,which was genetically correlated to geographic distribution.     

Molecular epidemiological investigation of infectious hypodermal and hematopoietic necrosis virus and Taura syndrome virus in <Emphasis Type="Italic">Penaeus Vannamei</Emphasis> cultured in China

The Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV) are two important shrimp viruses in cultured shrimp in America. These two viruses were transmitted to China at the beginning of the 21^st century. In this study, 214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection. The results showed that there were a high prevalence of IHHNV (65.42%) and low prevalence of TSV (3.27%) in the tested samples. Several samples were found to be co-infected with these two viruses. A 3 kb fragment of 7 positive IHHNV samples and a structure protein region (ORF2) of three TSV positive samples were amplified and sequenced. The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii. Phylogenetic analysis showed that TSV isolates were clustered into two groups, Asia and America group, which was genetically correlated to geographic distribution. Foundation item: This work was supported by 973 project (2006CB101801)     

多重RT-PCR同时检测鉴别三种对虾病毒的研究与应用

根据基因库中对虾桃拉综合征病毒（TSV）、白斑综合征病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）的基因序列,分别设计了三对特异性引物,通过对多重RT—PCR扩增条件的优化,研究建立了可同时检测鉴别TSV、WSSV和IHHNV的多重RT—PCR。该技术对同一样品中的TSV RNA、WSSV DNA和IHHNV DNA模板进行扩增,结果均同时得到3条大小与实验设计相符的231bp（TSV）、593bp（WSSV）和356bp（IHHNV）的特异性多重RT—PCR扩增带,对其它对虾病原核酸的扩增结果为阴性。敏感性试验结果表明,该技术最低能检测到10pgTSV RNA、100pg WSSV DNA和100pg IHHNV DNA。临床检测试验结果表明,该技术对TSV、WSSV和I—HHNV的检出率明显高于传统的临床症状观察和组织病理学检查,提示该技术适用于这三种病毒的临床快速检测和鉴别诊断。     

南美白对虾传染性皮下及造血组织坏死病毒抗性相关基因的微卫星标记研究

传染性皮下及造血组织坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)是南美白对虾养殖中最普遍的虾病毒之一.它能引起生长缓慢和畸形,通称矮小畸形综合症(RDS).分子标记是研究抗病基因的有效手段之一,微卫星(microsatellite)标记是新发展的遗传标记.介绍用微卫星技术对南美白对虾抗病和感病群体筛选出的IHHNV抗性相关基因进行研究.     

对虾白斑综合症病毒与虾鳃细胞膜特异性结合关系的确立

对虾白斑综合症病毒(White spot syndrome virus,WSSV)是养殖对虾的一个主要病原,也是目前发现的基因组最大的动物病毒(基因组约290kDa,双链环状)。WSSV病毒粒子为卵形杆状,外被囊膜,囊膜在尾部延伸成一长尾。它不仅能感染对虾,还能感染其它淡水及海水甲壳类。养殖对虾被感染后,3—10d内累积死亡率可达100％,给对虾养     

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immun-ological diagnosis methods for WSSV infection.     

斑点杂交检测对虾白斑综合征病毒青岛株在螯虾体内的动态分布

近年来 ,我国学者对人工养殖对虾暴发性病毒病的病原进行了较为系统的研究[1～ 5] ,本试验应用螯虾这一动物模型[6] ,利用斑点杂交方法 ,研究了白斑综合征病毒 (ＷＳＳＶ ,前称无包埋体对虾病毒Ｎｏｎ -Ｏｃｃｌｕｄｅｄ -ＳｈｒｉｍｐＶｉｒｕｓＮＯＳＶ )青岛株在螯虾体内的动态分布 ,为研究该病毒的传播途径、增殖致病机理提供了参考。1　材料与方法1.1　实验动物克氏原螯虾 (Ｃａｍｂａｒｕｓｐｒｏｃｌａｒｋｉｉ ,以下简称螯虾 ) 40尾 ,购自南京某农贸市场 ,实验室饲养一周以上 ,健康存活。1.2　种毒处理及接种白斑综合征病毒青岛株 (…     

对虾白斑综合症病毒囊膜蛋白VP28的表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp28的序列,设计并合成引物,PCR扩增得到vp28基因,成功构建重组表达载体pET22b-vp28并转化大肠杆菌BL21(DE3)。基因工程菌株37℃IPTG诱导,表达产物经Western-blot和SDS-PAGE检测显示有与预期大小32kDa相符合的目的蛋白。用Ni2 -柱纯化的目的蛋白分别直接注射螯虾和包被饲料投喂螯虾,实验结果表明vp28在大肠杆菌中的表达产物有显著提高虾体抗WSSV感染力的作用,而且注射效果更好。     

对虾白斑综合症病毒囊膜蛋白VP19的融合表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp19的序列,设计并合成引物,PCR扩增得到vp19基因并克隆到pGEM‐T载体中,经过BamHⅠ/HindⅢ酶切、连接并将vp19插入到pET32b表达载体中。用重组质粒pET32b-vp19转化大肠杆菌Origam(iDE3)pLysS,在IPTG诱导下,融合蛋白Trx-VP19以可溶性的形式得到表达,经SDS-PAGE和Western-blot检测显示其分子量与预期的大小相符合。目的蛋白经Ni2 柱纯化并定量后分别直接注射鳌虾和包被饲料投喂鳌虾。实验结果表明注射Trx-VP19可以提高鳌虾个体抗WSSV感染力的作用。     

对虾白斑综合征病毒的细胞因子受体基因的分析与表达

在对虾白斑综合征病毒(White spot syndrome virus,WSSV)的基因组中发现一个具有细胞因子受体特征的开放阅读框,该阅读框全长2022个核苷酸,编码674个氨基酸,蛋白质理论分子量为76kDa。该基因含有真核生物细胞因子gp130受体特征序列。为了研究该基因的功能,采用PCR方法从病毒基因组中扩增出基因片段,克隆到pGEM-T Easy载体中,经BamH I和Sal I双酶切后插入pET28b表达载体中。重组质粒转化到大肠杆菌BL21中,IPTG诱导后,经SDS-PAGE电泳表明在。76kDa处有目的蛋白表达。用冰浴超声波对诱导后的菌液进行处理以获得初步纯化的蛋白,作为抗原人工免疫实验兔子以获得含特异性抗体的抗血清。该基因的表达成功,为其功能的进一步深入研究奠定了基础。     

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。     

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.     

对虾白斑综合症病毒与虾鳃细胞膜特异性结合关系的确立

对虾白斑综合症病素养(White spot syndrome virus,WSSV)是养殖对虾的一个主要病原,也是目前发现的基因组最大的动物病素养(基因组约290kDa,双链环状).     

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。       

A thioredoxin response to the WSSV challenge on the Chinese white shrimp, Fenneropenaeus chinensis

Thioredoxin (TRX) is involved in cell redox homeostasis. In addition, it is responsible for maintaining proteins in their reduced state. In our study, a Fenneropenaeus chinensis thioredoxin (FcTRX) gene was identified from the Chinese white shrimp. The full length of FcTRX was 777 bp, including a 60 bp 5′ untranslated region (UTR), a 318 bp open reading frame (ORF) encoding a 105 amino acids protein, and a 399 bp 3′ UTR. FcTRX contained a TRX domain with a conserved motif of Cys-Gly-Pro-Cys (CGPC). No signal peptide was predicted by SMART analysis. The molecular mass and pI of FcTRX were 12 kDa and 4.62, respectively. FcTRX is a widely distributed gene, and its mRNA is detected in hemocytes, hearts, hepatopancreas, gills, stomach, and intestine from an unchallenged shrimp. The expression level of FcTRX was the highest in hepatopancreas, where it was down-regulated to the lowest level at 12 h white spot syndrome virus (WSSV) challenge. In the gills, it went up to the highest level at 6 h. Western blot showed that FcTRX protein in hepatopancreas challenged with WSSV was down-regulated from 2 h to 12 h and then restored to the level similar to that of unchallenged shrimp at 24 h. In the gills challenged with WSSV, the FcTRX protein was up-regulated from 6 h to 24 h. Our research indicated its possible role in the anti-WSSV innate immunity of shrimps.     

一种改良的对虾白斑综合征病毒的提纯技术

The envelope proteins of White spot syndrome virus (WSSV) are very fragile and easy to be destroyed during purification. It was difficult to obtain a large quantity of intact virions by routine sucrose gradient centrifugation. After modifying the sucrose gradient by adding citrate sodium, we can obtain a large quantity of intact virions and nucleocapsids. This purified virions and nucleocapsids were subsequently used for analyzing viral structural proteins and DNA extraction. The result showed that this modified techniaue is very efficient for virus purification.     

桃拉综合征病毒中国株ZHZC3全基因测序及分子结构预测

设计8对引物分片段扩增桃拉综合征病毒中国分离株ZHZC3全基因组,病毒两末端序列采用末端快速扩增方法(RACE)获取.扩增产物克隆到pMD18-T载体并测序,用DNAstar软件拼接全序列及同源性比较.结果显示ZHZC3全序列除去3' poly (A)尾,由10202个碱基组成,有两个开放阅读框,分别编码2107和1011个氨基酸的聚蛋白.与美国参考株HI94相比,在编码区没有核苷酸的缺失和插入,但在5' UTR缺失3个A,两者整体核酸同源性达97.9%.ORF1 中ZHZC3与HI94及巴西株(BLZ01)的核酸同源性分别为97.6%、97.7%,在ORF2中ZHZC3与HI94、BLZ01的核酸同源性则分别为98.3、97%.与国外株ORF2的部分序列比较发现ZHZC3和中国台湾株均与美国株HI94同源性最高.克隆分析6株TSV中国大陆株主要结构蛋白CP2基因,发现其编码的氨基酸存在三个高变区,中国大陆株更有其独特的氨基酸变异模式,312 (S), 449 (A), 451 (Q) 和468 (H).表明该病毒的整体变异性不高,但中国的流行株已形成其自己的遗传演变特征.在此基础上,利用生物学软件对CP2蛋白功能域和三维结构进行了预测,为进一步分析CP2蛋白结构与功能关系奠定了基础.ZHZC3株是第一个测定全序列的TSV中国株.     

RT—PCR法检测我国东南沿海凡纳滨对虾的桃拉综合征病毒

桃拉综合征病毒（Taura syndrome virus,TSV）引致养殖及野生对虾急性传染病,14d至40d的幼虾极易感。该病毒属微RNA病毒科（Picornaviridae）,近年来研究者认为应归类为类蟋蟀麻痹病毒属（Cricket paralysis—like viruses）,因其基因组特征更接近于前所未分类的昆虫单股RNA病毒,如丙型果蝇病毒。