2017年中国部分地区白斑综合征病毒高变异区的序列分析

白斑综合征病毒(WSSV)自暴发以来给全球范围内的对虾养殖产业带来了巨大的损失。为了解我国凡纳滨对虾(Litopenaeus vannamei)养殖区WSSV的流行变异情况,选取2017年中国部分地区的42个WSSV阳性样本,对ORF14/15、ORF23/24、ORF75、ORF94和ORF125共5个可变区进行PCR特异性扩增,分析其序列的缺失变异和重复单元(Ru)中单核苷酸多态性的变化。研究结果显示,在ORF14/15的扩增中共出现4种缺失片段; ORF23/24只出现11 945 bp的缺失片段; ORF75扩增中,总RUs数目为3、4、9,其中45 bp的RUs在12、27、80位点发生多核苷酸多态性; ORF94的RUs数目为6,其各重复单元在48位发生单核苷酸多态性; ORF125的RUs数目为4、6、7不等,其各重复单元分别在20、27、50、53、61位发生碱基突变。研究结果表明,2017年样本中,WSSV在中国大部分地区均出现一定程度的缺失变异,其中部分可变区表现出缺失情况的稳定性,某些可变区的重复单元数目及SNP表现出地区的差异性以及不稳定性。     

2013年中国典型对虾养殖区白斑综合征病毒流行株高变异区序列的分析比较

为了解中国不同地区白斑综合征病毒的流行变异情况,本研究取用2013年3—12月从7个省市发病地区采集到的64份WSSV阳性样本,以特异的引物扩增目的片段,通过测序分析不同样本的缺失及变异差异。结果显示,在开放阅读框ORF14/15的扩增中,分别有6530 bp、6533 bp和5138 bp的片段缺失,而在ORF23/24扩增中有12070bp大片段的缺失,不同地区样本中未能成功扩增ORF75,而ORF94的重复单元数目分别为0、3、4、12不等,ORF125的重复单元数目为0、7。SNP分析表明,含有3个重复单元的ORF94在48位的碱基为T、T、T,重复单元数为4的在48位的碱基为T、T、T、T,重复单元数为12的在48位的碱基分别为T及11个A。而ORF125所有重复单元数为7的情况在8、18、25、66、69位置的碱基均为G、G、G、G、A,在9、50、53、61位的碱基也普遍出现了变异。结果表明,WSSV在中国不同地区存在一定程度的变异,其在序列中的缺失、重复单元数目以及SNP的差异较为明显。     

2015年中国典型对虾养殖区WSSV流行株高变异区序列的分析比较

为了解我国白斑综合征病毒(WSSV)流行变异特征，本研究对2015年4~10月期间在山东、江苏、天津、浙江、海南和广东6省市采集到的57份WSSV阳性的样本，通过特异性的扩增目的片段，根据测序结果分析比较不同地区、不同分离株之间在ORF14/15、ORF23/24上的缺失变异情况，以及ORF75、ORF94和ORF125上的重复单元(Repeat unit, RU)数目差异。结果显示，在ORF14/15扩增中，分别有6530、5908和5725 bp的片段缺失，而在ORF23/24扩增中均有12070 bp大片段的缺失，ORF75的45 bp的RU数目分别为1、2和3，102 bp的RU数目均为1，而ORF94的RU数目分别为4、5、10和12不等，ORF125的RU数目为3、5和6。结果表明，WSSV在部分开放型阅读框上表现出明显的变异差异，而在某些开放型阅读框上的缺失情况则有显著的稳定性。     

湖北地区克氏原螯虾白斑综合征病毒变异区ORF14/15、ORF23/24基因序列比较分析

试验扩增、克隆了在湖北地区采集的19份克氏原螯虾(Procambarus clarkii)白斑综合征病毒(White spot syndrome virus,WSSV)阳性样品的变异区ORF14/15和ORF23/24基因,通过测序比较分析了湖北各WSSV毒株与Gen Bank公布的标准毒株间在变异区ORF14/15及ORF23/24基因的差异性。结果显示,19份WSSV阳性样品中有部分样品在变异区扩增出ORF14/15、ORF23/24基因片段,变异区基因序列分析发现,与Gen Bank已公布的标准毒株相比,存在大片段缺失。在变异区ORF14/15,有3个毒株扩增出1 442 bp的片段,4个毒株扩增出630 bp的片段,基于变异区ORF14/15构建的系统进化树显示,这些毒株归属两个不同的分支。在变异区ORF23/24,有2个毒株扩增出大小为2 096 bp的片段,进化分析发现这2个毒株在变异区ORF23/24的遗传距离较近。     

2014年中国不同地区对虾白斑综合征病毒ORF14/15和ORF23/24缺失区序列比较

对虾养殖面临诸多病害威胁，对虾白斑综合征病毒(White spot syndrome virus, WSSV)是养殖对虾主要病原之一，WSSV不同地理株的变异可能导致WSSV毒力的变化。为了解2014年中国大部分地区WSSV ORF14/15和ORF23/24的变异情况，本研究选择2014年1月–8月期间采集的48份WSSV阳性样本，用特异引物扩增ORF14/15和ORF23/24片段，连接于T载体，转化至Top10中，筛选阳性克隆，测序分析不同样本之间的缺失差异。结果显示，能够扩增ORF14/15和ORF23/24样品的比例分别为43.75%和33.33%。在ORF14/15扩增中，分别扩增出1260 bp、1270 bp、1892 bp和2662 bp片段，与TH-96-Ⅱ比对共有4种缺失情况，即缺失6540 bp、6530 bp、5908 bp和5138 bp。而在ORF23/24扩增中，分别扩增出1140 bp和1146 bp片段，与中国台湾株(TW)比对有两种缺失情况，即缺失12070 bp和12064 bp。研究结果表明，WSSV在中国大部分地区存在一定程度的变异，而不同毒株之间在ORF14/15可变区差异比较明显，在ORF23/24可变区差异不大，但均具有大片段缺失。     

罗氏沼虾白斑综合症病毒WSSV ORF147片段的克隆、表达与序列分析

白斑综合症病毒WSSV(White Spot Syndrome Virus),是严重危害虾类养殖业的主要病原之一.本实验根据已知南美白对虾WSSV ORF147序列设计1对特异性引物,从患疑似白斑病毒病的罗氏沼虾中提取总DNA,用PCR法扩增得到1特异性片段.将该片段克隆进pET-28a( )载体,测序表明该片段全长1 475 bp,最大开放式阅读框为1 380 bp,编码459个氨基酸,预计其相对分子质量为51.9 kDa;与GenBank登录的WSSV ORF147序列(登录号AF369029)进行比对,核苷酸同源性为99%,证实为WSSV ORF147片段.将该片段在大肠杆菌E coli中进行表达,能获得相应的特异多肽条带.根据测序结果推导WSSV ORF147多肽在N端有信号肽序列,并且在氨基酸序列的122～144区间形成跨膜螺旋区.     

安徽省克氏原螯虾白斑综合征病毒缺失区序列分析

为了解安徽省克氏原螯虾(Procambarus clarkii)白斑综合征病毒(white spot syndrome virus,WSSV)缺失区ORF23/24和ORF14/15的遗传差异及其与世界各地WSSV的遗传进化关系,2016年4月—8月,在安徽省6个市采集了9个养殖克氏原螯虾样本进行WSSV套式PCR检测,扩增病毒缺失区ORF23/24和ORF14/15,将获得的序列进行比较分析。结果显示,9个样本均在第1轮PCR扩增中获得阳性结果,其ORF23/24区与中国台湾株(TW)比对,缺失5 892 bp或9 310 bp,其ORF14/15区与WSSV祖先株(TH-96-Ⅱ)比对,缺失5138 bp或5948 bp。其中8个样本中WSSV与2008至2010年在江苏的克氏原螯虾中检测到的一些毒株的ORF23/24和ORF14/15区缺失情况相同,且这些病毒ORF14/15区均缺失5 138 bp,与TW株缺失情况相同。     

1个白斑综合症病毒基因的克隆及其在大肠杆菌M15中的表达

根据对虾白斑综合症病毒（white spot syndrome virus，WSSV）1个可能编码蛋白的开放读码框（open reading frame，ORF）的序列（WSSV A基因），结合pQE30载体的多克隆位点，设计1对引物进行PCR，扩增出大小为0.28kb的A基因片段。把目的片段克隆到pGEM-T Easy载体上，构建出重组质粒pGT-A，再用引物两端酶酶切出目的片段，并按正确的读码框顺序插入到pQE30表达载体上的乳糖操纵子中，构建出带有目的片段的重组质粒pQE30-A，然后将重组质粒转化到大肠杆菌M15细胞，经IPTG诱导，SDS-PAGE和Western blot显示有与A基因预期大小11kD相吻合的融合蛋白带。结果表明，来源于WSSV的这一ORF是1个可表达的基因。     

同时检测两种对虾病毒和4种弧菌的同步PCR方法的建立

通过检索、多重比对、分析和筛选GenBank中对虾白斑综合征病毒(WSSV)、传染性皮下和造血器官坏死病毒(IHHNV)、副溶血孤菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌的基因序列,设计了10对特异性引物,以已知毒株和菌株的DNA为模板进行PCR,均能扩增出与实验设计相符合的DNA片段,对PCR扩增条件进行优化,建立了可同时检测鉴别WSSV、IHHNV、副溶血弧菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌,并且能同时区分WSSV不同地理毒株的同步PCR方法.研究结果表明,该方法检测特异性好,检测通量大,适合于对虾多种病原的同时检测.     

白斑综合征病毒江苏分离株变异区和缺失区基因的序列比较

为了解白斑综合征病毒(White spot syndrome virus,WSSV)在养殖克氏原螯虾(Procambarus clarkii)中的感染流行情况和分子流行病学特征,以及探讨不同病毒株间的遗传进化差异,2008年7月至2010年6月在江苏各主要克氏原螯虾养殖产区共采集了83份样品,应用环介导等温扩增技术(LAMP)检测42份样品为病毒感染阳性,阳性率达50.6%。在此基础上考虑地理和时间因素选取了兴化、盱眙等地的6份样品,PCR扩增、克隆了样品中所携带病毒的变异区和缺失区基因并对其进行了测序。分析结果显示,在变异区除兴化毒株为1 829 bp之外,其余各分离株间差异微小,均在1 440 bp左右;而在缺失区各毒株大小均为384 bp,且核苷酸序列完全一致。随后将各分离株序列与GenBank已公布的毒株做进一步的序列比较分析,结果表明在江苏境内流行的兴化毒株与其余毒株间存在明显差异,在遗传上相距较远,并且推测WSSV变异区和缺失区可能是各自独立进化变异的;同时,缺失变小的病毒基因组更易于适应宿主和环境的变化。     

Low numbers of repeat units in variable number of tandem repeats (VNTR) regions of white spot syndrome virus are correlated with disease outbreaks

White spot syndrome virus (WSSV) is the most important pathogen in shrimp farming systems worldwide including the Mekong Delta, Vietnam. The genome of WSSV is characterized by the presence of two major ‘indel regions’ found at ORF14/15 and ORF23/24 (WSSV‐Thailand) and three regions with variable number tandem repeats (VNTR) located in ORF75, ORF94 and ORF125. In the current study, we investigated whether or not the number of repeat units in the VNTRs correlates with virus outbreak status and/or shrimp farming practice. We analysed 662 WSSV samples from individual WSSV‐infected Penaeus monodon shrimp from 104 ponds collected from two important shrimp farming regions of the Mekong Delta: Ca Mau and Bac Lieu. Using this large data set and statistical analysis, we found that for ORF94 and ORF125, the mean number of repeat units (RUs) in VNTRs was significantly lower in disease outbreak ponds than in non‐outbreak ponds. Although a higher mean RU number was observed in the improved‐extensive system than in the rice‐shrimp or semi‐intensive systems, these differences were not significant. VNTR sequences are thus not only useful markers for studying WSSV genotypes and populations, but specific VNTR variants also correlate with disease outbreaks in shrimp farming systems.     

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.     

Virulence and genotypes of white spot syndrome virus infecting Pacific white shrimp Litopenaeus vannamei in north‐western Mexico

White spot syndrome virus (WSSV) has caused substantial global economic impact on aquaculture, and it has been determined that strains can vary in virulence. In this study, the effect of viral load was evaluated by infecting Litopenaeus vannamei with 10‐fold serial dilution of tissue infected with strain WSSV Mx‐H, and the virulence of four WSSV strains from north‐western Mexico was assessed along with their variable number of tandem repeat (VNTR) genotypes in ORF75, ORF94 and ORF125. The LD₅₀ of the Mx‐H strain was a dilution dose of 10^?7.5; the mortality titre was 10^9.2 LD₅₀ per gram. In shrimp injected with 10^2.5 to 10^6.5 LD₅₀, no significant virulence differences were evident. Using mortality data, the four WSSV strains grouped into three virulence levels. The Mx‐F strain (intermediate virulence) and the Mx‐C strain (high virulence) showed more genetic differences than those observed between the Mx‐G (low‐virulence) and Mx‐H (high‐virulence) strains, in ORF94 and ORF125. The application of high‐viral‐load inocula proved useful in determining the different virulence phenotypes of the WSSV strains from the Eastern Pacific.     

Indian isolates of white spot syndrome virus exhibit variations in their pathogenicity and genomic tandem repeats

To detect genomic variation of white spot syndrome virus (WSSV) isolates from different geographical regions of India, the variable number of the tandem repeat (VNTR) region of the ORF 94 (Thailand WSSV isolate – GeneBank Accession No. AF369029 ) was analysed using five specific sets of primers. Analysis of 70 WSSV‐positive samples showed the presence of 14 different genotypes of WSSV with VNTRs ranging from 2 to 16 tandem repeats with the majority (85.47%) having 6–12 tandem repeats. Occurrence of different genotypes of WSSV was found to be neither correlated to any specific geographical region nor to the different growth stage of the tiger shrimp, Penaeus monodon. Pathogenicity studies conducted with 25 isolates of WSSV revealed the presence of virulent and avirulent strains of WSSV in Indian shrimp farms. However, an unambiguous link could not be established between the different genotypes and their virulence.     

New genetic recombination in hypervariable regions of the white spot syndrome virus isolated from Litopenaeus vannamei (Boone) in northwest Mexico

The white spot syndrome virus (WSSV) is a pathogen of great concern to the worldwide shrimp culture. In comparative studies on the WSSV genome, regions such as the open reading frame (ORF) 14–15 and ORF 23–24, prone to deletions and recombination, had been useful to study the evolutive relationships among viral strains. When looking for the WSSV strains infecting Litopenaeus vannamei (Boone) in northwest Mexico, we found evidence of a genetic similarity in ORF 14–15 to a strain from India and a recombination involving ORFs 78, 79 and 80. Two genotypes were found involving the insertion of a 265 base‐pair segment of ORF 108 into ORF 78 with inversions and deletions within ORFs 78, 79 and 80. The WSSV has an Asian origin and the mutations found could be an adaptation strategy to infect L. vannamei and other crustacean species of the American continent.     

A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus

Since it first appeared in 1992, white spot syndrome virus (WSSV) has become the most threatening infectious agent in shrimp aquaculture. Within a decade, this pathogen has spread to all the main shrimp farming areas and has caused enormous economic losses amounting to more than seven billion US dollars. At present, biosecurity methods used to exclude pathogens in shrimp farms include disinfecting ponds and water, preventing the entrance of animals that may carry infectious agents and stocking ponds with specific pathogen-free post-larvae. The combination of these practices increases biosecurity in shrimp farming facilities and may contribute to reduce the risk of a WSSV outbreak. Although several control methods have shown some efficacy against WSSV under experimental conditions, no therapeutic products or strategies are available to effectively control WSSV in the field. Furthermore, differences in virulence and clinical outcome of WSSV infections have been reported. The sequencing and characterization of different strains of WSSV has begun to determine aspects of its biology, virulence and pathogenesis. Knowledge on these aspects is critical for developing effective control methods. The aim of this review is to present an update of the knowledge generated so far on different aspects of WSSV organization, morphogenesis, pathology and pathogenesis.