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。     

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。     

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。     

白斑综合征病毒2014年中国毒株变异区的序列比较

为研究白斑综合征病毒(White spot syndrome virus,WSSV)在中国不同地区的分子流行病学变异特征,对2014年1-8月期间在病害暴发区采集到的48份PCR检测阳性样本,用ORF75、ORF94和ORF125引物扩增目的片段,连接转化已克隆目的片段,测序分析不同样本ORF75、ORF94和ORF125重复序列数目的差异.结果显示,不同地区毒株ORF75的重复单元数目有4、10、11、12、13不等,ORF94的重复单元数目有4和14,而ORF125的重复单元数目有0、3、5、6、7不等.结果表明,流行在中国大部分地区的WSSV存在一定程度变异,毒株间的变异在ORF75、ORF94和ORF125上比较明显.     

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。     

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.     

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。     

对虾白斑综合征病毒蛋白质组的研究进展

白斑综合征病毒WSSV(White Spot Syndrome Virus,WSSV)是引起对虾大规模死亡的主要病毒性病原,且该病毒感染的宿主广泛。自WSSV基因组全序列公布以来,国内外的学者对WSSV的研究热点转移到该病毒基因产物功能的相关研究上面来。弄清WSSV感染机理是有效防治病毒的基础,而对其感染机理的阐释则主要是搞清病毒各蛋白在感染中的作用,这些是WSSV蛋白质组学的研究内容之一。分析WSSV蛋白同时可以在分子水平上为研究海洋和陆地生物病毒的进化趋势提供有力的基础。     

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.     

Effect of low water temperature on viral replication of white spot syndrome virus in Procambarus clarkii

This study evaluated the effect of low water temperature (10 ± 1 °C) on viral infection and replication of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, under standardized conditions. Crayfish were (i) maintained at 24 ± 1 °C before challenge and 10 ± 1 °C afterwards, or (ii) maintained at 10 ± 1 °C before challenge and 24 ± 1 °C afterwards. No mortality was observed when crayfish were held at 10 ± 1 °C after challenge, but mortality reached 100% when they were transferred to 24 ± 1 °C. Competitive PCR showed that viral levels at 10 ± 1 °C rose from 10⁶ to 10⁸ copies/mg of gill tissues, while at 24 ± 1 °C levels increased from 10⁶ to 10¹⁰ copies/mg of gill tissues during the same time interval. These results showed that a low water temperature of 10 ± 1 °C could reduce viral replication when compared to 24 ± 1 °C but could not prevent it.     

克氏原螯虾自噬相关基因PcAtg2的克隆及其在白斑综合征病毒胁迫下的表达分析

为了解自噬相关基因Atg2在克氏原螯虾(Procambarus clarkia)先天免疫中的作用,本研究克隆了克氏原螯虾Atg2 (PcAtg2)基因全长序列。生物信息学分析显示,PcAtg2蛋白编码序列全长为9 966 bp,推测其编码2 189个氨基酸。组织定量表达分布显示,PcAtg2在克氏原螯虾的各个组织中均有表达,其中在肝胰腺中表达最高,在眼柄中表达最低。在白斑综合征病毒(WSSV)感染实验中,PcAtg2基因表达量在不同组织中均呈现显著上调趋势。RNA干扰(RNAi)实验显示,PcAtg2基因沉默后,WSSV在克氏原螯虾体内的增殖明显被抑制,同时,自噬相关基因的表达量上调。透射电镜分析结果显示,在WSSV感染后,PcAtg2基因沉默组中克氏原螯虾肝胰腺组织中的自噬小体多于对照组。本研究结果可为了解克氏原螯虾应对WSSV胁迫下的调控机制提供理论参考。     

Virulence status,viral accommodation and structural protein profiles of white spot syndrome virus isolates in farmed Penaeus monodon from the southeast coast of India

The objective of this study was to investigate the reason for variation in the virulence of white spot syndrome virus (WSSV) from different shrimp farms in the Southeast coast of India. Six isolates of WSSV from farms experiencing outbreaks (virulent WSSV; vWSSV) and three isolates of WSSV from farms that had infected shrimps but no outbreaks (non‐virulent WSSV; nvWSSV) were collected from different farms in the Southeast coast of India. The sampled animals were all positive for WSSV by first‐step PCR. The viral isolates were compared using histopathology, electron microscopy, SDS‐PAGE analysis of viral structural proteins, an in vivo infectivity experiment and sequence comparison of major structural protein VP28; there were no differences between isolates in these analyses. A significant observation was that the haemolymph protein profile of nvWSSV‐infected shrimps showed three extra polypeptide bands at 41, 33 and 24 kDa that were not found in the haemolymph protein profile of vWSSV‐infected shrimps. The data obtained in this study suggest that the observed difference in the virulence of WSSV may not be due to any change in the virus, rather it could be due to the shrimp defence system producing certain factors that help it to accommodate the virus without causing any mortality.     

Effect of hot water extracts of brown seaweeds Sargassum spp. on growth and resistance to white spot syndrome virus in shrimp Penaeus monodon postlarvae

An experiment was conducted to evaluate the effect of a hot water extract of brown seaweeds Sargassum duplicatum and Sargassum wightii on the growth and white spot syndrome virus (WSSV) resistance in shrimp Penaeus monodon postlarvae (PL). Artemia nauplii (instar II) were enriched with both seaweed extracts at various concentrations (250, 500 and 750 mg L^?1) and fed to the respective P. monodon (PL15–35) group for 20 days. A control group was also maintained without seaweed extract supplementation. The weight gain of the experimental groups was significantly higher (0.274–0.323 g) than the control group (0.261 g). Similarly, the specific growth rate was also significantly higher (16.27–17.06%) in the experimental groups than in the control group (16.03%). After 20 days of the feeding experiment, the shrimp PL were challenged with WSSV for 21 days. During the challenge test, the control shrimp displayed 100% mortality within 8 days. In contrast, the mortality percentage of the highest concentration (750 mg L^?1) of seaweed extract enriched Artemia nauplii fed shrimp was 54–79%. Comparatively, low mortality was observed in S. wightii extract‐enriched Artemia nauplii fed shrimp. The polymerase chain reaction analysis indicated the concentration‐dependent infection of WSSV in P. monodon PL.