2016年中国部分地区白斑综合征病毒高变异区序列的分析比较

Variation analysis of highly variable regions of white spot syndrome virus from partial areas of China in 2016

为探究中国白斑综合征病毒(white spot syndrome virus,WSSV)的分子流行病学变异情况,选取2016年1-7月从我国3省9市采集到的47份WSSV阳性样本,提取WSSV核酸后,使用特定引物对目的片段进行扩增,继而进行测序并分析比较不同地区样本的ORF14/15和ORF23/24序列缺失情况,ORF75、ORF94和ORF125的VNTR及SNPs变化情况。结果显示,在ORF14/15的扩增中,有4种缺失片段,分别是6 540 bp、6 530 bp、5 950 bp和5 140 bp,而在ORF23/24扩增中则有3种大片段缺失,分别是12 073 bp、12 070bp和11 945 bp,在所有样本中ORF75的RUs数目分别为11、8、10、3,ORF94的RUs数目分别为5、7、8、11、14,ORF125的RUs数目为4、5、6。SNPs分析结果表明,ORF94片段中含有5个和8个RUs的在48位的碱基均为T,含有7个RUs的在48位的碱基为T、G、G、G、G、T、T,含有11个RUs的在48位的碱基为T、T、T、T、T、T、G、T、G、T、T,含有14个RUs的在48位的碱基为T、T、T、T、G、G、T、G、G、T、T、T、T、T,含有所有RUs数目的ORF125片段在8、18、25、66和69位置的碱基均为G、G、G、G和A,,而在9、50、53、61和63位的碱基则出现了4种变异。研究结果表明,2016年的样本中,WSSV毒株存在一定程度的变异情况。主要表现在ORF14/15和ORF23/24均出现了新的缺失片段,ORF75、ORF94和ORF125的VNTR以及SNPs变异情况也存在明显差异。

White spot syndrome virus（ WSSV）,hosting a wide range of infections,is one of the major pathogens in shrimp culture industry,and has become the most dangerous and widespread virus to aquatic crustaceans,especially shrimp population. The variations in WSSV ORF14/15,ORF23/24 and the variable number of tandem repeats（ VNTR） and SNPs located in ORF75,ORF94,and ORF125 have been used as molecular markers to study the molecular epidemiology of the virus.In order to study the molecular epidemiology of WSSV in China,we investigated 47 WSSV-positive samples collected in partial disease outbreak areas from January to July,2016. WSSV-positive samples were amplified by PCR used specific primers and the amplified fragments were ligated to a T-vector and transformed into TOP10,and then the positive clones were screened and sequenced. Finally,the DNAMAN8 software was used to analyze the results. The fragments of ORF14/15 and ORF23/24 were respectively aligned with TH-96-II（ AY753327） and the China Taiwan strain [TW（ AF440570） ] to make analyses of the deletion fragments. The resultant sequences of ORF75,ORF94,and ORF125 from different samples were analyzed in VNTR and SNPs. The results showed that there were four deletion fragments in ORF14/15 amplification,which were 6 540 bp,6 530 bp,5 950 bp and 5 140 bp,while three large fragments were deleted in ORF23/24 amplification,which were 12 073 bp,12 070 bp and 11 945 bp. Among all the samples,ORF75 amplification had four kinds of VNTRs,which contained 11,8,10 and 3 RUs,and there were five kinds of VNTRs in ORF94 amplification,including 5,7,8,11 and 14 RUs. The VNTRs of ORF125 amplification contained 4,5 and 6 RUs. The SNPs analyses showed that the bases at site 48 in ORF94 fragment containing5 and 8 RUs were all T,the bases in 48 containing 7 RUs were T,G,G,G,G,T,T,the bases containing11 RUs were T,T,T,T,T,T,G,T,G,T,T,and the bases containing 14 RUs were T,T,T,T,G,G,T,G,G,T,T,T,T,T. The bases at site 8,18,25,66,and 69 in ORF125 containing all VNTRs were the same,which were G,G,G,G,and A,respectively. It was noticed that there were variations in bases at site 9,50,53,61,and 63 in ORF125.The results indicated that there was certain degree of variation in WSSV strains in 2016. New deletion fragments were found in both ORF14/15 and ORF23/24,and there were also significant variations in VNTR and SNPs located in ORF75,ORF94 and ORF125.