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。

Variation sequence comparison of shrimp WSSV from different parts of China in 2013

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant eco-nomic losses in the aquaculture industry worldwide. With the constant expansion of the areas and scale of prawn farm-ing, variations between different strains of WSSV have emerged. The differences between isolates may cause variations in the virulence of WSS. Deletions in the ORF14/15 and ORF23/24 variable regions have been used as molecular markers to study the epidemiology of the virus. The repeat units of ORF75, ORF94, and ORF125 vary in different iso-lates and the numbers of repeat units in these three genomic regions are a useful marker for epidemiological studies and for genotyping strains of WSSV. However, each repeat unit may contain single-nucleotide polymorphisms (SNPs) that are important for differentiating haplotypes with the same numbers of repeat units. The results of epidemiological stud-ies may differ if the variability provided by these SNPs is not considered and only the number of repeat units is taken into account. To understand the variations in WSSV ORF14/15 and ORF23/24 and the variable number of tandem repeats (VNTR) and SNPs located in ORF75, ORF94, and ORF125 in different regions of China, we investigated 64 samples that were WSSV-positive on PCR, collected in a disease outbreak area between March and December 2013. The tested samples were from Hainan, Guangdong, Guangxi, Zhejiang, Shandong, Hebei, and Tianjin. Using specific primers, WSSV-positive samples were genotyped with PCR and the amplified fragments were ligated to a T-vector and used to transform Top10 cells. The positive clones were selected and sequenced. After sequencing, the fragments deleted from ORF14/15 and ORF23/24 in different samples were compared with the sequences of Th-96-II and TW respectively, and the three WSSV VNTR regions and SNPs in ORF75, ORF94, and ORF125 from different samples were analyzed with the DNAMAN software. The results showed that there were five samples which had the products of ORF14/15 ampli-fication, and one sample had the products of ORF23/24 amplification. The lengths of the amplified fragments of ORF14/15 were 1267 bp, 1270 bp, and 1892 bp, corresponding to deletions of 6533 bp, 6530 bp, and 5908 bp, respec-tively, compared with Th-96-II. One type of ORF23/24 was confirmed in this experiment, and the amplified fragment was 1140 bp, corresponding to a deletion of 12070 bp compared with the TW isolate. No fragment was amplified from ORF75. Four different ORF94 VNTRs, including 0, 3, 4, or 12 repeat units, were identified. The ORF125 VNTR, which included 0 or 7 repeat units, was identified. The SNP analysis showed that the bases on the special site in this study were different from the reported types. The bases at site 48 in ORF94 were T, T, T(Type I: 3 RUs), or the bases at site 48 were T, T, T, T(Type II: 4 RUs), and the base were T and 11 A(Type III: 12RUs), respectively. The bases at site 8, 18, 25, 66, and 69 in ORF125(contained 7 RUs), were G, G, G, G, and A, respectively. It is noteworthy that there were also some variations at sites 9, 50, 53, and 61 in ORF125. These results suggest that there are certain degrees of preva-lence and variation in WSSV, which causes white spot disease in most parts of China, with clear variations between the different strains in ORF14/15, ORF23/24, ORF75, ORF94, and ORF125.