白斑综合征病毒广西株缺失区基因的比较分析

为了解广西养殖凡纳滨对虾中白斑综合征病毒(white spot syndrome virus,WSSV)的感染流行情况,并探讨WSSV广西株缺失区ORF23/24基因的遗传进化差异及其与各地WSSV毒株间的遗传进化关系,2010年5月至2013年9月在广西凡纳滨对虾主产区北海、钦州和防城港共采集306份患病虾样品,应用世界动物卫生组织(OIE)推荐的套式聚合酶链式反应(nested-PCR)方法进行WSSV检测;选取第一轮PCR为阳性的53份样品进行缺失区ORF23/24基因的PCR扩增,并将12份阳性PCR产物进行克隆、序列测定与比较分析。结果显示:306份患病虾样品中有82份为WSSV感染阳性,阳性率为26.8%;12株WSSV广西株缺失区ORF23/24基因大小均为2 096 bp,相对于此区域最为完整的WSSV-TW株,广西株均在中间缺失了10 970 bp;12株WSSV广西株的缺失区ORF23/24基因差异小,核苷酸同源性均在99.6%以上,仅存在少数碱基的替换,没有缺失和插入,其中BH-3、QZ-1、FCG-1和FCG-2的核苷酸序列完全一致;基于缺失区构建的系统发育进化树显示,WSSV广西地方株在遗传上相距较近,全部与印度株IN-05-Ⅰ聚在一个大的分支,而与台湾株、中国株及韩国株在遗传上相距较远。研究表明,WSSV感染在广西凡纳滨对虾主要养殖地区存在一定程度的流行;广西WSSV毒株缺失区ORF23/24基因的变异类型为中间大片段缺失,各毒株间无明显的地域和时间差异;WSSV广西株与印度株IN-05-Ⅰ的遗传进化关系最近。

Comparative analysis of deletion region of white spot syndrome virus genome among isolates in Guangxi

To understand the prevalence of white spot syndrome virus （WSSV） of cultured Penaeus vannamei （P. vannamei） in Guangxi Province and to evaluate the genetic diversity of deletion region ORF23/24 and the genetic evolution lineage among WSSV isolates of Guangxi Province and other places, we collected 306 diseased P. vannamei samples between May 2010 and September 2013 from Beihai, Qinzhou and Fangchenggang City in Guangxi Province, and the detection of WSSV among these samples was carried out using the nested polymerase chain reaction （nested PCR） recommended by Office International Des Epizooties （OIE）. We selected 53 positive samples found by first step PCR for amplification of the deletion regions ORF23/24 of the WSSV genome, and 12 PCR amplifications were used to clone and sequence. We then conducted a pairwise and multiple alignment analysis to evaluate the degree of genetic divergence among different strains. The results were as follows： 82 of 306 samples were positive for WSSV, and the positive rate was 26.8%; 12 Guangxi strains were 2096 bp in length and carried a 10970 bp deletion in the ORF23/24 region relative to WSSV-TW, which has the longest nucleotide sequence in this region. By comparison of nucleotide sequences of ORF23/24 region, we found that 12 Guangxi strains were above 99.6% homology, only existed nucleotide substitution but no deletion and insert, and BH-3, QZ-1, FCG-1 and FCG-2 strains were complete homology. In the phylogenetic tree of WSSV deletion region ORF23/24, the Guangxi strains were near in genetics and formed a separate branch with India strain IN-05-Ⅰ, while far from WSSV-TW, WSSV-CN and WSSV-Korea. In conclusion, there was some extent of prevalence of WSSV infection in cultured P. vannamei of Guangxi; the results showed that the Guangxi WSSV strains had large deletion in middle region of the ORF23/24 gene, and there were no differences in spatio-temporal and they were closer in genetics to India strain IN-05-Ⅰ than other strains in GenBank.