2.1c亚群猪瘟病毒GXF29/2013株全基因组克隆测序及遗传进化分析

Cloning,Sequencing and Phylogenetic Analysis of Complete Genome of Classical Swine Fever Virus Isolate GXF29/2013 Belonging to a New Subgenotype 2.1c

参考GenBank 登录的2.1 亚群猪瘟病毒（CSFV）的核苷酸序列,设计并合成4 对引物,应用RTPCR 技术,扩增出了CSFV 广西流行毒株GXF29/2013的全基因组,并进行核苷酸测序.结果GXF29/2013株的全基因组长度为12296个核苷酸.用MEGA5.0软件对GXF29/2013与GenBank上登录的20株不同亚型（1.1、2.1、2.2、2.3和3.4）CSFV 参考株进行全基因组核苷酸序列及开放阅读框编码的氨基酸序列比较,发现GXF29/2013 与参考株核苷酸序列同源性在83.2% ～98.0% 之间,氨基酸序列同源性在91.2%～98.8%之间.用E2基因进行系统发生树分析,结果表明GXF29/2013属于最近出现的2.1c亚群.对E2蛋白进行分析,结果表明GXF29/2013株有2.1c亚群毒株的特征,与2.1b毒株的遗传进化关系比与1.1亚群毒株的近.该毒株既保留了CSFV E2蛋白的一些关键特征,又与疫苗毒株的一些关键抗原位点不同.GXF29/2013病毒株全基因序列的获得为下一步进行CSFV 反向遗传操作平台的建立奠定了基础.

A Classical swine fever virus(CSFV)was isolated from the lymph nodes of CSFV infected pig in a farm in Guangxi province in 2013 and was named GXF29/2013.In order to obtain the genome sequence and know the revolution relationship of GXF29/2013 with the currently epidemic strains,four pairs of overlapping degenerate primers were designed according to the sequences of subgenotype 2.1 CSFVs in the GenBank,and then four fragments of the genome of GXF29/2013 were amplified by RT-PCR.The four amplified fragments were sequenced. The genomic nucleotide sequence and the deduced amino acid sequence of the whole reading frame of GXF 2 9/2 0 1 3 was compared with 2 0 reference strains of different subtypes of CSFVs in GenBank using MEGA5.0.The genetic evolution relationship of GXF29/2013 with 20 reference strains was analyzed.At the same time,the variation of amino acids in some key positions of E2 protein was demonstrated .The results showed that the complete genome of GXF29/2013 had 12296 nucleotides.The nucleotide homologies were 83.2%-98.0% and the amino acid homologies of the whole reading frame were 91.2%-98.8% compared with the 20 reference strains.The phylogenetic tree construc-ted with reference strains showed GXF29/2013 was classified into subgenotype 2.1c which is a new emer-ging subgenotype in China.GXF29/2013 had the characteristics of subgenotype 2.1c strains which was closer to subgenotype 2.1b than subgenotype 1.1.The E2 protein of GXF29/2013 still remained some im-portant features,but the amino acids in some key position were different compared to vaccine strain.The specific biological meanings of these variations had not known now.The results in analyzing the genetic e-volution of GXF29/2013 showed that CSFVs had been evolving in China's Mainland,and suggested a re-search on the characteristics of subgenotype 2.1c viruses should be taken on so that the new subgenotype i-solates could well be controled.