【摘要】 为阐明云南省西南边境地区登革2型病毒(DENV-2)流行株的全基因组序列特征及流行病学特点,采用C6/36细胞培养法分离病毒,用RT-PCR法扩增新分离DENV-2的全基因组序列,采用Clastal X1.83和MEGA6等生物信息学软件进行核苷酸同源性及系统进化分析。结果从登革热患者血清中分离到10株DENV-2,其中德宏州瑞丽市6株(2013年3株和2015年3株),西双版纳州景洪市2015年4株。经RTPCR和序列测定,获得这10株DENV-2的全基因组序列。基于结构蛋白和非结构蛋白基因的系统进化和同源性分析表明,这10株病毒中5株为亚洲基因Ⅰ型(AsianⅠGenotype,AG-Ⅰ),5株为全球基因型(Cosmopolitan genotype,CG)。其中2013和2015年瑞丽流行株均为AG-Ⅰ,景洪市2015年流行株均属CG,它们均与东南亚相同基因型流行株具有较高的同源性和较近的亲缘关系。云南株与DENV-2原型株(New Guinea-C)的E和NS5基因核苷酸同源性分别为93.38%~93.77%和92.80%~94.74%。所有云南株和东南亚参考株与New Guinea-C株在结构蛋白或非结构蛋白的氨基酸位点均存在一定程度的改变。本研究证实,云南省西南边境地区存在DENV-2两种基因型流行,瑞丽和景洪流行株分别为AG-Ⅰ和CG,但它们的输入来源均为相邻东南亚国家。云南株与DENV-2原型株New Guinea-C株间存在明显差异,但决定病毒抗原、致病性和毒力的关键位点未发现明显变化。更多还原

【Abstract】 To understand the molecular characteristics of the full-length genome of dengue serotype 2 virus( DENV-2)strains isolated in southwestern border region of Yunnan province,China,in 2013 and 2015 and their epidemiological feature. Dengue virus was cultured in C6/36 cell line. Viral RNA was extracted from isolates,then the full-length genome was amplified by RT-PCR. The homology and phylogenetic analyses were made on the nucleotide sequences by bioinformatics softwares including Clastal X1. 83 and MEGA6 etc. Ten strains of DENV-2 were harvested from dengue fever cases,of these,6 strains from Ruili city of Dehong prefecture and 4 from Jinghong city of Xishuangbanna prefecture,Yunnan province. Phylogenetic tree and homology analysis based on structural and non-structural protein genes showed that5 strains of the experimental virus belonged to Asian I genotype( AG-I) and the other 5 strains belonged to Cosmopolitan genotype( CG). Among them,the AG-I strains were isolated from Ruili in 2013 and 2015,and CG strains were isolated from Jinghong in 2015 as well as they have a closer genetic relationship with the strains isolated from Southeast Asia.Compared with prototype strain( New Guinea-C) of DENV-2,E and NS5 nucleotide homology of the Yunnan strains were93. 38% ~ 93. 77% and 92. 80% ~ 94. 74% respectively. Yunnan strains compared with US_Hawaii strain,there were 44 and 150 different sites in amino acid of structural and non-structural proteins respectively. Only 29 and 86 different sites in amino acid of structural and non-structural proteins were found when compared to New Guinea-C strain,respectively.DENV-2 AG-I and CG transmitted in southwestern border region of Yunnan and AG-I was prevalent in Ruili in 2013 and2015,and CG was prevalent in Jinghong in 2015. They have genetic diversity but multiple transmission sources were from neighboring countries of Southeastern Asia. It is obvious different in amino acid sites of structural and non-structural proteins between Yunnan strains and New Guinea-C strain. Their main amino acid sites for antigenicity,pathogenic and virulence of both AG-I and CG genotypes were shown without significant difference.更多还原