云南省登革4型病毒全基因组序列特征研究

摘要

We investigated the molecular characteristics of the full-length genome of 5 dengue serotype 4 virus (DENV-4) strains isolated in Yunnan Province,China,2015 and their molecular epidemiological feature.Isolation of dengue virus was using C6/36 cell culture method.Viral RNA was extracted from virus isolates,then the full-length genome was amplified by RT-PCR.The homology and phylogenetic analysis was made on the nucleotide and deduced amino acid sequences by bioinfor matics softwares including ClastalX1.83 and MEGA6 etc.Results showed that five strains of DENV-4 isolated from dengue fe ver cases in Ruili City of Yunnan Province in 2015,of these,2 strains from indigenous cases,3 from imported cases from Lashio and Nam Can cities of Myanmar to Ruili of China.RT-PCR and sequencing indicated that the full-length genes (10 661 nt) of 5 DENV-4 strains were obtained,and their open reading frame (103-10 264) were coded 3 386 amino acid residues.Phylogenetic tree and homology analysis based on the comeplete genome or structural and non-structural protein genes showed that the 5 DENV 4 isolates were highly homologous and gathered in an evolution as well as they have a closer genetic relationship with the DENV-4 genotype Ⅰ (G-Ⅰ) strains isolated from Thailand.Results indicated that the Yunnan strains belonged to G-Ⅰ.Yunnan strains and Thailand strains compared with DENV 4 prototype strain (H241,Philippines 1956) and Guangzhou strain (B5,1990) of China and showed low homology and evolutionary relationship.When Yunnan strains compared with H241 strain,there were 21 and 45 different sites in amino acid of structural and non-structural proteins,respectively.This is the first time in Yunnan to obtain full-length genomes sequence of DENV-4 and they have closer evolutionary relationship with DENV 4G-Ⅰ strains from Southeast Asia region in recent years.The autochthonous DENV-4 epidemic in Yunnan was detected for the first time,and the virus transmission sources were from neighboring northern Myanmar.It is necessary to further study that change of the amino acid sites of Yunnan strains of DENV-4 is related to its antigenicity and virulence.%目的 阐明云南省2015年5株登革4型病毒(DENV-4)流行株的全基因组序列特征及分子流行病学特点.方法 采用C6/36细胞培养法分离病毒,用RT-PCR法扩增新分离DENV-4的全基因组序列,采用ClastalX1.83和MEGA6等生物信息学软件进行核苷酸和推导氨基酸序列同源性及系统进化分析.结果 从2015年云南省瑞丽市登革热患者血清中分离到5株DENV-4(本地病例2株,来自缅甸腊戍和南坎市输入性病例3株).经RT-PCR和序列测定,获得这5株DENV 4的金基因组序列(10 661 nt),其开放读码框(103-10 264)编码3 386个氯基酸.全基因组或结构蛋白和非结构蛋白基因序列的系统进化和同源性分析表明,云南分离株间高度同源并聚集为一个进化支,并与泰国不同年代DENV-4基因Ⅰ型(G-Ⅰ)流行株具有较近的进化关系和较高的同源性,同属G-Ⅰ.云南株和泰国株均与同基因型的DENV-4原型株(H241,1956年菲律宾)和中国广州1990年B5株亲缘性和同源性都较低.云南株与H241株在结构蛋白或非结构蛋白中分别存在21和45个氨基酸位点差异.结论 首次获得云南省DENV-4分离株的全基因组序列并发现它们与近期东南亚DENV-4 G-Ⅰ流行株亲缘关系较近.首次证实云南省存在DENV-4本地流行,传播来源为相邻缅甸北部边境地区.云南分离株某些氨基酸位点的改变是否与其抗原性和毒力有关尚需进一步研究.