云南中缅边境登革1型病毒全基因组序列特征研究

摘要

目的 阐明云南省中缅边境地区2013-2015年14株登革1型病毒(DENV-1)全基因组序列特征.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-1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 从登革热患者血清中分离到14株DENV-1,其中瑞丽市9株,临沧市3株,昆明市2株.经RT-PCR和序列测定,获得这14株DENV-1的全基因组序列(10 735nt),其开放读码框(95-10 271)编码3 392个氨基酸.系统进化和同源性分析表明,13株为基因Ⅰ型(G-Ⅰ),其中瑞丽和临沧本地病例7株,缅甸输入性病例6株;1株为G-Ⅲ(昆明的印度输入性病例).云南13株G-Ⅰ可分为2个进化群,但均与缅甸、泰国等东南亚流行株具有较近的亲缘关系.云南13株G-Ⅰ的E基因的核苷酸和氨基酸同源性分别为97.02％-100％和98.78％-100％,它们与6株东南亚G-Ⅰ参考株的核苷酸和氨基酸同源性分别为96.53％-99.53％和97.33％-100％,与DENV-1原型株US_Ha-waii核苷酸和氨基酸同源性分别为93.76％-94.45％和95.86％-96.91％.所有云南株和东南亚参考株与US_ Hawaii株在结构蛋白或非结构蛋白的氨基酸位点分别存在44和150个位点差异.结论 云南中缅边境地区2013-2015年流行的DENV-1均为G-Ⅰ,并具有基因多样性特点但均为来自缅甸的多个传播来源.%We investigated the molecular characteristics of the full-length genome of 14 dengue serotype 1 virus (DENV-1)strains isolated in Sino-Myanmar border region in Yunnan Province,China during 2013-2015.Isolation of dengue virus was using C6/36 cell culture method.Viral RNA was extracted from virus isolates,and 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 bioinformatics software including ClastalX1.83 and MEGA6 etc.Results showed that fourteen strains of DENV-1 isolated from dengue fever cases,of these,9 strains from Ruili City of Dehong Prefecture,3 from Lincang Prefecture,2 from Kunming City.RT-PCR and sequencing indicated that the full-length genes (10 735 nt) of 14 DENV-1 strains were obtained,and their open reading frame (95-10 271) were coded 3 392 amino acid residues.The genotypes of DENV-1 were revealed by homology and phylogenetic analysis based on structural and non-structural proteins.Thirteen were genotype Ⅰ (G-Ⅰ) (7 from indigenous cases in Ruili and Lincang and 6 from imported case from Myanmar to Ruili,Lincang and Kunming),and 1 G-Ⅲ from imported case from India to Kunming.The phylogenic analysis indicated that the 13 isolates from Yunnan divided into 2 phylogenic subgroups,and they had a closer genetic relationship with the strains isolated from Southeast Asia.The gene sequences of the 13 G-Ⅰ strains have been acquired,the rate of their nucleotide homology and amino acid homology were 97.02 ％-100 ％ and 98.78 ％100 ％ respectively.Compared with 6 strains from Southeast Asia,nucleotide homology and amino acid homology were 96.53％-99.53％ and 97.33％-100％ respectively.Compared with prototype strain (US_Hawaii) of DENV-1,nucleotide homology and amino acid homology were 93.76％-94.45 ％ and 95.86 ％-96.91％ respectively.Compared with US_Hawaii strain,there were 44 and 150 different sites in amino acid of structural and non-structural proteins,respectively.The G-1 of DENV-1 have been popular in Sino-Myanmar border region in Yunnan,2013-2015.They have genetic diversity but multiple transmission sources were from Myanmar,and should strengthen control cross-border spread of dengue fever in this region.It is necessary to further study that change of the amino acid sites of Yunnan strains of DENV-1 is related to its antigenicity and pathogenicity.