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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的全基因组序列(10 661nt),其开放读码框(103-10 264)编码3 386个氨基酸。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-4G-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。
Objective To elucidate the whole genome sequence and molecular epidemiological characteristics of five strains of Dengue 4 virus (DENV-4) in Yunnan Province in 2015. Methods The virus was isolated by the method of C6 / 36 cell culture, and the whole genome sequence of DENV-4 was amplified by RT-PCR. The bioinformatics softwares such as ClastalX1.83 and MEGA6 were used for nucleotide and deduced amino acid sequence homology And system evolutionary analysis. Results Five strains of DENV-4 were isolated from the sera of dengue fever patients in Ruili City, Yunnan province in 2015 (two from local patients, three from imported patients in Lashio and Nam-Kan, Myanmar). The complete genome sequence (10 661 nt) of these 5 strains of DENV-4 was obtained by RT-PCR and sequence analysis. The open reading frame (103-10 264) encoded 3 386 amino acids. Phylogenetic analysis and homology analysis of whole genome or structural protein and non-structural protein gene sequences showed that Yunnan isolates were highly homologous and clustered into one clade and were associated with the prevalence of type I (GI) DENV-4 gene in different generations in Thailand Strains have a closer evolutionary relationship and higher homology, belong to GI. Both Yunnan and Thai strains had lower affinity and homology with the isogenic DENV-4 prototype strain (H241, 1956) and Guangzhou, China, 1990, B5 strain. Yunnan strains and H241 strains in the structural protein or non-structural proteins were 21 and 45 amino acid differences. Conclusion The complete genome sequences of DENV-4 isolates in Yunnan Province were obtained for the first time and found that they are closely related to the recent DENV-4G-I epidemic in Southeast Asia. For the first time confirmed that there is a local epidemic of DENV-4 in Yunnan Province, the source of transmission is adjacent to the northern border region of Myanmar. Whether the change of certain amino acid sites in Yunnan isolate is related to its antigenicity and virulence needs further study.