五株登革病毒的分离鉴定及E基因序列分析

Isolation and Identification of Five Dengue Virus Strains and Sequence Analysis of The E Gene

登革病毒作为一种重要的蚊媒传播病毒,威胁全球人类健康,及时甚至实时了解病毒的变异和流行病学规律,对科学防控登革热具有重要意义。因此,本研究利用广东省深圳市发现的登革热患者血清进行病毒分离、鉴定及囊膜蛋白E基因的扩增、测序及生物信息学分析病毒E基因进化特点。结果显示,应用Vero和C6/36细胞培养法从登革热病患血清中分离获得5株登革病毒,利用MEGA7软件对囊膜蛋白E的核苷酸和氨基酸序列进行分析显示,5株均属血清Ⅱ型登革病毒,其中输入性SZ926株和本地株SZ38株同源性为99.5%,均与泰国16681经典毒株关系密切,可能衍生于同一祖先;SZ868株和SZ871株为境外输入性病患分离株,两株病毒核苷酸同源性达到99.9%,说明两株病毒可能源于同一毒株;SZ29株与新几内亚的New Guinea C毒株同源性为99.8%,显示出进化保守、稳定传播的特点。基因型分析显示,SZ926和SZ38为基因型Ⅲ型,SZ868和SZ871为基因型Ⅳ型,SZ29为基因型Ⅰ型。本研究成功分离获得5株血清Ⅱ型登革病毒,分属于3个基因型,且不同基因型的E基因整体变异较低,而且我国存在多种基因型感染的潜在威胁,必须加强防范意识和研究力度。

Dengue virus(DENV),as an important mosquito borne virus,threatens human health all over the world.In order to provide theoretical basis for prevention and control of dengue fever,it is necessary to catch the variation and epidemiology of dengue virus in time or even in real time.Therefore,we isolated the virus from patients'serum and identified the serotypes and genotypes of the isolated strains.The E gene of the isolated strains were amplified and sequenced,and their evolution characteristics were analyzed through bioinformatics methods.The results showed that five strains of dengue virus were isolated from the sera of dengue patients by cell culture in C6/36 or Vero cells.The nucleotide and amino acid sequences of envelope protein E were analyzed by MEGA7 software.Results showed that the homology of imported SZ926 strain and local SZ38 strain was 99.5%,which were closely related to the classic strain of Thailand 16681 and probably derived from the same ancestor.The nucleotide homology of the imported SZ868 and SZ871 strains was 99.9%,indicating that the two strains may be from the same strain.The nucleotide homology of SZ29 and New Guinea C strain was 99.8%,showing the characteristics of evolutionary conservation and stable transmission.Genotypic analysis showed that SZ926 and SZ38 were genotype III,SZ868 and SZ871 were genotype IV,and SZ29 was genotype I.In this study,five strains of dengue virus were successfully isolated and identified as the serotypeⅡ.The mutation rate of E gene was relatively low among different genotypes.Obviously,there are potential threats posed by DENV of various serotypes or genotypes in China,so we must strengthen the consciousness of prevention and research.