云南省登革3型病毒全基因组序列特征研究

Molecular Characteristics of the Full-length Genome of the Dengue Serotype 3 Virus Strains Isolated from Dengue Fever Cases in Yunnan Province,China

为阐明云南省2013年和2016年登革3型病毒（DENV-3）流行株的全基因组分子进化特征及流行病学特点。采用C6/36细胞培养法从登革热患者血清中分离病毒,用RT-PCR法扩增新分离DENV-3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氨基酸同源性及系统进化分析。结果从云南省本地登革热患者血清中分离到10株DENV-3,其中西双版纳州2013年5株（简称版纳分离株）,德宏州瑞丽市2016年5株（简称瑞丽分离株）。经RT-PCR和序列测定,获得这10株DENV-3的全基因组序列（10 707nt）,其开放读码框（95~10 265）编码3 389个氨基酸。基于全基因组或各种结构蛋白和非结构蛋白基因的系统进化分析表明,版纳分离株均为基因II型（genotype-II）,瑞丽分离株均为基因I型（genotype-I）,并各自高度聚集为同一进化群并与东南亚流行株具有较近亲缘关系。版纳分离株间和瑞丽分离株间的全基因组核苷酸（氨基酸）同源性分别为99.75%~99.91%（99.40%~99.91%）和99.21%~99.68%（98.78%~99.57%）,并与DENV-3原型株（H87）的核苷酸（氨基酸）同源性分别为94.21%~94.34%（97.88%~98.14%）和93.81%~93.98%（97.12%~97.67%）。与H87株比较,本次云南分离株结构蛋白和非结构蛋白分别存在26和63个氨基酸位点的改变。本研究证实,云南省西双版纳州2013年流行的DENV-3为基因II型,瑞丽市2016年流行的DENV-3为基因I型,它们的传播来源分别为老挝和缅甸北部边境地区。本次云南DENV-3分离株与H87株间存在明显差异,但决定病毒抗原性和毒力的关键位点未见明显变化。

To study the molecular-evolution characteristics and the molecular epidemiological feature of the full-length genome of 10 dengue serotype 3 virus（DENV-3）strains isolated from Yunnan Province,China in 2013 and 2016.Dengue viruses were isolated through C6/36 cell culture.The full-length genome of DENV-3 isolates were amplified by reverse transcription-polymerase chain reaction（RT-PCR）and sequenced.The homology and phylogenetic analysis was made upon the nucleotide and deduced amino acid sequences using bioinformatics softwares including ClastalX1.83 and MEGA6 etc.Ten DENV-3 strains were isolated from indigenous cases of dengue fever in Yunnan Province.Of these,five strains（Banna isolates）from Xishuangbanna Prefecture,Yunnan Province in 2013 and five strains（Ruili isolates）from Ruili City of Dehong Prefecture,Yunnan Province in 2016.RT-PCR and sequencing indicated that the fulllength genomes（10,707 nt）of the ten DENV-3 strains were obtained,and the open reading frame（95~10,265）code for 3,389 amino acid residues.Phylogenetic tree analyses based on structural and non-structural protein genes or complete genome showed that five Banna isolates were genotype-II（G-II）and five Ruili isolates were genotype-I（G-I）.Whether Banna isolates or Ruili isolates are highly gathered in a evolution.They have close genetic relationship with the strains isolated from Southeast Asia.Complete genome nucleotide（amino acid）homology of Banna isolates and Ruili isolates were 99.75% ~99.91% （99.40% ~99.91% ）and 99.21% ~99.68% （98.78% ~99.57% ）respectively,and compared with prototype strain（H87）of DENV-3 nucleotide（amino acid）homology were 94.21% ~94.34% （97.88% ~98.14% ）and 93.81% ~93.98% （97.12% ~97.67% ）respectively.Comparing the ten strains from Yunnan Province with H87 strain,there were 26 and 63 different sites in amino acid of structural and nonstructural proteins,respectively.The present study indicated that the DENV-3 G-II was prevalent in Xishuangbanna Prefecture in 2013,and the DENV-3 G-I was prevalent in Ruili City in 2016.The transmission sources of DENV-3 G-II and DENV-3 G-I were from Laos and north Myanmar border respectively.There was a significant difference between H87 strain and Yunnan isolates of DENV-3.However their main amino acid sequence of antigenicity and virulence of both G-II and G-I had no significant change.