盐城市流感样病例核酸阴性标本中肠道病毒病原谱及VP1基因特征

The pathogenic spectrum and he VP1 gene characteristics of enterovirus in negative influenza-like illness samples from Yancheng City

目的研究盐城市流感样病例(influenza-like illness,ILI)核酸阴性标本中肠道病毒病原谱构成,以及不同基因型肠道病毒的分子进化特征。方法采集2017年9月—2018年1月盐城市ILI咽拭子559份进行流感病毒核酸检测,采用半巢式PCR扩增进行肠道病毒检测,扩增PCR产物经电泳确认后测序,拼接序列与GenBank数据库中的序列BLAST比对进行肠道病毒的分型,采用一步法RT-PCR方法扩增不同基因型肠道病毒的VP1基因并进行测序分析,采用生物信息软件分析不同血清型肠道病毒核苷酸和氨基酸同源性以及基因分子进化特征。结果559份ILI咽拭子中流感病毒核酸阳性率为11.45%;495份流感病毒核酸阴性标本中,肠道病毒核酸阳性检出率7.88%(39份),有CVA2、CVA5、CVA10等3个血清型,以CVA2型为优势血清型(占82.05%)。分子进化分析显示:CVA2型肠道病毒盐城株聚集成簇单独构成一传播分支,位于分支Lineage2基因群,与我国香港重组株431306株(JX867333)不在同一进化分支;CVA5型肠道病毒盐城株聚集成簇,与浙江温州代表株(KP289362)位于同一进化分支;CVA10型肠道病毒盐城株和盐城市2016—2017年手足口病代表株聚集成簇,与国内主流代表株亲缘关系相近,位于进化分支Lineage2基因群。结论盐城市流感流行高峰期流感病毒阴性标本中肠道病毒感染占一定比例,应加强动态监测、完善病毒谱,并重点关注能引起呼吸道重症的血清型。

Objective To study the pathogenic spectrum of the enterovirus in negative influenza like illness(ILI)samples in Yancheng City;to analyze the molecular evolutional characteristics of different serotypes of enterovirus.Methods The throat swab specimens of ILI were obtained from 559 patients from Sep 2017 to Jan 2018 for influenza nucleic acid analysis.The enterovirus was detected by semi nested PCR amplification,the PCR products were confirmed by electrophoresis and sequencing analysis.The spliced sequences were subjected to BLAST analysis in Genbank database for enterovirus typing.The VP1 genes of different serotypes of enterovirus strains were amplified by one step RT-PCR and the PCR products were sequenced.The nucleotide and amino acid homology and gene molecular evolution of different serotypes of enteroviruses were analyzed by bioinformatics software.Results The positive rate of influenza virus nucleic acid in 559 ILI throat swabs was 11.45%.Among the 495 negative specimens,the enterovirus positive rate was 7.88%(39 samples),and three serotypes such as CVA2,CVA5 and CVA10 were identified,and the CVA2 was the dominant serotype(accounted for 82.05%).Phylogenetic trees analysis showed that the clustering Yancheng strains of enterovirus CVA2 consisted of a transmission branch located in the evolutionary branch Lineage2 gene group and were not in the same evolutionary sub-branch of the Hongkong recombinant strain 431306(JX867333).The Yancheng strains of enterovirus CVA5 with Wenzhou representative strain in Zhejiang(KP289362)were clustered in the same evolutionary branch.The Yancheng strains of enterovirus CVA10 and representative strains of hand foot and mouth disease from 2016 to 2017 clustered together,demonstrating close relationship with the domestic mainstream representative strains.Conclusion Enterovirus infection accounted for a certain proportion of influenza negative cases in the peak season of influenza epidemic in Yancheng City.The dynamic enterovirus surveillance in negative ILI samples in Yancheng City should be strenghthend,the pathogenic spectrum should be improved,the enterovirus subtypes cuasing severe respiratory tract infections should be focused in the future.