2009-2015年青岛市手足口病病原中柯萨奇病毒A4型VP1区基因特征分析

Genetic characteristics of the VP1 region of coxsackievirus A4 in HFMD in Qingdao city of China from 2009 to 2015

目的阐明2009-2015年引起青岛市手足口病(hand,foot and mouth disease,HFMD)的病原之一柯萨奇病毒A4型(CV-A4)VP1编码区的基因特征。方法用荧光RT-PCR法对青岛市2009-2015年HFMD患者的咽拭子标本进行总肠道病毒(EV)、柯萨奇病毒A16(CV-A16)型和肠道病毒71(EVA71)型检测,以半巢式反转录PCR扩增非EV-A71,非CV-A16型肠道病毒VP1区部分基因序列,结合序列分析鉴定CV-A4血清型。对全部CV-A4阳性分离株进行VP1基因全长序列扩增及基因序列测定。用MEGA 5.0软件进行遗传进化及分子特征分析。结果青岛市2009-2015年7 967份HFMD患者的咽拭子标本中共分离到CV-A4 39株,分别占各年度肠道病毒总构成比的0.18%(1/541)、0.21%(2/944)、0.29%(2/681)、1.37%(4/292)、2.25%(12/534)、0.75%(7/928)和1.86%(11/591)。遗传进化分析显示,94.87%(37/39)的青岛地区CV-A4分离株位于C2b亚型,5.13%(2/39)位于C1亚型;青岛C2b亚型病毒株VP1区核苷酸一致性为90.7%~100.0%,氨基酸一致性为96.9%~100.0%。结论 CVA4不是引起HFMD的优势病原,C2b亚型毒株是青岛市CV-A4的优势流行株,同时伴有C1亚型毒株的流行。

Objective To analyze the molecular characteristics of the coxsackievirus A4 （CV-A4） isolated from HFMD patients in Qingdao city of China from 2009 to 2015. Methods Throat swabs of HFMD cases were assayed for total enteroviruses （EVs）, EV-A71 and CV-A16 by multiplex real time RT-PCR. RD and hep-2 cells were used for virus proliferation of Non-EV-A71 and non-CV-A16 samples and serotypes of CV-A4 were identified through the sequencing analysis of the partial VP1 gene. The full-length of VP1 gene of the CV-A4 was amplified and sequenced. The sequences were phylogenetically analyzed using MEGA 5.0 software pack- age. Results A total of 39 CV-A4 strains were identified from HFMD patients in Qingdao city. Of all EV specimens, and the proportions of CV-A4 detected from 2009 to 2015 were 0. 18% （1/541）, 0.21% （2/944）, 0.29% （2/681）, 1.37% （4/292）, 2.25% （12/534）, 0.75% （7/928） and 1.86% （11/591）, respective- ly. Phylogenetic analysis of the complete VP1 sequences of all the 39 CV-A4 strains in this study revealed that 94.87% （37/39） CV-A4 strains were sub-genotype C2b while 5.13 % （2/39） sub-genotype C1. The nucleotide identity between CV-A4 strains in sub-genotype C2b were 90.7% to 100.0% and the amino acid identity were 96.9% to 100.0%. Conclusions Sub-genotype C2b was the predominant genotype of CV-A4 circulated in Qingdao city of China in HFMD cases from 2009 to 2015. Sub-genotype C1 co-circulated during this period.