东莞市2014年手足口病病原检测与基因特征分析

Etiology detection and gene characterization analysis of hand-foot-mouth disease in Dongguan in 2014

目的对东莞市2014年手足口病（HFMD）疑似病例样本进行病原学检测,并对引起HFMD的肠道病毒71型（EV71）和柯萨奇病毒A16型（CA16）进行基因特征分析,为东莞市HFMD的防控提供基础资料。方法采用Real timeRT-PCR（实时荧光PCR）方法对HFMD疑似病例样本进行检测,核酸阳性样本上送广东省疾病预防控制中心,进行病毒分离及鉴定,基因扩增并测定VP1基因序列。从Gen Bank中选取EV71和CA16不同基因型参考毒株,利用DNA-STAR软件进行核苷酸、氨基酸序列分析和同源性比较,并用Mega4.0.2软件构建亲缘性进化树。结果共检测HFMD疑似病例样本693份,核酸阳性结果614份,总阳性率为88.60%。其中EV71阳性结果241份,占39.25%,CA16阳性194份,占31.60%,其他肠道病毒（EV）179份,占29.15%。共获得11株EV71分离株和8株CA16分离株的VP1基因全长序列。11株EV71分离株的核苷酸和氨基酸序列同源性分别为94.3%~100.0%和98.3%~100.0%,8株CA16分离株的核苷酸和氨基酸序列同源性分别为89.9%~100.0%和99.3%~100.0%。系统进化树分析显示,11株EV71全部属于C4a基因亚型;8株CA16分离株中有6株属于B1b基因亚型,2株属于B1a基因亚型。结论 2014年引起东莞市HFMD的EV71型病毒流行株为C4a基因亚型,CA16型病毒流行株B1a和B1b亚型并存,并兼有一定比例的其他肠道病毒。

Objective To reveal the pathogen spectrum of hand- food- mouth disease（HFMD）and geneticcharacterization of Enterovirus 71（EV71）and Cosackievirus A16（CA16）isolated from Dongguan in 2014. Methods Specimens of HFMD cases were detected by Real time RT-PCR method. The entire VP1 genes of EV71 and CA16 strains wereamplified by RT-PCR and then sequenced,all by Guang Dong CDC. The phylogenetic analysis between strains isolated andthose downloaded from Gene Bank was done by using the software DNASTAR and MEGA 4.0.2. Results Among 693 samplesof HFMD cases,the overall positive rate of enterovirus was 88.60%（614/693）.Of them,241 samples were positive for EV71（39.25%）,194 were positive for CA16（31.6%）and 179 were positive for other Enterovirus（EV）（29.15%）. Complete genomefrom 11 EV71 strains and 8 CA16 strains were isolated from HFMD patients. The homogeneity of 11 EV71 strains shared94.%-100.0% in nucleotide acid identity and 98.3%-100.0% in amino acid identity,and all strains belonged to EV71 C4 asubgenotype. The homogeneity of 8 CA16 strains shared 89.9%-100.0% in nucleotide acid identity and 99.3%-100.0% inamino acid identity,6 strains belonged to CA16 B1 b subgenotype and 2 belonged to CA16 B1 a subgenotype. Conclusion C4 a subgenotype of EV71,B1 b and B1 a subgenotype of CA16 were the major pathogens for the epidemics of HFMD inDongguan in 2014,while there was also other enterovirus co-existed.