惠州市重症手足口病病原谱及EV71型VP1区基因特征分析

Pathogen spectrum analysis of severe HFMD and genetic characterization of VP1 region of enterovirus 71 in Huizhou

目的分析惠州市手足口病重症病例的病原谱构成，了解惠州市肠道病毒71型分离株的VPl区基因特征．为科学防治手足口病提供科学依据。方法采集300例重症手足口病（HFMD）患者标本，采用实时荧光RT-PCR检测肠道病毒核酸，并对肠道病毒7l型（EV71）和柯萨奇病毒A16型（CoxA16）进行分型检测；选择8株EV71分离株进行VPl区基因全长序列测定，测序结果利用DNASTAR软件进行核苷酸、氨基酸序列分析和同源性比较。并用Mega5．0软件构建亲缘性进化树。结果通过实时荧光RT-PCR特异性检测，EV71阳性结果154份，阳性率为51．33％；CoxAl6阳性结果38份，阳性率为12．67％。测序结果表明，8株EV71之间的VPl基因核苷酸同源性为96．9％～99．2％。氨基酸同源性为99．3％～100％。VPI区基因遗传进化分析表明。8株EV71分离株与c4基因亚型的代表株处于同一分支，均属于C4基因亚型的C4a进化分支。结论惠州市手足口病疫情主要病原EV71病毒均属于C4a基因亚型，与2004年以来的中国大陆EV71病毒流行的基因型一致，未产生明显的抗原漂移及变异。

Objective To study the etiological agent of severe hand-foot-and-mouth disease （HFMD）, genetically characterize enterovirus 71 （EV71） isolated from clinical specimens of HFMD patients in Huizhou, and provide scientific basis for control and prevention of HFMD. Methods The virus RNA was extracted from 300 severe HFMD cases. Real-time RT-PCR was performed to detect EV genes, and to identify EV71 and CoxA16. VP1 genes of the eight EV71 isolates were sequenced. DNAStar was used to analyze the degree of nucleotide sequence and amino acid sequence homology, and Mega 5.0 was used to construct the phylogenetic tree. Results Of the RT-PCR, enterovirus 71 （EV71） was detected in 51.33% of the patients （154 of 300）, and CoxA16 was detected in 12.67% （38 of 300）. There was little difference in the nucleotide and the amino acid sequences among the 8 EV71 strains, and the nucleotide and amino acid homogeneity ranged from 96.9% to 99.2% and 99.3% to 100%, respectively. Phylogenetic tree analysis revealed that 8 stains were clustered with reference stains of subgenotype C4 and the Huizhou EV71 isolates were belonged to subgenotype C4a. Conclusion The genotype of Huizhou EVT1 strains belonged to the C4a subgenotype. The genotype of these isolates is in line with the EV71 circulating in China's Mainland since 2004 and there was no obvious antigenic drift or variation.