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目的分析柳州市重症手足口病病原谱构成,了解引起重症手足口病的肠道病毒71型(EV71)VP1的基因特征。方法收集柳州市2015年临床诊断重症手足口病病例标本,使用逆转录聚合酶链反应技术(RT-PCR)和DNA测序技术对病例标本进行肠道病毒核酸检测,并用MEGA 5.0对EV71 VP1基因进行分析。结果 67例临床诊断重症手足口病病例标本中,肠道病毒核酸阳性49例,其中EV71阳性11例、Cox A16阳性3例、Cox A2阳性17例、Cox A4阳性2例、Cox A6阳性9例、Cox A10阳性3例及其它肠道病毒4例;11株EV71毒株VP1核苷酸同源性为94.3%~100.0%,氨基酸同源性为98.3%~100.0%;通过与各亚型代表株进行同源性比较及构建系统进化树发现,11株EV71毒株均属于C4a亚型,在VP1区存在共变异现象的6个氨基酸位点上模式为KADSTV,在3个中和抗原决定簇上,除毒株15LZ-58在163位存在差异外,其余10株毒株均高度保守。结论柳州市重症手足口病病原谱复杂,主要病原体为Cox A2及EV71,其次为Cox A6;病原体EV71均属于C4a亚型,VP1基因片段较保守,没有发生较大的有意突变。
Objective To analyze the composition of pathogenic spectrum of severe hand-foot-mouth disease in Liuzhou and to understand the genetic characteristics of VP1 gene of enterovirus 71 (EV71) that causes severe hand-foot-mouth disease. Methods The clinical samples of severe HFMD in 2015 in Liuzhou City were collected and tested for the presence of enterovirus nucleic acid using reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing techniques. The EV71 VP1 gene was tested using MEGA 5.0 analysis. Results Among the 67 clinically diagnosed cases of severe HFMD, 49 were EV71 positive, of which EV71 was positive in 11, Cox A16 was positive in 3, Cox A2 was positive in 17, Cox A4 was positive in 2 and Cox A6 was positive in 9 , 3 cases of Cox A10 positive and 4 cases of other enteroviruses. The homology of VP1 of 11 strains of EV71 was 94.3% -100.0% and the amino acid homology was 98.3% -100.0% Homology comparison and phylogenetic tree analysis showed that 11 strains of EV71 belonged to C4a subtype. The pattern of KADSTV at 6 amino acid sites with covariate in VP1 was KADSTV. Among 3 neutralizing epitopes On the other hand, the remaining 10 strains were highly conserved except that strain 15LZ-58 was different at position 163. Conclusions The pathogenic spectrum of HFMD is complex in Liuzhou City. The main pathogens are Cox A2 and EV71, followed by Cox A6. The pathogen EV71 belongs to C4a subtype and the VP1 gene fragment is more conservative with no significant intentional mutation.