2020年文山州手足口病病原监测及柯萨奇病毒A6型、A16型的基因特征分析

Pathogen surveillance and genetic characteristics of coxsackievirus A6 and A16 in children with hand,foot and mouth disease in Wenshan Prefecture of Yunnan Province,2020

目的了解2020年文山州手足口病患儿病原学特征并对柯萨奇病毒A组6型(coxsackievirus A6,CVA6)和CVA16 VP1区基因特征进行分析。方法直接从2020年云南省手足口病实验室监测系统收集的文山州手足口病患儿粪便标本中提取病毒核酸,先用MD91/OL68-1引物扩增肠道病毒(enterovirus,EV)VP4/VP2结合区基因并进行测序,确定病毒型别,再用各型病毒的相关引物扩增VP1区全序并测序,进行基因特征分析。按参考文献下载基因库中CVA6和CVA16 VP1区参考序列,用MEGA5.2软件构建系统进化树,进行基因特征和分子流行病学分析。结果共从317份标本中检测到32株EV,1株星状病毒MLB1型(astrovirus MLB1)。病毒总体检出率为10.41%(33/317),EV检出率为10.09%(32/317),其中肠道病毒A组(enterovirus species A,EVA)31株,占EV阳性数的96.88%(31/32),EVB组病毒1株,占3.12%(1/32),未检测到EVC和EVD组病毒。EV中,检出率最高的病毒是CVA6,占62.50%(20/32),其次是CVA16(18.75%,6/32)、CVA4(9.37%,3/32)、CVA10(3.12%,1/32),未检测到EVA71病毒。基因进化分析表明20株CVA6为D3a亚型,并可分为3个进化分支。6株CVA16为B1a亚型,是中国一直流行的基因型,文山州6株B1a亚型可分为两个进化分支。结论2020年云南省文山州手足口病的病原检测率为10.41%,EV中CVA6是主要的流行毒株,其次是CVA16。基于VP1区基因特征分析表明,2020年文山州手足口病的主要病原是CVA6 D3a亚型,其次是CVA16 B1a亚型,未检测到EVA71。

Objective To analyze the etiological characteristics of hand,foot and mouth disease(HFMD)cases and to investigate the genetic characteristics of VP1 region of coxsackievirus A6(CVA6)and CVA16 strains in Wenshan prefecture of Yunnan Province in 2020.Methods Virus RNA was extracted directly from stool samples of children with HFMD in Wenshan prefecture of Yunnan Province in 2020.Enterovirus(EV)VP4/VP2 junction region was amplified using MD91/OL68-1 primers and sequenced,and then the serotypes of EV isolates were preliminarily identified.Amplification and sequencing of the complete VP1 gene were performed.A phylogenetic tree was constructed by MEGA 5.2 software with the reference strains from GenBank.Genetic and molecular epidemiological characteristics of CVA6 and CVA16 were analyzed.Results Thirty-two strains of EV and one strain of astrovirus MLB1 were detected in 317 specimens with an overall virus detection rate of 10.41%(33/317).Among the 32 EV strains,31(96.88%)were enterovirus species A(EVA)and one(3.12%)was EVB.EVC and EVD were not detected.CVA6 was the predominant EV,accounting for 62.50%(20/32),followed by CVA16(18.75%,6/32),CVA4(9.37%,3/32)and CVA10(3.12%,1/32).EVA71 was not detected.The phylogenetic analysis showed 20 CVA6 strains belonged to D3a sub-genotype and could be further divided into three clusters.Six CVA16 strains belonged to B1a sub-genotype,which was one of the predominant genotypes circulating in China,and could be divided into two clusters.Conclusions The detection rate of pathogens causing HFMD in Wenshan prefecture in 2020 was 10.41%and the most common etiologic agents were CVA6 and CVA16.Based on the genetic analysis of the VP1 gene,the predominant genotype circulating in Wenshan prefecture in 2020 was CVA6 D3a sub-genotype,followed by CVA16 B1a sub-genotype.EVA71 was not detected.