5株在L20B细胞复制的非脊髓灰质炎肠道病毒的鉴定及基因特征分析

Identification and Genetic Characteristics of Five Non-polio Enteroviruses(NPEVs) Multiplicated on L20B Cell Line

目的鉴定L20B细胞[转人脊髓灰质炎(脊灰)病毒受体基因的小鼠肺细胞系]支持生长的非脊灰肠道病毒(Non-polio Enteroviruses,NPEV)的血清型,并分析病毒基因特征,探讨其对脊灰病毒(Poliovirus,PV)分离率的影响。方法收集宁夏回族自治区从健康人群和广西壮族自治区从急性弛缓性麻痹病例粪便标本中分离的5株L20B细胞支持生长的NPEVs,对病毒分离物采用逆转录-聚合酶链反应进行VP4编码区基因扩增和核苷酸序列测定和分析,根据序列比对结果确定血清型,再进行全长VP1编码区序列测定和分析,并和基因数据库下载的相应血清型NPEVs的VP1编码区核苷酸序列进行比对和分析,构建基因亲缘性关系树。结果 5株NPEVs均鉴定为柯萨奇A组病毒(Coxsackie Virus GroupA,CVA),其中1株为CVA4,另1株为CVA8,其余3株均为CVA10,均属A组人肠道病毒(Human Enteroviruses,HEV);通过比较全长VP1编码区核苷酸同源性和构建亲缘关系树发现,3个血清型的中国分离株都分别独立成簇,与其他国家分离的相应血清型的NPEVs及原型株病毒差异都较大,是本土流行病毒株。结论所研究的L20B细胞支持生长的5株NPEVs均属A组HEV,包括CVA4、CVA8、CVA10三个血清型。由于L20B细胞对PV的最初接种具有很高的敏感性,因此L20B细胞即使支持某些CVA生长,也不会影响PV分离率。但在近年来中国发生较大规模手足口病(Hand,Foot and Mouth Disease,HFMD)的背景下,由于这些A组HEV同时是HFMD的病原体,对这些病毒的鉴定对PV的鉴别诊断和HFMD的预防控制都有实际意义。

Objective To identify and describe the genetic characteristics of non-polio enteroviruses（NPEVs） which could multiplicated on L20B cell line,and discuss the impact to isolation rate of polioviruses.Methods5 L20B positive isolates were collected from stool samples of patient with acute flaccid paralysis（AFP） and health children in Guangxi Zhuang autonomous region and Ningxia Hui autonomous region.These isolates were analysed using VP4 nucleotide sequencing method.The completed VP1 coding regions were amplified and sequenced.Finally,phylogenetic tree were constructed base on completed VP1 coding regions sequences of these L20B positive isolates and those downloaded form GenBank database.ResultsThe 5 NPEVs were all identified as coxsackieviruses group A（CVA）.Among them,1 was identified as CVA4,1 was identified as CVA8 and 3 were identified as CVA10,and all of them belonged to human enterovirus species A（HEV-A）.Nucleotide homology analysis and phylogenetic analysis base on completed VP1 coding regions sequences showed that all these 3 serotypes of NPEVs clustered independently,and they were all native circulated viruses in China's Mainland.Conclusions5 NPEVs in this study belonged to HEV-A,covered 3 serotypes,CVA4,CVA8 and CVA10.Due to the high sensitivity of primarily inoculation of poliovirus onto L20B cell,the isolation rate of polioviruses could not be affected so much.Hand,foot,and mouth disease（HFMD） outbreaks in China's Mainland in recent years,and it was found that HEV-A is also pathogen of HFMD.Identification of these viruses have current significance to differential diagnosis of poliovirus infection and HFMD control and prevention.