云南省1株分离自手足口病病例的新型肠道病毒EV-A89的分子生物学鉴定
Molecular identification of a new enterovirus EV-A89 from hand,foot and mouth disease patient in Yunnan province
寸建萍 1田炳均 1姜黎黎 1周晓芳 1曹亿会 1杨溪 1李楠 1伏晓庆 1李桂满2
作者信息
- 1. 云南省疾病预防控制中心,云南昆明 650022
- 2. 昆明市疾病预防控制中心,云南昆明 650228
- 折叠
摘要
目的 对云南省1株从手足口病病例中分离得到的新型肠道病毒EV-A89进行分子生物学鉴定,并对其基因特征进行分析.方法 对2022年1-12月云南省手足口病实验室监测网络收集的1448份手足口病病例粪便标本进行病毒分离,将经处理的便悬液接种于人横纹肌肉瘤(human rhabdomyosarcoma,RD)细胞和人喉表皮样癌(human epidermoid carcinoma,Hep-2)细胞,在出现典型的细胞病变效应(cytopathic effect,CPE)后,采用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)对阳性分离物进行VP4/VP2结合区序列和VP1区基因扩增,对扩增产物进行基因测序(Sanger法).用Sequencher 5.4.5软件对测序图谱进行拼接和编辑,分别得到病毒VP4/VP2结合区序列和VP1区序列.对病毒株VP4/VP2结合区序列和VP1区序列进行序列鉴定,得到1株EV-A89(标本编号:B36).用Mega5.2软件的邻位相接法(neighbor jorning method)和Kimura 2参数法(Kimura 2 parameter)构建B36株病毒的基因进化树.结果 1448份手足口病病例粪便标本中,共分离得到126株病毒.对病毒株VP4/VP2结合区和VP1区序列鉴定,得到1株EV-A89.VP4/VP2结合区的序列同源性分析表明,该病毒最接近分离自新疆的1株EV-A89病毒(KSYPH-TRMH22F-XJ-CHN-2011,KT277550,以下简称 KSYPH-TRMH22F),二者的核苷酸(nucleotide,nt)相似性为100.00％,氨基酸(amino acid,aa)同源性为100.00％;该病毒与EV-A89原型株BAN00-10359-BAN-2000(基因库编号:AY697459)的核苷酸相似性为93.82％,氨基酸同源性为99.38％;在VP1区,该病毒也最接近分离自新疆的EV-A89病毒KSYPH-TRMH22F;该病毒与KSYPH-TRMH22F的核苷酸相似性为99.66％,氨基酸同源性为98.99％;该病毒与EV-A89原型株BAN00-10359的核苷酸相似性为97.90％,氨基酸同源性为98.31％.结论 在云南省2022年手足口病病例中检测到1株EV-A89病毒,该病毒为肠道病毒A组的一种新型病毒.
Abstract
Objective To analyze molecular typing and genetic characteristics for a new enterovirus EV-A89 detected from hand,foot and mouth disease(HFMD)patients in Yunnan province.Methods Virus isolation was done for 1 448 stool samples collected from HFMD patients from laboratory surveillance network of Yunnan province from January to December in 2022.The stool suspension was inoculated onto human rhabdomyosarcoma(RD)and human epidermoid carcinoma(Hep-2)cells.After the occurrence of typical cytopathic effects(CPE),the VP4/VP2 junction region and VP1 gene sequences were amplified by RT-PCR and sequenced using Sanger method for the positive samples.The sequences were splited and edited by Sequencher 5.4.5 software and virus sequences in VP4/VP2 junction region and VP1 gene were obtained,from which a new enterovirus EV-A89 was detected(sample ID:B36).The phylogenetic tree of B36 was constructed by Mega softwere version 5.2 using the neighbor joining method and Kimura 2 parameter.Results Altogether,126 virus strains were isolated from 1 448 stool samples and one strain,EV-A89 virus,was identified by sequencing on both VP4/VP2 junction region and VP1 gene.In VP4/VP2 junction region,this virus was mostly closed to an EV-A89 strain isolated from Xinjiang(KSYPH-TRMH22F-XJ-CHN-2011,KT277550,hereafter referred to as KSYPH-TRMH22F).The nucleotide(nt)similarity and amino acid(aa)homology between B36 and KSYPH-TRMH22F were 100.00％and 100.00％respectively.The nt similarity and aa homology between the B36 and the prototype strain of EV-A89(BAN00-10359-BAN-2000,AY697459)were 93.82％and 99.38％respectively.In VP1 gene,the B36 was also mostly closed to the EV-A89 strain isolated from Xinjiang(KSYPH-TRMH22F).The nt similarity and aa homology between B36 and KSYPH-TRMH22F were 99.66％and 98.99％respectively.The nt similarity and aa homology between the B36 and the prototype strain of EV-A89(BAN00-10359-BAN-2000)were 97.90％and 98.31％,respectively.Conclusion A new enterovirus EV-A89 is detected from HFMD patients in Yunnan province,2022.
关键词
手足口病/新型肠道病毒/EV-A89/基因特征分析Key words
Hand,foot and mouth disease/New enterovirus/EV-A89/Genetic characteristics analysis引用本文复制引用
基金项目
国家科技重大专项(2017ZX10103010)
出版年
2024
NETL
NSTL
万方数据