ECHO20病毒株KM/EV20/2010的分离鉴定及其VP1基因序列特征分析

目的分离并鉴定2010年云南省昆明市甲型肝炎患儿粪便标本中混存的ECHO20(Echovirus 20,ECHO20)KM/EV20/2010病毒株,分析其VP1基因遗传特征。方法采用KMB17细胞对大便标本进行病毒分离;采用微量细胞病变法,经WHO肠道病毒组合血清及单价血清对该分离株进行鉴定;采用RT-PCR法扩增病毒VP1基因并进行序列测定,并运用Mega 4.0等软件进行分析处理。结果分离毒株经微量中和试验鉴定为ECHO20血清型,编号为KM/EV20/2010;经RT-PCR法扩增获得为867bp的VP1基因,与国内分离株核苷酸和氨基酸的同源性分别为79.5%~91.7%和98.9%~99.6%,与国外分离株的核苷酸和氨基酸同源性分别为80.2%~83.2%和96.4%~99.6%;在进化树上云南分离株与GenBank中登录的中国国内2001年山东分离株01352和1998年法国分离株94CF1666形成一个进化分支。结论 KM/EV20/2010分离株为肠道病毒ECHO20型病毒,可用KMB17人二倍体胚肺成纤维细胞分离增殖。

Analysis of the complete VP1 nucleotide sequence of the KM/EV20/2010 strain of echovirus 20 isolated from Kunming in 2010

Objective To analyze the genetic characteristics of the VP1 gene of Echovirus 20 isolated from the fecal specimens of a child infected with hepatitis A virus in Kunming in 2010. Methods The KM/EV20/2010 strain was iso- lated from fecal specimens using human KMB17 diploid cells, and its serotype was identified via a neutralization test using a WHO panel of sera for enterovirus typing and sera with a single strain of enterovirus. Viral RNA was extracted and then the VP1 gene was amplified using RT-PCR and sequenced. The sequence of the VP1 gene was compared to that of other strains of Echovirus 20 in GenBank using Mega4.0 software. Results The isolate was identified as an ECHO 20 serotype of enterovirus and the isolate was designated KM/EV20/2010. Its complete VP1 gene had 867 bp. There were no missing and inserted uucleotides. The strain＇s nucleotides had similarity of 79.5 %- 91.7% to those of the Echovirus 20 strains isolated from Shandong Province, China and its amino acids had similarity of 98.9%--99.6% to those of the Echovirus 20 strains isolated from Shandong Province. Phylogenetic analysis based on the complete VP1 sequence revealed that the complete VP1 sequence of KM/EV20/2010 was clustered in a distinct group with strains 94CF1666 and 01352, which were respectively isolated from France in 1998 and Shandong, China in 2001. Conclusion KM/EV20/2010 is a strain of Echovirus 20. This strain can be isolated and grown in human KMB17 diploid cells.