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目的分析2009年昆明市病毒性脑炎患儿粪便中分离获得的埃可病毒30型(Echovirus30,Echo30)分离株A363/KM/2009全长VP1基因序列特征。方法从疑似病毒性脑膜炎患儿粪便样本中分离Echo30,提取病毒RNA,采用RT-PCR法扩增病毒VP1基因,并进行测序。采用NCBI BLAST软件对所测定的序列进行数据库比对;采用Geneious软件对Echo30分离株全长VP1基因的核苷酸及氨基酸同源性进行分析;采用Mega 5.1软件对A363/KM/2009分离株进行VP1基因分析,并与20个参考株的VP1序列进行比较。结果分离株为A363/KM/2009,其VP1基因的核苷酸长度与其他Echo30一致,均为867 bp;与其他Echo30分离株核苷酸同源性在76.9%~95.7%之间,氨基酸同源性在88.7%~99.7%之间;与中国株的核苷酸和氨基酸的同源性分别为84.5%~95.7%和96.9%~99.7%,其中与中国浙江分离株Echo30/zhejiang/10/4的同源性最高,为95.7%;与广西分离株GX10/15的同源性最低,为84.5%。氨基酸序列分析显示,与其他中国株包括脑脊液分离株相比,未见特异的突变位点;与国外分离株的核苷酸和氨基酸同源性分别为76.9%~86.3%和88.7%~97.3%;基因进化树分析显示,与其他中国株分属不同的两个分支,而与Echo30/zhejiang/10/4和Echo30/zhejiang/4/04(CSF)株亲缘关系较近。结论 A363/KM/2009分离株为肠道病毒Echo30,属于中国两个分支中的一支。
Objective To analyze the sequence of the full-length VP1 gene of A363 / KM / 2009 isolated from the stool of children with viral encephalitis in Kunming in 2009. Methods Echo30 was isolated from stool samples of children with suspected viral meningitis and the viral RNA was extracted. The VP1 gene was amplified by RT-PCR and sequenced. NCBI BLAST software was used for database comparison. The nucleotide and amino acid homology of VP1 gene of Echo30 was analyzed by Geneious software. The A363 / KM / 2009 isolates were analyzed by Mega 5.1 software The VP1 gene was analyzed and compared to the VP1 sequence of 20 reference strains. Results The nucleotide sequence of the VP1 gene was A363 / KM / 2009, which was 867 bp in length. The nucleotide homology with other Echo30 isolates was between 76.9% and 95.7% The nucleotide and amino acid homologies with Chinese isolates ranged from 84.5% to 95.7% and from 96.9% to 99.7%, respectively. Among them, the homologies of Echo30 / zhejiang / 10 / 4 had the highest homology of 95.7%. The homology with Guangxi isolate GX10 / 15 was the lowest, 84.5%. Amino acid sequence analysis showed no specific mutation sites compared with other Chinese strains, including CSF strains. The nucleotide and amino acid homologies with foreign isolates were 76.9% -86.3% and 88.7% -97.3% Phylogenetic tree analysis showed that there are two branches which are different from other Chinese strains, but have closer genetic relationship with Echo30 / zhejiang / 10/4 and Echo30 / zhejiang / 4/04 (CSF) strains. Conclusion The isolate A363 / KM / 2009 is the enterovirus Echo30, belonging to one of two branches in China.