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目的分析2010年昆明市引起手足口病(Hand,foot and mouth disease,HFMD)的病原埃可病毒9型(Echovirus 9,E9)分离株MSH-KM812 VP1基因的遗传特征。方法分别采用RD细胞和Hep-2细胞对3份HFMD患儿粪便标本进行病毒分离,应用RT-PCR法分2段扩增病毒VP1基因,并进行序列测定。应用NCBI BLAST软件对所测定的节段序列进行比对;Omiga软件对所测定的节段序列的核苷酸及推导的氨基酸序列进行编辑、比对和拼接;应用Mega 5.05软件进行序列分析,并与15个E9参考株的VP1基因序列进行比较,构建基因系统进化树。结果从3份HFMD患儿粪便标本中分离到1株E9,命名为MSH-KM812,其VP1区的核苷酸长度为888 bp。MSH-KM812株与其他15个E9参考株的核苷酸和氨基酸序列同源性分别为78.6%～98.0%和87.5%～99.3%,与中国分离株的核苷酸和氨基酸序列同源性分别为92.7%～98.0%和97.0%～99.3%,其中与中国江苏分离株M10MF37 China2010核苷酸和氨基酸序列同源性最高,分别为98.0%和99.3%,而与德国分离株Hill的核苷酸和氨基酸序列同源性最低,分别为78.6%和87.5%。基因进化树分析显示,MSH-KM812分离株与中国江苏分离株M10MF37 China 2010和中国山东分离株05189-SD-CHN-2005-E9属于同一个进化分枝。结论 MSH-KM812分离株为E9,与中国其他分离株同属一个进化分枝。
Objective To analyze the genetic characteristics of the VP1 gene of the MSH-KM812 strain of Echovirus 9 (E9) causing hand, foot and mouth disease (HFMD) in Kunming in 2010. Methods The stool samples from 3 HFMD children were isolated by RD cells and Hep-2 cells respectively. The virus VP1 gene was amplified by RT-PCR and sequenced. The NCBI BLAST software was used to compare the determined segment sequences. Omiga software was used to edit, align and splicing the determined nucleotide sequences and deduced amino acid sequences of the segment sequences. Sequence analysis was performed using Mega 5.05 software. The VP1 gene sequences of 15 E9 reference strains were compared to construct the phylogenetic tree. Results One strain of E9 was isolated from 3 stool samples of HFMD patients and named as MSH-KM812. The nucleotide length of VP1 region was 888 bp. The nucleotide and amino acid sequence homologies of MSH-KM812 and other 15 E9 reference strains were 78.6% -98.0% and 87.5% -99.3%, respectively. The nucleotide and amino acid sequence homologies with MSH-KM812 strains in China were Were 92.7% -98.0% and 97.0% -99.3%, respectively. Among them, the nucleotide and amino acid sequences of M10MF37 China2010 with the highest homologies of 98.0% and 99.3%, respectively, With the lowest amino acid sequence identity of 78.6% and 87.5%, respectively. Phylogenetic tree analysis showed that MSH-KM812 isolates belonged to the same evolutionary branch with China Jiangsu isolate M10MF37 China 2010 and China Shandong isolate 05189-SD-CHN-2005-E9. Conclusion The MSH-KM812 isolate is E9, which belongs to the same evolutionary branch as other Chinese isolates.