摘要
目的了解手足口病相关病原CVB5病毒安徽分离株VP1基因遗传进化特征。方法用RD和Hep-2两种细胞对采集的手足口病临床病例咽拭子标本进行病毒分离,出现CPE后培养物上清,采用微量中和试验鉴定毒株血清型;抽提病毒RNA,用RT-PCR扩增VP1基因,对扩增产物进行基因序列测定,采用ClustalX2.0和Mega5.0生物软件对测序结果与国内外参考毒株进行基因亲缘性进化分析。结果 2株毒株微量中和试验鉴定为CVB5,基因测序显示VP1区核苷酸长度均为891bp,其核苷酸和氨基酸序列同源性均为100%;基因进化分析显示,安徽分离株与山东、河南和浙江分离毒株亲缘性较近,处于同一进化分支CladeⅠ,核苷酸同源性为92.56%-98.94%,氨基酸同源性为92.93%-100%;与国外参考株及CVB5的原型株（Faulkner/）比对,核苷酸和氨基酸序列同源性分别为78.89%-83.84%和96.81%-98.59%。结论分离到的2株病毒均为肠道病毒CVB5,其VP1区基因和氨基酸序列变异较小,与国内流行参考株同源性高。
Objective To phylogenetically characterize the VP1 gene of Coxsackie B5virus（CVB5）,which is responsible for hand,food,and mouth disease（HFMD）in Anhui Province. Methods Hep-2and RD cells were used to isolate CVB5 from throat swab specimens from patients with HFMD.Cultures exhibiting a cytopathic effect（CPE）were collected and viral nucleic acids were extracted.Reverse transcription-PCR（RT-PCR）was performed to amplify the VP1 gene of CVB5.The PCR products were directly sequenced by Shanghai Giant Lifetech,Co.（Shanghai）.Sequence alignments were analyzed with ClustalX2.0software,and a phylogenetic analysis was performed with the software Mega5.0using the neighbor-joining method. Results Two CVB5 strains were confirmed with a micro-neutralization test（MNT）.The entire VP1 gene was successfully amplified using RT-PCR with a predicted product of 907 bp from cell cultures.Furthermore,sequence determination revealed that two viral isolates exhibited 100% similarity in their nucleotide and amino acid sequences.Phylogenetic analysis indicated that the CVB5 strains isolated from Anhui and Shandong were closely related to isolates from Henan and Zhejiang.The CVB5 strains isolated from Anhui and Shandong were in the same evolutionary branch,clade I.The CVB5 strains isolated from Anhui and Shandong had a nucleotide similarity of 92.56%-98.94% and an amino acid similarity of 92.93%-100%.In comparison to foreign reference strains and the prototype strain of CVB5（Faulkner）,the CVB5 strains isolated from Anhui and Shandong had a nucleotide similarity of 78.89%-83.84%and an amino acid similarity of 96.81%-98.59%. Conclusion Two viral strains isolated from Anhui Province were enterovirus CVB5.There was little variation in the VP1 gene and amino acid sequence of these strains,and these strains are highly homologous to reference CVB5 strains that are prevalent in China.
出处
《中国病原生物学杂志》
CSCD
北大核心
2014年第11期979-981,1028,共4页
Journal of Pathogen Biology
关键词
手足口病
CVB5
VP1基因
遗传分析
Hand
foot
and mouth disease（HFMD）
Coxsackie B5
VP1gene
phylogenetic
