从手足口病病例分离的两株柯萨奇病毒A组4型毒株的VP_4～VP_2区基因特征分析

Genetic Characterization of VP_4-VP_2 of Two Coxsackievirus A_4 Isolated From Patients with Hand,Foot and Mouth Disease

目的研究2008年甘肃省从手足口病(Hand,Foot and Mouth Disease,HFMD)患儿分离的柯萨奇病毒A组4型(Coxsackievirus A4,CVA4)的VP4～VP2区特征。方法采集门诊就诊的两例HFMD患儿的临床标本,接种人横纹肌肉瘤细胞(Human Rhabdomyosarcoma,RD细胞)分离病毒,然后对阳性病毒分离物使用逆转录-聚合酶链反应法扩增病毒VP4～VP2区,并进行核苷酸序列测定和分析,通过生物信息学的方法进行分子定型,最后与其它16株基因数据库中登录的CVA4株构建亲缘进化树图。结果使用RD细胞成功地从两例HFMD患儿的临床标本中分离到病毒,分子定型鉴定为CVA4。2株CVA4在VP4～VP2区核苷酸同源性为94.6%,氨基酸同源性为100%;与CVA4原型株High Point/USA/1948株的核苷酸和氨基酸同源性分别为85.5%～85.8%和99.3%。在亲缘进化树图中显示,2株CVA4分离株位于独立的进化支中。结论与基因数据库中登录的在日本、蒙古分离的CVA4株相比,从甘肃省2008年HFMD病例分离的两株CVA4,在VP4～VP2区核苷酸序列上差异很大。同源进化分析结果表明,甘肃省CVA4属于独立的进化支。

Objective To study the genetic characteristic of VP4-VP2 of Coxsackievirus A4 （CVA4） isolated from clinical specimens of patients with Hand,foot and mouth disease （HFMD） in Gansu in 2008. Methods Two clinical samples were collected from HFMD patients in outpatient service,and then viral isolation was performed by inoculating RD cells. VP4-VP2 region of two viral isolates were ampliied by using reverse transcription-polymerase chain reaction methods,and then the PCR products were sequenced; molecular typing was used to identify the serotype of these two viral isolates. Finally phylogenetic tree was constructed among these two Gansu viral isolates and 16 CVA4 strains downloaded from GenBank database. Results Two Viruses were isolated from the clinical specimens collected from two HFMD patients by inoculating RD cells,and were identif ied as CVA4 by using molecular typing methods. The homology of the nucleotide sequences in VP4-VP2 region between the two Gansu CVA4 strains was 94.6%,and t.he homology of the amino acid sequences was 100%. Compared with the prototype strain of CVA4 （Strain High Point/USA/1948）,the homologies of nucleotide sequences and amino acid sequences were 85.5%-85.8% and 99.3%,respectively. It revealed that two Gansu CVA4 strains lie in the independence group. Conclusion Compared with CVA4 strains isolated in Japan and Mongolia downloaded from GenBank database,twoGansu CVA4 were found to have great difference on VP4-VP2 region,and phylogenetic analysis revealed that they belong to an independence evolution lineage.