重庆地区2009年手足口病病原分离鉴定及病毒基因组特征

Identification of enterovirus type 71 isolated from Chongqing children with hand-foot-mouth disease in 2009 and its genomic characteristics

目的对2009年重庆市手足口病(hand-foot-mouth disease,HFMD)患儿粪便标本的病原进行分离鉴定及全基因组序列测定,并同相关毒株序列进行同源性比对和进化分析,以了解新分离病毒基因组序列特征及可能的传播来源。方法将47例手足口病患儿临床标本接种人横纹肌肉瘤细胞(human rhabdomyosarcoma,RD)进行分离培养,采用EV、EV71、Cox A16特异性引物对出现细胞病变的细胞上清进行PCR鉴定,同时电镜观察病毒颗粒形态,将鉴定的病毒基因组分为4个片段进行RT-PCR扩增,扩增产物纯化后进行PCR测序。通过末端重叠序列拼接成全长病毒基因组序列,利用生物信息学软件对序列进行比对分析,绘制进化树。结果在47例HFMD患儿临床标本接种RD细胞培养中,有7份观察到明显的细胞病变,通过RT-PCR检测有3份呈EV特异性引物及EV71特异性引物阳性,而所有标本Cox A16特异性引物检测为阴性,EV71特异性引物的PCR产物测序结果证实为EV71病毒,同时用电镜观察形态病毒感染的RD细胞,为圆形无包膜病毒颗粒,证实所分离病毒为EV71。测序获得的3株EV71病毒基因组全长分别为7 409、7 406 nt和7 404 nt,其VP1氨基酸序列同源性达99%以上。Blast分析表明重庆毒株Chongqing-2-09-China(GQ994990.1)、Chongqing-3-09-China(GQ994991.1)均与安徽阜阳2008年分离的3株EV71毒株同源性较高,重庆毒株Chongqing1-09-China(GQ994989.1)与2005年台湾分离984 polyprotein、1235 polyprotein序列毒株同源性最高,进化分析表明属于C4基因亚型。结论新分离的重庆EV71毒株基因组序列符合肠道病毒特征,与国内2008年安徽阜阳及台湾2005年分离的EV71病毒可能具有相同来源。

Objective To study the genomic sequence characteristics and possible origins of enterovirus type 71 （EVT71） isolated from feces of patients with hand-foot-mouth disease （HFMD） from Chongqing in 2009, and compare the evolution of 3 EV71 strains with similar genome sequences. Methods HFMD specimens were inoculated into human rhabdomyosarcoma （RD） cells for isolation of enteroviruses （EV）. Supernatant of diseased cells was detected by RT-PCR with specific primers of EV, EV71 and Coxsackie A16, respectively. Morphology of virus particles was observed under an electron microscope. The identified virus gene was cut into 4 fragments and amplified by RT-PCR. Complete genome sequences were detected by PCR, analyzed using bioinformatic programs, and plotted into an evolution tree. Results Significant diseased cells were observed in 7 out the 47 specimens from patients with HFMD that were inoculated into RD cells to isolate EV. RT-PCR showed that 3 out of the 7 specimens were positive for the specific primers of EV and EV71, while all the samples were negative for the specific primers of Coxsackie A16. EV71was confirmed to be an EV71 virus by sequencing of the PCR products of its specific primers. The virus particles in infected RD cells were round in shape with no envelope under a scanning electron microscope. The length of 3 EV71 strains was 7 409 nt, 7 406 nt, and 7 404 nt, respectively. Their homologous VP1 sequence reached over 99%. Blast analysis showed that the homology of strains isolated in Chongqing （Chongqing2-09-China and Chongqing3-09-China）was similar with that of strains isolated in Fuyang, Anhui Province （2008）, China. The homology of Chongqingl-O9-China was identical with that of 984 polyprotein and 1 235 polyprotein isolated in Taiwan （2005, China）. Phyiogenefie analysis showed that these strains belonged to subtype of CA gene. Conclusion The newly isolated EV strains have the characteristics of enterovirus and may be of the same origin of those isolated in Fuyang County, Anhui Province （2008） and Taiwan （2005）, China.