论文部分内容阅读
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 isolate and identify the pathogen of stool specimens from hand-foot-mouth disease (HFMD) patients in Chongqing in 2009 and compare the homologous sequences and phylogenetic analysis with the related strains. To understand the sequence characteristics of the newly isolated virus genome and possible sources of transmission. Methods 47 cases of hand, foot and mouth disease in children were inoculated with rhabdomyosarcoma (RD) isolated from clinical samples. PCR was used to identify the cytopathic cell supernatants by using EV, EV71 and Cox A16 specific primers. Meanwhile, electron microscopy The morphology of the virus particles was observed. The identified virus genome was divided into four fragments and amplified by RT-PCR. The PCR products were purified and sequenced. The full-length viral genome was spliced by the overlapping sequences of the end and the sequences were aligned by bioinformatics software to draw the phylogenetic tree. Results In 47 cases of HFMD inoculated with clinical samples of RD cell culture, 7 were observed significant cytopathic effect, by RT-PCR detected 3 EV-specific primers and EV71-specific primers positive, and all specimens Cox A16 Specific primers were negative, EV71-specific primers PCR products sequencing results confirmed EV71 virus, while morphological virus infected RD cells observed by electron microscopy, round non-enveloped virus particles, confirmed that the virus isolated EV71. The total length of the three strains of EV71 viruses sequenced were 7 409,7 406 nt and 7 404 nt, respectively, and the homology of VP1 amino acid sequence was over 99%. Blast analysis showed that Chongqing strains Chongqing-2-09-China (GQ994990.1) and Chongqing-3-09-China (GQ994991.1) shared high homology with the three strains of EV71 isolated from Fuyang in Anhui in 2008, The Chongqing strain Chongqing1-09-China (GQ994989.1) shared the highest homology with the isolates of 984 polyprotein and 1235 polyprotein in Taiwan in 2005, and the evolutionary analysis indicated that it belonged to C4 subtype. Conclusion The genome sequence of newly isolated Chongqing EV71 strain is consistent with the characteristics of enteroviruses and may be of the same origin as the EV71 virus isolated in Fuyang, Anhui Province and Taiwan in 2005.