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目的:对一例重症手足口病死亡患儿肠道病毒型别进行鉴定,分析VP4区基因特征,了解其变异情况。方法:提取病毒RNA,用肠道病毒VP4区分型引物进行半巢式RT-PCR(RT-nPCR),通过测序和Blast比对确定肠道病毒型别;并对其VP4区进行序列和进化分析。结果:RT-nPCR扩增目的片段测序结果经Blast比对确定为柯萨奇病毒B5型(CoxB5),命名为BJ09-237(Genbank登录号为JF430480)。其VP4区全长207bp,与CoxB5参考序列在VP4区的核苷酸同源性为79%～98%,氨基酸同源性为95%～100%,与广东株HQ005438及俄罗斯株GQ281602在同一进化树分支上。BJ09-237在VP4区无氨基酸的缺失和插入,未发现其所特有的氨基酸置换。结论:该重症手足口病死亡患儿CoxB5的VP4区基因序列未发生明显变异。
Objective: To identify the type of enterovirus in a child with severe HFMD, analyze the gene characteristics of VP4 and understand its variation. Methods: The viral RNA was extracted and the semi-nested RT-PCR (RT-nPCR) was performed with the VP4 differentiating type of enterovirus. The enterovirus type was determined by sequencing and Blast comparison. The sequence and evolution of VP4 . Results: The sequencing results of RT-nPCR amplification were identified as CoxB5 by Blast comparison and named BJ09-237 (GenBank Accession No. JF430480). The full-length VP4 region of 207bp shared 79% -98% homology with the CoxB5 reference sequence in the VP4 region and 95% -100% homology with the Guangdong strain HQ005438 and the Russian strain GQ281602 Tree branch. BJ09-237 in the VP4 region without amino acid deletion and insertion, did not find its unique amino acid replacement. Conclusion: The sequence of VP4 gene of CoxB5 did not change significantly in children with HFMD.