北京、上海严重急性呼吸道感染儿童中肠道病毒D68的检出与基因特征分析

Molecular Detection and Genomic Characterization of Enterovirus D68 among Children with Severe Acute Respiratory Infection in Beijing and Shanghai

为了解肠病毒D68（EV-D68）在北京、上海地区严重急性呼吸道感染（SARI）儿童中的流行情况和分子特征。本研究采集2008～2010年北京儿童医院SARI儿童呼吸道样品259份,2013～2014年上海复旦大学附属儿科医院SARI儿童呼吸道样品441份,采用套式RT-PCR结合测序方法检测筛选EV-D68阳性样品,设计EV-D68全基因组通用引物,对EV-D68阳性样品进行全基因组扩增,收集NCBI核酸数据库中目前所有的EV-D68全基因组序列进行序列分析,比较EV-D68中国和美国流行株的差异。结果发现：北京地区检测EV-D68阳性样品1例,阳性率为0.4%,上海地区检测EV-D68阳性样品4例,阳性率为0.9%。结合目前中国已上传的所有EV-D68相关序列,分析表明：目前中国地区流行的EV-D68主要属于Clade A2或Clade B2亚型,不同于美国的Clade A1、Clade B1、Clade B4和Clade B5亚型,另外EV-D68部分基因进化分析时出现树形结构冲突现象;上述结果表明目前EVD68在北京、上海地区的SARI儿童群体中的感染率较低（5/700;〈1%）;中国与美国流行的EV-D68毒株属于不同的分支,差异较大,部分序列分析表明EV-D68病毒可能存在组内重组现象。

To understand the prevalence and molecular typing of enterovirus D68 among children with severe acute respiratory infection（SARI）in Beijing and Shanghai,259 respiratory samples were collected from in Beijing during 2008-2010,and 441 respiratory samples were collected in Shanghai city between 2013and2014.All the samples were used for the screening of EV-D68 by nest RT-PCR and sequencing,and then EV-D68-positive samples were used for the complete genome sequencing through overlapping PCR.All available EV-D68full-length genomes collected from GenBank were used for phylogenetic analysis and comparison of EV-D68 types prevalent in China and America.One（0.4%）from 259 respiratory samples in Beijing was positive for EV-D68,and 4（0.9%）among the 441 samples from Shanghai were positive for EV-D68.Phylogenetic analysis of full length genome indicated that the EV-D68 prevalent in Beijing belong to Clade A2 and Clade B2,different from the American popular strains（Clade A1,Clade B1,Clade B4 and Clade B5）.Partial sequence analysis declared phylogenetic conflict among different gene sequences.We concluded that the prevalence rate of EV-D68 among SARI Children in Beijing and Shanghai currently was lower（5/700;〈1%）,and the EV-D68 genotype prevalent in China and America belong to different clusters.Partial sequence analysis indicated that intratypic recombinant events may occur in EV-D68 prevalent in China.