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目的对引起聊城地区手足口病(HFMD)流行的肠道病毒CVA16进行生物学分析,掌握其基因特征,为聊城地区手足口病的防控提供病原学资料。方法对2010~2013年聊城市各辖区采集手足口病患儿标本采用Real-time PCR法检测肠道病毒CVA16,用人横纹肌瘤(RD)细胞对核酸检测阳性标本进行病毒分离,对分离到的CVA16病毒分离株采用RT-PCR方法进行VP1区基因扩增,并对扩增产物进行序列测定,利用DNAStar和MEGA 4.0分析软件进行同源性分析和构建系统进化树。结果 2010~2013年聊城市各辖区共送检手足口病标本862份,639份标本检测为通用肠道病毒核酸阳性,其中CVA16阳性标本181份,分离到109株毒株;对毒株进行基因序列分析显示,2010~2013年聊城市流行的CVA16病毒株为B1基因型,2010和2011年为B1a和B1b两种基因亚型共循环,2012和2013年为B1b型,不同毒株VP1区基因序列在核苷酸和氨基酸水平上同源性分别为91.0%~97.0%和98.3%~100%,与近源的亚型代表株相比,聊城市优势毒株的氨基酸变异一般发生在VP1第23位(L→M)。结论聊城市2010~2013年手足口病CVA16流行毒株为B1基因型,存在B1a、B1b两个基因亚型,同源性分析表明辖区部分CVA16分离株B1a亚型与我国台湾及周边国家的分离株关系较近,B1b亚型与来自国内北方分离株形成进化树上关系较近的一群。聊城市存在不同的传播链,流行的CVA16相对保守,但已出现一定程度的变异。
OBJECTIVE: To analyze the biological characteristics of CVA16, an enterovirus that causes HFMD in Liaocheng area, and to study its genetic characteristics to provide etiological data for prevention and control of HFMD in Liaocheng. Methods Real-time PCR was used to detect CVA16 in children with hand-foot-mouth disease in different districts of Liaocheng from 2010 to 2013. The positive samples of human rhabdomyosarcoma (RD) were isolated by virus isolation. The isolated CVA16 The VP1 gene was amplified by RT-PCR and sequenced. The DNAStar and MEGA 4.0 softwares were used to analyze the homology and construct the phylogenetic tree. Results A total of 862 hand-foot-mouth disease samples were sent from Liaocheng in 2010-2013. A total of 639 samples were tested positive for universal enterovirus, of which 181 were positive for CVA16 and 109 were isolated. Sequence analysis showed that the CVA16 strain prevailing in Liaocheng City in 2010-2013 was B1 genotype, B1a and B1b were sub-circulated in 2010 and 2011, B1b in 2012 and 2013, and the VP1 gene of different strains The nucleotide and amino acid homology of the sequence was 91.0% -97.0% and 98.3% -100%, respectively. Compared with the near-subtype representative strains, the amino acid variation of dominant strains in Liaocheng generally occurred in VP1 23 (L → M). Conclusion The prevalence of B1 and B1b genotypes in CVA16 from 2010 to 2013 in Liaocheng city is high, and the homology analysis shows that the subtypes B1a subtype of CVA16 isolates in the area are separated from Taiwan and its neighboring countries in China The strain B1b is closely related to the phylogenetic tree from the northern isolates in China. Liaocheng City, there are different transmission chain, the prevalence of CVA16 is relatively conservative, but there have been some degree of variation.