2010年内蒙古自治区手足口病病原谱及人肠道病毒71型分子特征研究

The Pathogenic Spectrum of Hand,Foot and Mouth Disease and Molecular Characterizations of Human Enterovirus 71 in Inner Mongolia Autonomous Region in 2010

研究2010年中国内蒙古自治区引起手足口病(Hand foot and mouth disease,HFMD)的病原谱及人肠道病毒71型(Human enterovirus,HEV71)的分子特征。采集内蒙古自治区12个盟市门诊就诊的HFMD患者粪便和咽拭子标本共921份,进行病毒分离,然后利用三通道实时荧光定量PCR法[同时检测HEV71,柯萨奇病毒A16型(CVA16)和人肠道病毒(HEV)]对阳性分离物进行鉴定,对鉴定为其它HEV的阳性分离物进行VP4和VP1编码区扩增及核苷酸序列测定和分析。921份标本共分离出153株病毒,阳性率为16.61%,其中61株为HEV71,占39.87%,82株为CVA16,占53.59%,7株为其它HEV(分别为6株CVB4和1株Ⅱ型脊髓灰质炎疫苗病毒株),占6.53%,3株为腺病毒。重症病例中分离到9株病毒,其中6株为HEV71,3株为CVA16。选取从临床诊断分别为普通型病例、重型病例的HFMD患者临床标本中分离到的32株HEV71代表株进行VP1编码区基因扩增及核苷酸序列测定和分析,与HEV71其它各基因型和基因亚型的代表株构建亲缘性进化树。32株内蒙古HEV71代表株与1998年以来中国大陆HEV71分离株的VP1区核苷酸和氨基酸水平上的同源性都较高,尤其与2008年的北京代表株同源性最高,与C4基因亚型代表株聚为一支,属于C4基因亚型C4a进化分支,但它们之间的核苷酸和氨基酸的同源性略有差异,分别为96.4%～100%和98.14%～100%,与2007年的内蒙古代表株存在一定的差异,核苷酸同源性为96.95%～97.87%。亲缘进化关系树显示,这些HEV71处于不同的簇中,属于多个病毒传播链。2010年内蒙古HFMD的病原谱以CVA16和HEV71为主,重症病例中以HEV71居多。内蒙古流行的HEV71属于C4基因亚型C4a进化分支,并且存在多个传播链,与2008年北京代表株亲缘关系比2007年内蒙古代表株亲缘关系近,说明内蒙古流行的HEV71不是独立进化的,而是与中国流行的HEV71在共同进化。

To study the pathogenic spectrum of hand, foot and mouth disease （HFMD） and the molecular characterizations of human enteroviruses 71 （HEVT1） isolated from the clinical specimens of HFMD pa- tients in Inner Mongolia in 2010. A total of 921 clinical specimens including stools and throat swabs were collected from HFMD patients in outpatient service in Inner Mongolia and then viral isolation was per- formed, the positive viral isolates were identified by using the real-time PCR method （detecting EV, HEV71 and CVA16 in a single tube）, and VP4 and VP1 coding region amplification and sequencing was performed with the viral isolates that were identified as non-HEV71, non-CVA16 HEVs. A total of 153 viruses were isolated form 921 clinical specimens, the positive rate was 16.61%, of which 61（39.87%） were HEV71, 82（53.59%0） were CVA16, 7（6.53%） were other HEVs（6 were CVB4 and 1 was polio vac- cine virus type Ⅱ ） and 3（1.96%） were adenoviruses. Nine viruses were isolated from severe cases, of which 6 were HEVT1 and 3 were CVA16. Thirty two HEV71 isolates were selected from the patients presenting mild symptoms and the patients presenting severe symptoms randomly, and the VP1 coding re- gions of represented HEV71 isolates were amplified and sequenced. Finally the phylogenetic tree was con- structed among the VP1 coding regions of the different genotypes and subgenotypes of HEVT1 strains. The nucleotide acid and amino acid of 32 represented HEV71 strains in Inner Mongolia were closed to HEV71 strains isolated from China's Mainland since 2007, especially from Beijing in 2008, and it showed that all HEV71 strains clustered within the C4a evolution branch of C4 subgenotype. There was slight difference in the nucleotide and the amino acid sequence in VP1 region among the 32 Inner Mongolia HEVT1 strains, the identity were 96.4%100% and 98.14%100%, respectively, and there was a little difference in the nucleotide acid sequence between the HEV71 strains from Inner Mongolia in 2010 and in 2007, the identity was from 96.95%to 97. 87%. thirty two HEV71 strains were in different lineages in the phylogenetic tree, and it indicated that these strains belonged to many different viral transmission chains. HEV71 and CVA16 were the main pathogens of HFMD in Inner Mongolia in 2010 and most severe cases were caused by HEV71. All the HEV71 strains circulated in Inner Mongolia belonged to C4a evolu- tion branch within C4 subgenotype. Phylogenetic analysis revealed that 2010 Inner Mongolia HEV71 strains were located in different lineages, and had more nucleotide identity with 2008 Beijing HEV71 strains than with 2007 Inner Mongolia HEV71 strains. This indicated that Inner Mongolia HEV71 strains had not evolved independently, but co-evolved with the HEV71 strains in other provinces in mainland Chi- na.