目的 阐明2013年青岛地区手足口病(HFMD)病原谱及肠道病毒71(EV71)型、柯萨奇病毒A16(CVA16)型和柯萨奇病毒A6(CVA6)型青岛分离株的基因特征. 方法 用荧光RT-PCR法检测2013年青岛地区HFMD患者咽拭子标本中的总肠道病毒( EV)、EV71、CVA16和CVA6,对阳性分离株进行VP1基因全长序列扩增、基因序列测定及系统进化分析. 结果 2013年共检测841个手足口病轻症病例和107 个重症病例,肠道病毒阳性率64 .3%,主要病原构成为EV71 ( 44 .8%)、CVA6(28.2%)和CVA16(9.5%). CVA6超过CVA16成为手足口病的第二大致病原,占3岁以下儿童肠道病毒构成的42 .7%,占重症病例分离病原的22 .2%,在手足口病病原构成中所占比例随年龄增长呈下降趋势( P<0 .001 ). VP1全长的系统发生分析显示,2013年EV71属于C4基因亚型中的C4a,CVA16属于B1亚型的B1a和B1b,与其他地区同期参考株无明显差别;CVA6的VP1序列分为A~F 6个基因组,青岛地区CVA6分离株分处A和D基因组,2013年CVA6青岛分离株83 .9%位于A基因组,16 .1%位于D基因组;2012年CVA6青岛分离株66 .7%位于A基因组,33 .3%位于D基因组;2008—2011年CVA6青岛分离株全部位于D基因组. 结论 EV71、CVA6和CVA16是2013年青岛地区手足口病的主要病原;CVA6所占病原构成随年龄增长呈下降趋势;EV71 流行亚型为C4a, CVA16流行亚型为B1a和B1b;CVA6优势毒株在2008—2013年间显示出由D基因组向A基因组的改变.%Objective To investigate the etiology spectrum of hand , foot and mouth disease ( HFMD) and to analysis the molecular characteristics of three predominant human enterovirus stains in Qingdao in 2013.Methods The total enterovirus (EV) strains and strains of EV71, CVA16 and CVA6 in throat swabs of HFMD cases were detected by using multiplex real time RT-PCR.The full-length of the viral VP1 genes of the EV strains were amplified and sequenced .The sequences were phylogenetically analyzed by using the MEGA5.0 software package .Results A total of 841 patients with mild HFMD and 107 patients with serious HFMD were recruited in this study and 64 .3%of them were positive for EV .The predominant pathogens were EV71 (44.8%), CVA6 (28.2%) and CVA16 (9.5%) in 2013.CVA6 replaced CVA16 as the second most common pathogen for HFMD , accounting for 42.7% of all pathogens in children aged less than 3 years and 22.2%of all pathogens in the serious patients .The proportions of CVA6 in the etiology spectrum showed a downtrend along with the increasing age of the patients (P<0.001).Phylogenetic analy-sis of the complete VP1 gene sequences showed that all of the EV 71 strains identified in this study belonged to the subgenotype C4 (evolutionary branch C4a) and all of the CVA16 strains belonged to the subgenotype B1 (evolutionary branches B1a and B1b).There were 6 genogroups (A to F) regarding to the VP1 gene of CVA6 and all of the CVA6 strains identified in this study belonged to genogroups A and D .Among the CVA6 strains isolated in Qingdao in 2013, 83.9% belonged to genogroup A, while the rest 16.1% belonged to genogroup D.66.7%of the CVA6 strains isolated in 2012 belonged to genogroup A, while the rest 33.3%belonged to genogroup D .All of the CVA6 strains isolated from year 2008 to 2011 in Qingdao belonged to genogroup D.Conclusion EV71, CVA6 and CVA16 were the prevalent pathogens responsible for the de-velopment of HFMD in Qingdao in 2013.The proportions of CVA6 strains in the etiology spectrum showed a downtrend with the increasing age in children .C4a was the major subtype of EV71 strains circulating in Qingdao in 2013, while B1a and B1b were the major subtypes of CVA16 strains.The pattern of endemic cir-culation of CVA6 strains showed a trend of changing from genogroup D to A from year 2008 to 2013 .
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