摘要
目的：调查2011—2015年安阳地区手足口病（ hand,foot and mouth disease, HFMD）相关人肠道病毒B种（HEV-B）病原组成,对柯萨奇病毒B2（CVB2）和柯萨奇病毒B5（CVB5）VP1基因特征进行研究。方法使用实时荧光逆转录 PCR 和半巢式逆转录 PCR 法对柯萨奇病毒 A16（CVA16）、肠道病毒71（EV71）以及其他肠道病毒进行鉴定,以获得完整的手足口病病原组成,统计出HEV-B病原种类及阳性比例。针对HEV-B病原中阳性病例数较多的CVB2和CVB5病原,分别以其VP1基因为靶段设计引物 PCR扩增测序后获得 VP1基因核苷酸序列全长。使用 MEGA7.0和BioEdit7.2软件对本研究获得的CVB2、CVB5序列以及GenBank数据库已收录的全部相关VP1序列构建系统发育树并进行系统发育分析。使用DateMonkey在线分析程序对我国近年来流行的CVB2和CVB5毒株的VP1基因进行选择压力分析。结果2011—2015年安阳地区共鉴定出手足口病相关HEV-B病原14种,HEV-B阳性标本57份。 HEV-B病原占手足口病全部病原种类的比例为56%, HEV-B阳性标本占总肠道病毒阳性标本的比例为3.06%。绝大多数HEV-B病原引起的手足口病呈高度散发,个别病原如 CVB2、CVB5在特定时间内的病例较为聚集。经过测序获得安阳地区8条CVB2和9条CVB5 VP1序列。构建的系统发育树显示,CVB2毒株可分为A-D四个基因型,各基因型的组间平均进化距离介于0.191-0.208,核苷酸序列相似性介于79.7%-85.8%。 CVB5毒株可分为A-F六个基因型,各基因型的组间平均进化距离介于0.170-0.285,核苷酸序列相似性介于76.0%-86.8%。 CVB2 VP1基因氨基酸序列第157、263位,CVB5 VP1基因氨基酸序列第75、90、95位在不同基因型毒株中的残基有较大差异。安阳地区CVB2序列均属于D基因型,且集中于1个分支内；CVB5序列均属于F基因型,分布于其中2个分支内。中国大陆地区近年流行的CVB2（ D基因型）和CVB5（F基因亚型）毒株的进化选择压力ω值分别为0.037、0.036,未发现CVB2 VP1基因的正选择氨基酸位点,但在CVB5 VP1基因中发现6处正选择氨基酸位点。结论 HEV-B是安阳地区2011—2015年手足口病病原谱的重要组成。 CVB2、CVB5在安阳地区手足口病HEV-B病原中占有一定优势,其中CVB5 VP1基因比CVB2 VP1基因具有更为复杂和活跃的进化特征。
Objective To investigate the serotypes of human enterovirus B （ HEV-B） species cau-sing hand, foot and mouth disease （ HFMD） and to analyze the genetic characteristics of VP1 region in cox-sackievirus B2 （ CVB2 ） and coxsackievirus B5 （ CVB5 ） strains circulating in Anyang area during 2011 to 2015. Methods Real-time RT-PCR and semi-nested RT-PCR were performed to identify coxsackievirus A16 （CVA16）, enterovirus 71 （EV71） and other serotypes of enterovirus in order to obtain the complete etiologic composition of HFMD. The numbers of HEV-B serotypes and the percentages of specimens positive for every serotype in all enterovirus-positive specimens were calculated. As CVB2 and CVB5 were the pre-dominant serotypes of HEV-B species, five pairs of primers targeting the VP1 regions of CVB2 and CVB5 were designed to obtain the complete nucleotide sequences of CVB2 and CVB5 VP1 regions. The phylogenet-ic trees were constructed based on the VP1 sequences obtained in this study and those submitted to GenBank by using MEGA7. 0 and BioEdit7. 2. The selection pressures on VP1 regions of CVB2 and CVB5 strains cir-culating in China in recent years were evaluated with the online program of DataMonkey. Results A total of 57 specimens that belonged to 14 serotypes of HEV-B species were detected in Anyang area from 2011 to 2015. The 14 serotypes of HEV-B species accounted for 56% of all serotypes of enterovirus and the speci-mens positive for HEV-B species accounted for 3. 06% of all enterovirus-positive specimens. The HFMD ca-ses caused by most of the HEV-B serotypes were sporadic cases. Small outbreaks of HFMD could also be caused by some serotypes of HEV-B such as CVB2 and CVB5. The complete sequences of VP1 region were obtained from 8 CVB2 strains and 9 CVB5 strains. The phylogenetic trees based on the VP1 sequences dem-onstrated that the CVB2 strains were classified into four genotypes （ A-D） . The mean evolutionary distances between different genotypes ranged from 0. 191 to 0. 208 and the similarities in nucleotide sequences ranged from 79. 7% to 85. 8%. The CVB5 strains were classified into 6 genotypes （A-F）. The mean evolutionary distances and the similarities in nucleotide sequences between different genotypes of CVB5 strains ranged from 0. 170 to 0. 285 and 76. 0% to 86. 8%, respectively. Strains of different genotypes varied significantly in the residues on positons 157 and 263 in the VP1 region of CVB2 strains and on positions 75, 90 and 95 in the VP1 region of CVB5 strains. All of the CVB2 strains isolated in Anyang area belonged to D genotype and located intensively in one lineage. The CVB5 strains circulated in Anyang area belonged to F genotype and located in two lineages. The selection pressures on CVB2 strains of D genotype and CVB5 strains of F geno-type circulating in China in recent years were 0. 037 and 0. 036, respectively. Six positively selected amino acid sites were found in the VP1 region of CVB5 strains, but no positively selected amino acid site was found in the VP1 region of CVB2 strains. Conclusion HEV-B species was an essential component of the etiologic spectrum of HFMD in Anyang area during 2011 to 2015, of which CVB5 and CVB2 were the predominant se-rotypes. The VP1 region of CVB5 was more complex and active than that of CVB2 over the course of evolution.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2016年第6期435-441,共7页
Chinese Journal of Microbiology and Immunology
基金
河南省科技攻关计划项目（122102310268）
河南省医学科技重大攻关项目（201001015）
河南省卫生科技创新型人才工程中青年科技创新人才
河南省安阳市科技攻关项目（2013042）
关键词
手足口病
肠道病毒
柯萨奇病毒B2
柯萨奇病毒B5
系统发育树
正向选择压力
Hand, foot and mouth disease
Enterovirus
Coxsackievirus B2
Coxsackievirus B5
Phylogenetic tree
Positive selection pressure
