急性出血性结膜炎病原学分子特征分析

Molecular characteristics of etiologic agent of acute hemorrhagic conjunctivitis

目的检测分析引发浙江省急性出血性结膜炎(AHC)爆发流行的病原学及毒株的分子特征。方法采用荧光定量逆转录-聚合酶链反应方法直接从AHC患者眼拭子样本中检测肠道病毒(EV)和柯萨奇病毒A24变异株(CA24v)核酸;用Hep-2细胞分离病毒,对阳性分离物扩增病毒VP1基因和3C区片段,测定核苷酸序列,应用DNA-MAN和MEGA软件进行同源性和进化分析。结果 3株浙江CA24v分离株VP1基因全长915个核苷酸(nt),3C区全长549 nt,均没有nt的插入和缺失;浙江分离株Zhejiang/13/08与CA24v原型株EH24/70在VP1和3C区核苷酸同源性分别为85.3%和86.5%,与2005年新加坡分离株Singapore/DSO-26/05和2007年广东分离株Guangdong/332/07株在VP1区和3C区的核苷酸同源性分别为97.0%～99.1%和96.7%～99.5%;浙江CA24v分离株在3C区进化树的Ⅲ基因亚型B分支上(GⅢ/B),在VP1进化树的CA24v Group2分支上。结论引起2007-2008年浙江省急性出血性结膜炎爆发流行的病原为CA24v GⅢ亚型,该病毒最有可能来源于新加坡。

Objective To identify the etiologic agent of acute hemorrhagic conjunctivitis（AHC） epidemic in Zhejiang province in 2007-2008.Methods The virus nucleotide（nt） of enterovirus（EV） and a variant of coxsackievirus A24（CA24v） were detected with real-time（RT）-PCR.The viruses were isolated from the swab samples of suspected conjunctivitis patients by the Hep-2.The RNAs of the viruses were extracted and amplified by RT-PCR for VP1 gene and 3C.The genes were sequenced and their phylogenetic and homology trees were constructed with DNAMAN and MEGA softwares.Results The complete VP1 gene of CA24v in 3 virus strains is 915 nt.The complete 3C is 549 nt.Both VP1 and 3C nucleotides does not have any insert or deletion.The identity of VP1,3C of Zhejiang/13/08 strain and prototype strain EH24/70 is 85.3% and 86.5%,and that of the strain and Singapore/DSO-26/05 in 2005 and Guangdong/332/07 in 2007 is 97.0%-99.1% and 96.7%-99.5%,respectively.The phylogenetic tree of 3C indicated that CA24v isolated in Zhejiang located in the CA24v Ⅲ genotype B branch（GⅢ/B）.The phylogenetic tree of VP1 gene belonges to the CA24v Group 2.Conclusion AHC epidemic in Zhejiang province in 2007-2008 was caused by CA24v GⅢ.They originated most likely from Singapore.