江苏省2012年柯萨奇病毒B组3型分离株全基因序列分析

Sequence analysis of coxsakievirus B3 strains isolated from Jiangsu Province of China in 2012

目的探讨2012年江苏省手足口病（HFMD）患者临床样本中分离得到的3株柯萨奇病毒B组3型（CVB3）病毒的全基因序列特点。方法采集江苏省东海县和邳州市2012年HFMD患儿的咽拭子或肛拭子样本16份,经Vero细胞分离培养得到3株CVB3病毒,利用RT-PCR扩增CVB3全长基因。利用DNAStar软件包的MegAlign进行核苷酸序列分析,构建系统进化树,并对CVB3的毒力位点进行比较。结果扩增得到覆盖CVB3全基因组的3个片段,拼接后为CVB3全基因组,分别命名为DH09Y/JS/2012、DH16G/JS/2012和PZ23Y/JS/2012。同源性分析结果显示,该3株分离株之间同源性为87.6%~98.9%,与原型株（Nancy）的核苷酸同源性分别为80.5%、80.4%和81.2%。3个毒株均属于D2亚型。邳州市分离株和东海县2株毒株VP1区的同源性为91.8%~92.3%,分属于D2a和D2b两个不同分支。3株分离株除5′UTR的GUAA/GCAA模序和VP2的151位氨基酸外,其余毒力位点与近年引起心肌炎、无菌性脑膜炎等重症CVB3流行株一致。结论引起2012年江苏省HFMD的CVB3为D2亚型,不同地区流行D2a和D2b两个分支,此次分离株主要毒力位点与近年来CVB3流行株一致。

Objective To study the genetic characteristics of Coxsakievirus B3（CVB3）viruses isolated from hand foot mouth disease（HFMD）patients of Jiangsu province of China,2012.Methods A total of 16 throat swab or stool swab samples were collected from children with HFMD.Three virus strains were cultured and isolated in Vero cells.Viral RNA was amplified by RT-PCR to obtain the whole genome sequence.The nucleotide identity and sequence alignments were done to generate phylogenetic trees using MegAlign in DNAStar software.Results Three PCR products covering the complete genome of CVB3 and 3virus strains named DH09Y/JS/2012,DH16G/JS/2012 and PZ23Y/JS/2012 were obtained.Sequence alignments showed 87.6%-98.9% homology at the nucleotide level among these 3strains and 80.5%,80.4%,81.2% when comparing with the sequence of Nancy strain,respectively.All 3strains were categorized into D2 subtype.The 2strains（D2a）from Donghai county share 91.8%-92.3% nucleotide homology with the one（D2b）from Pizhou city.The main virulence sites of the 3isolates are identical to the epidemic isolates causing myocarditis,aseptic meningitis or other severe diseases in recent years,except the 5′UTR GUAA/GCAA motif and the VP2 amino acid at position 151.Conclusions The CVB3 strains caused HFMD in Jiangsu province of China belong to D2 subtype.The main virulence sites of the viral strains from Jiangsu province of China are identical to other CVB3 isolates over the years.