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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 investigate the gene sequence of three strains of Coxsackievirus B (CVB3) isolated from clinical samples of HFMD in Jiangsu Province in 2012. Methods 16 samples of throat swabs or anal swabs from 2012 HFMD children in Donghai County and Pizhou City of Jiangsu Province were collected. Three CVB3 viruses were isolated and cultured in Vero cells, and the full-length CVB3 gene was amplified by RT-PCR. Using DNAStar software package MegAlign nucleotide sequence analysis, phylogenetic tree construction, and CVB3 virulence sites were compared. Results Three fragments of whole genome of CVB3 were amplified and cloned. The whole genome of CVB3 was named as DH09Y / JS / 2012, DH16G / JS / 2012 and PZ23Y / JS / 2012 respectively. Homology analysis showed that the homology between the three isolates was 87.6% ~ 98.9%, and the nucleotide homologies with the original Nancy were 80.5%, 80.4% and 81.2%, respectively. All three strains belong to D2 subtype. The homology of VP1 in Pizhou city and Donghai county was 91.8% -92.3%, belonging to D2a and D2b two different branches. In addition to the GUAA / GCAA motifs of 5’UTR and 151 amino acids of VP2, the other three isolates were consistent with the severe CVB3 epidemic strains causing myocarditis and aseptic meningitis in recent years. Conclusions CVB3 of HFMD in Jiangsu Province was D2 subtype in 2012 and D2a and D2b were endemic in different regions. The main virulence sites of this isolate were consistent with those of CVB3 in recent years.