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目的了解龙岩市手足口病病原体柯萨奇B5病毒(Coxsackie virus B5,CVB5)VP1区基因特征及变异情况。方法对2011年采自确诊手足口病患者的Real-time RT-PCR法检测肠道病毒通用引物阳性而肠道病毒71型(enterovirus 71,EV 71)和柯萨奇病毒A 16型(Coxsackie virus A 16,CVA16)阴性未分型样品,经RD细胞分离肠道病毒。从培养上清中提取RNA核酸,经RT-PCR法初步鉴定病毒型别。应用187/222引物对扩增病毒株的VP1区,回收产物经克隆、筛选后测序。采用ClustalX、Mega5.0进行核苷酸及氨基酸序列同源性分析和进化树构建。结果 104份未分型样品中分离到3株CVB5。3株CVB5分离株VP1区长度为330~357个核苷酸,编码110~119个氨基酸。3株间的核苷酸同源性为97%~99%,氨基酸同源性为84%~99%。比较推导的氨基酸序列,FJLY2011126h株VP1与另外2株龙岩分离株相比有18个位点差异,核苷酸序列287~288位发生插入突变,第290、291、338、339位发生颠换,与2010年长春株JN695051的同源性最高(核苷酸同源性95%~98%,氨基酸同源性84%~99%)。结论 2011年福建省龙岩市手足口病患者中存在CVB5感染,病毒与2005年法国株和国内山东、长春、昆明、浙江病毒株属同一型别。
Objective To investigate the characteristics and variation of VP1 gene of Coxsackie virus B5 (CVB5), a pathogen of HFMD in Longyan City. Methods Real-time RT-PCR was used to detect the positive results of enterovirus 71 and enterovirus 71 (EV 71) and Coxsackie virus A 16, CVA16) negative undifferentiated samples were isolated by RD cells enterovirus. RNA nucleic acid was extracted from the culture supernatant and the virus type was initially identified by RT-PCR. The VP1 region of the virus strain was amplified by primer pair 187/222. The recovered product was cloned and screened for sequencing. ClustalX, Mega5.0 nucleotide and amino acid sequence homology analysis and phylogenetic tree construction. Results Three isolates of CVB5 isolated from 104 undifferentiated samples of CVB5 were 330 to 357 nucleotides in length and encoded 110 to 119 amino acids. The homology between the three strains was 97% -99%, and the amino acid homology was 84% -99%. Compared with the deduced amino acid sequence, VP1 of FJLY2011126h strain had 18 loci differences compared with the other two Longyan isolates, the insertion mutation of nucleotide sequence from 287 to 288 occurred, the mutation of 290,291,338,339 occurred, And 2010 Changchun strain JN695051 the highest homology (nucleotide homology 95% ~ 98%, amino acid homology 84% ~ 99%). Conclusions CVB5 infection is present in HFMD patients in Longyan City, Fujian Province in 2011. The virus belongs to the same genus as the 2005 French strain and the domestic Shandong, Changchun, Kunming and Zhejiang strains.