龙岩市手足口病病原体柯萨奇B5病毒基因特征分析

Genetic characteristics of Coxsackie virus B5 isolated from patients with HFMD in 2011 in the City of Longyan

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 examine the variation and phylogenetic features of the VP1 region of coxsackie virus B5（CVB5）isolated from patients with HFMD. Methods One hundred and four specimens from patients with HFMD were collected in 2011.All samples were subjected to real-time RT-PCR with universal or type-specific primer pairs.Samples that tested positive for enterovirus but negative for enterovirus 71（EV71）and Coxsackie virus A16（CVA16）in preliminary nucleotide detection were chosen and cultured in RD cells for viral recovery.Viral RNA was extracted from the culture supernatant and was used for viral typing via RT-PCR.The entire VP1 region of CVB5 was amplified using RT-PCR with primers 187/222 and it was then cloned and sequenced.The homology of nucleotide and amino acid sequences was analyzed and a phylogenetic tree was constructed with the software Clustal X and Mega5.0. Result Result Three strains of CVB5 were isolated from 104 specimens testing negative for EV71 and CVA16.The length of the viral VP1 region ranged from 330 to 357nucleotides（nt）,and the region encoded 110-119 amino acids（aa）.The three CBV5 isolates had an nt identity of 97%-99% and an aa identity of 84%-99%.Based on the derived amino acid sequences,the VP1 of FJLY2011126hhad mutations at 18 sites compared to the VP1 of the other two isolates,and these mutations included insertion mutations at positions 287-288 and transversion mutations at positions 290,291,338,and339.The VP1 of FJLY2011126hwas most homologous to a 2010 Changchun strain（JN695051）,as indicated by an nt identity of 95%-98%and an aa identity of 84%-99%. Conclusion The CVB5 virus was present in patients with HFMD in the City of Longyan,Fujian Province.The virus studied is the same type as the strain isolated in France in 2005 and domestic strains isolated in Shandong,Changchun,Kunming,and Zhejiang.