摘要目的 对柯萨奇病毒A组16型(coxsackievirus A16,CA16)毒株进行分离、鉴定,并分析其生物学特性,为CA16疫苗候选毒株的筛选奠定基础.方法 从手足口病患者的咽拭子或疱疹液样本中分离CA16毒株,经噬斑纯化后,用逆转录-聚合酶链反应进行病毒鉴定.将病毒连续传代后,对其VP1基因进行序列测定,分析VP1基因和氨基酸序列的同源性和遗传稳定性.结果 通过Vero细胞适应传代和噬斑纯化,获得遗传稳定的CA16毒株,感染Vero细胞后引起典型的细胞病变.病毒分离株经CA16特异性引物扩增后可见208 bp的特异性条带,经VP1基因测序进一步确定这些分离株为CA16.同源性和系统进化树分析表明,各毒株与shzh04-J31株的同源性最高,为92.4％～96.1％,均属于C基因亚型.病毒分离株在Vero细胞上连续传12代后,仅1株病毒的1个氨基酸发生改变.结论 成功分离到CA16毒株,其生物学特性稳定,可用于CA 16疫苗的研发.

abstractsObjective To isolate and identify coxsackievirus A16 (CA16) strains,analyze their biological characteristics,and lay a foundation for screen of candidate CA16 vaccine strains.Methods The CA16 strains were isolated from throat swab or herpes liquid samples of patients with hand,foot and mouth disease,and then determined for specificity by reverse transcription (RT)-PCR after plaque cloning.After continuous subculture,the homology and genetic stability of the isolated strains was evaluated by analysis of VP 1 gene and amino acid sequences.Results Genetically stable CA16 strains were obtained by subculture and plaque cloning in Vero cells.The strains caused a typical cytopathic effect on Vero cells.The product of RT-PCR with CA16-specific primers showed a specific band at 208 bp and VP1 gene sequencing further confirmed that the isolated strains were CA16.The strains were identified as C subtype of CA16 by homology and phylogenetic analysis.The homology of VP1 gene between the isolated strains and shzh04-J31 strain was 92.4％-96.1％.After subculture in Vero cells for 12 passages,only one amino acid variation was found in one strain.Conclusion The CA16 strains with stable biological characteristics are isolated successfully and can be used for development of CA16 vaccine.