33株柯萨奇病毒A组16型分离株的全基因序列分析

Whole gene sequencing of 33 human Coxsackie virus A16 isolates

目的对2008～2010年北京和青岛地区流行的33株柯萨奇病毒A组16型(Coxsackie virus A16,CA16)分离株的全基因序列进行分析。方法收集2008～2010年北京和青岛地区CA16感染患者咽拭子标本,采用Vero细胞对病毒进行分离,并经噬斑纯化。提取病毒RNA,采用RT-PCR法分段扩增CA16全长基因,经序列测定和拼接后,利用DNAStar和MEGA5.10软件分析全基因序列。结果经病毒分离和噬斑纯化,共获得33株CA16分离株;33个分离株之间的核苷酸序列同源性大于90.9%,与CA16国际标准株G10各区段的核苷酸序列同源性为72.7%～89.0%,氨基酸序列同源性为79.3%～100.0%;与近年中国分离的CA16 SZ/HK08-7株同源性较高,全基因组同源性大于91.5%,各区段核苷酸序列同源性均大于83.7%;在基于VP1序列的种系进化树中,BJWG16、BJWG17、BJWG20等23个分离株属于C1亚型,其余10个分离株属于C3亚型。结论 2008～2010年北京和青岛地区流行的33株CA16分离株均为C基因型。本研究对我国CA16分子流行病学、毒力位点的研究以及疫苗株的选择具有重要意义。

Objective To analyze the whole gene sequence of 33 human Coxsackie virus A16 （CA16） strains isolated in Beijing and Qingdao Regions, China in 2008 ~ 2010. Methods Throat swabs of patients infected with CA16 were collected in Beijing and Qingdao from 2008 to 2010, from which CA16 strains were isolated with Vero cells and purified by plaque. Viral RNA was extracted, from which full-length CA16 gene was amplified by RT-PCR then sequenced, spliced, and analyzed by DNAStar and MEGA5. 10 software. Results Thirty-three CA16 strains were isolated and purified, between which the homology of nucleotide sequences was more than 90. 9%. However, the homologies of nucleotide and amino acid sequences of the 33 strains were 72. 7% - 89. 0% and 79. 3% - 100% respectively to those of international standard CA16 strain G10. The homology of whole genome sequences of the 33 strains was more than 91. 5% to that of CA16SZ/HK08-7 strain isolated in China in recent years, while that of nucleotide sequences were more than 83. 7%. Phylogenetic tree of CA16 strains based on VP1 sequences showed that 23 CA16 strains including BJWG16, BJWG17 and BJWG20 belonged to C1 genotype, while the other 10 strains belonged to C3 genotype. Conclusion All the 33 CA16 strains isolated in Beijing and Qingdao from 2008 to 2010 belonged to C genotype, which was of an important significance in study on molecular epidemiology and virulence sites of CA16 as well as selection of vaccine strain in China.