柯萨奇病毒A16型VP1基因和病毒生物物理学分析

Characterization of the VP1 region and biophysics of Coxsackievirus A16

目的分析柯萨奇病毒A组16型（CoxA16）西安分离株VP1基因序列,探讨西安分离株基因亚型以及病毒颗粒的生物学特性。方法采集临床诊断为手足口病患儿的粪便标本,RTPCR方法检测粪便标本,CoxA16核酸检测阳性的粪便标本经处理后接种于RD细胞分离病毒。扩增CoxA16分离株VP1基因序列并测序。利用DNA Man、ClustalX 1.83和MEGA 4.0软件分析VP1基因和氨基酸序列。纯化CoxA16病毒颗粒,福尔马林灭活后电镜观察病毒颗粒的形状。结果成功从粪便标本中分离CoxA16病毒株,并获得CoxA16分离株VP1基因序列,该核苷酸编码区序列长度为891 bp,编码297个氨基酸序列;VP1基因比对显示,该分离株与其他不同地区的代表株核苷酸和氨基酸同源性分别为76.76%-97.57%和92.16%-100.0%;种系进化分析显示分离株属于B1a基因亚型的一个分支,与BJ10-03同属一个分支。透射电镜下病毒颗粒为20面体立体对称,病毒颗粒大小均匀,直径为20-30nm。结论西安CoxA16分离株属于B1a基因亚型,分离株在形态上与其他肠道病毒相似。

Objective To analyze the characteristics of VP1 gene sequences of Coxsackievirus A16（CoxA16） isolated from Xi＇an in Shanxi Province People＇s Hospital in 2011,and to identify the genotypes and investigate the virus biophysics of the CoxA16.Methods The stool samples were collected from children with hand,foot and mouth diseases（HFMD） in our hospital,and all of the stool samples were tested by RT-PCR.In order to obtain CoxA16 strains,CoxA16 positive samples were isolated by RD cell line.The nucleotide sequences of CoxA16 strain＇s VP1 were amplified and sequenced,then the nucleotide and amino acid sequences of CoxA16 strain＇s VP1 were analyzed by DNA Man,ClustalX 1.83 and MEGA 4.0.The CoxA16 virus particles were purified by ultracentrifugation,and the virus particles inactivated with formalin were analyzed by transmission electron microscopy（TEM）.Results CoxA16 strain was isolated from stool samples successfully,and the genome of VP1 region had 891 nucleotides in length and encoded for 297 amino acids.VPl gene comparison showed that the rates of homology of nucleotides and amino acids were76.76%-97.57%and 92.16%-100.0%,respectively,when compared with other reference strains from different regions.Phylogenetic analysis showed that CoxA16 isolates strain of Xi＇an was subgenotype B1 a,which was closely related to BJ10-03 strain.TEM analysis showed that CoxA16 strain were the icosahedral structure,and the diameters of CoxA16 strain were approximately 20-30 nm.Conclusions CoxA16 in Xi＇an isolates strain was subgenotype B1 a,and the biophysical characterization of CoxA16 virus particles were very similar to enterovirus of the piconaviridae family.