狂犬病病毒CVS-11株全基因序列测定及分析

Sequencing of Complete Genome of Rabies Virus CVS-11 Strain

目的对狂犬病病毒标准攻击毒株CVS-11株进行全基因序列测定,并就主要抗原位点与我国现用狂犬病疫苗生产毒株进行比较,评价CVS-11株作为抗狂犬病病毒中和抗体检测毒株的适用性。方法将CVS-11全基因组RNA分成8段进行RT-PCR扩增,分别将产物克隆入pGEM-TEasy载体中,测定全序列;用DNAStar软件包中的相应软件对基因全序列进行分析,并与5株生产用狂犬病病毒(CTN-1、aG、FluryLEP、PM和PV)进行基因比对分析和主要抗原位点比较。结果 CVS-11株基因组全长11927bp,其结构基因排列与已知其他狂犬病病毒相同,但G和M基因的非编码区偏长。CVS-11株与我国现用疫苗株的序列同源性在84.3%～99.5%之间。以相对保守的N基因作进化树分析,CVS-11株与PM株同源性最高,与CTN-1和aG株的亲缘关系较其他疫苗株远。各毒株G蛋白抗原位点存在一定的氨基酸差异。结论 CVS-11株与我国现用疫苗株具有较高的同源性;CVS-11株与不同疫苗株G蛋白抗原位点的差异可能对不同疫苗免疫效果评价产生不同程度的影响。

Objective To evaluate the adaptivity of CVS-11 strain as the challenge virus in the rapid fluorescent focus inhibition test（RFFIT）for determination of neutralizing antibody against rabies virus on the basis of sequencing the complete genome of CVS-11 strain and comparative research on important antigenic sites among CVS-11 and rabies vaccine strains used in China.Methods Eight overlapped gene fragments were amplified by RT-PCR in order to cover the complete genome and then were cloned into pGEM-T Easy vector respectively.Each gene fragment was sequenced on both strands,based on which sequences were analyzed with DNAStar software,and the results were compared with those of rabies vaccine virus strains CTN-1,aG,FluryLEP,PM and PV for analysis of homology and important antigenic sites.Results The full-length of genome of CVS-11 strain was 11 927 bp.The structural gene arrangement of CVS-11 strain was quite similar to those of other known rabies virus strains except the long non-coding sequences of G mRNA and M mRNA.The homologies of CVS-11 strain to the current rabies vaccine virus strains in China were 84.3% ～ 99.5%.Based on N gene phylogenetic tree,we found the highest homology of PM strain to CVS-11 strain,while CTN-1 and aG strains were more independent relative to CVS-11 strain than the other vaccine strains.Comparative analysis of the important antigenic sites of G protein also indicated a certain amino acid variation between CVS-11 strain and rabies vaccine virus strains.Conclusion CVS-11 strain is highly homologous to the current rabies vaccine virus strains in China.However,the variation of antigenic sites of G proteins between CVS-11 strain and various rabies vaccine virus strains may influence the evaluation of immune effects of various rabies vaccines at different degrees.