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目的研究人用狂犬病疫苗株aGV的生物学特性。方法取狂犬病病毒3aG株,接种于Vero细胞适应性传代,按照《中国药典》三部(2010版)要求建立毒种库,并进行全面检定:电镜观察毒种的形态结构,测定毒种的糖蛋白基因序列,直接免疫荧光法检测毒种的毒力,动物试验法和细胞培养法检测外源因子,免疫小鼠检测毒种的免疫原性,小鼠脑内中和试验法鉴定毒种的特异性,进行外周致病性试验,检测单次病毒收获液的效力。检测应用该毒株制备的疫苗的抗原性,并与上市疫苗进行比较。结果 aGV毒种在电镜下呈典型的子弹状,可见病毒包膜、刺突等结构;aGV株与3aG、PV、CTN-1、CVS株及街毒株糖蛋白基因序列的同源性分别为99%、92%、84%、91%、84%;病毒毒力逐代提高,第8～12代毒力稳定在108～109 FFU/ml;该病毒在Vero细胞上具有良好的适应性,外周致病性降低,无外源因子污染;aGV9和aGV12的中和指数分别为1 000和7 079,3个批次的aGV9免疫保护指数分别为38 019、7 943和13 489;中和抗体检测结果显示,试验组与对照组之间中和抗体效价差异无统计学意义(P>0.05)。结论本研究适应的狂犬病病毒aGV株有望作为人用狂犬病疫苗株应用于生产。
Objective To study the biological characteristics of rabies vaccine strain aGV for human use. Methods The rabies virus 3aG strain was inoculated into Vero cells for adaptive passage and the strain library was established according to the requirements of the Chinese Pharmacopoeia (2010 edition). The library was fully tested. The morphological structure of the virus species was observed by electron microscopy, Protein gene sequence, the direct immunofluorescence detection of virulent virulence, animal testing and cell culture assay of exogenous factors, immunized mice to detect the immunogenicity of the virus, mouse brain and neutralization test to identify species Specificity, peripheral pathogenicity test, test the efficacy of single virus harvest liquid. The antigenicity of the vaccine prepared using this strain was tested and compared with the listed vaccine. Results The aGV strains showed a typical bullet shape under electron microscopy. The envelopes and spikes of the aGV strains were observed. The homologies of the aGV strains with those of the 3aG, PV, CTN-1, CVS and street strains were The virulence of virus was increased from generation to generation, and the virulence of generation 8 to generation 12 was stable at 108-109 FFU / ml. The virus had good adaptability in Vero cells, The peripheral pathogenicity was reduced and no exogenous factor was contaminated. The neutralization indexes of aGV9 and aGV12 were 1 000 and 7 079, respectively. The immunoprotection index of aGV9 in three batches were 38 019, 7 943 and 13 489 respectively. Neutralizing antibody Test results showed that there was no significant difference in the titer of neutralizing antibody between test group and control group (P> 0.05). Conclusion The rabies virus aGV strain adapted to this study is expected to be used as a human rabies vaccine strain.