SA14-14-2乙型脑炎病毒Vero细胞疫苗候选株的筛选及其表型和基因型特征

Selection of Japanese encephalitis virus SA14-14-2 Vero cell live vaccine candidate and study on its phenotypic and genotypic characteristics

目的研制以Vero细胞为生产基质的SA14-14-2乙型脑炎(乙脑)减毒活疫苗候选毒株。方法将SA14-14-2株原始病毒在Vero细胞上传代并进行空斑克隆,对克隆株病毒进行Vero细胞培养,使用3周龄小鼠及3日龄乳鼠对脑内致病力进行测定,并进行乳鼠脑内回传、弱毒稳定性实验及免疫力实验等。结果挑选到10个病毒克隆株,通过全面对比后,筛选到1株病毒滴度高(>6.0 lgPFU/ml)且稳定的毒株SA14-14-2 VC_(6)。将该株与SA14-14-2原始株进行主要指标对比,结果表明,VC_(6)株全基因序列与原株相比仅有1个位点发生突变(S66L),对小鼠和乳鼠脑内致病力、弱毒稳定性、免疫原性均与原株相同。结论筛选到的SA14-14-2 VC_(6)株符合中国药典乙脑减毒活疫苗生产毒种的要求。各项主要指标不劣于SA14-14-2生产用毒种,是可应用Vero细胞培养生产的乙脑疫苗候选株。

Objective To develop Vero cell-adapted SA14-14-2 vaccine seed virus candidate for production of Japanese encephalitis(JE)live attenuated vaccine.Methods SA14-14-2 attenuated virus strain was passaged in Vero cell cultures for 3 generations followed by plaque cloning in Vero cell culture.Neuroattenuation,stability and immunogenicity were detected by infecting 3-week and 3-day mice with different strains of JE viruses.Results Ten individual plaque colons were picked up for evaluation and detection.One neuroattenuation stable and high titer(>6.0 lgPFU/ml)clone SA14-14-2 VC_(6) was selected after full comparison.The VC_(6) clone virus was compared with its parental SA14-14-2 strain on major aspects.The results indicated that the complete gene sequence of VC_(6) was similar to SA14-14-2 strain except one single nucleotide mutation(S66L),while the neuroattenuation and stability as well as immunogenicity were all the same as its parental virus SA14-14-2.Conclusion The selected virus strain SA14-14-2 VC_(6) meets requirements for JE live attenuated vaccine in Chinese pharmocopoeia with all major indices non-inferiority to its parental virus SA14-14-2 strain,and is a promising candidate seed virus for development of Vero cell JE vaccine.