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目的探究肠道病毒71型(EV-A71)FY23-K株在不同温度下连续传代对其生物学特性的影响,旨在获得EV-A71的减毒候选株。方法在33℃条件下进行FY23-K株克隆筛选,之后分别在33℃及35℃下连续传代,并进行病毒生长增殖特性、乳鼠致病性及在小鼠体内的免疫原性检测,对比分析其各项生物学特性的变化。结果挑选出感染性滴度较高的6个克隆,在33℃条件下连续传代后发现,其感染性滴度逐渐降低,且相同接种条件下,达到75%病变程度的收获时间随代次的增加逐渐延长,至26代后,接种至第8天细胞病变程度只达到40%。而在35℃条件下连续传代的病毒能够稳定增殖,保持良好的生长特性,毒株感染性滴度>6.5lg CCID50/ml;35℃传代后毒株的免疫原性良好,6株克隆株有4株样品的灭活抗原免疫小鼠后抗体的阳转率可达100.0%,GMT可达1∶128,最高的中和抗体效价为1∶512;活病毒免疫小鼠抗体阳转率达88.9%以上,中和抗体效价均>1∶16;传代样品攻击乳鼠后,乳鼠无发病、死亡情况,其脑、脊髓也未产生明显病理性损伤,仅在肌肉组织细胞间隙发现少量的炎性细胞浸润,在脑前额叶出现数个胶质小结。结论 FY-23K克隆株在35℃低温连续传代后有稳定的生长特性和良好的免疫原性,对乳鼠未表现出明显致病性,可作为EV-A71减毒活疫苗进一步研究的候选毒株。
Objective To investigate the effect of continuous passage of Enterovirus 71 (EV-A71) strain FY23-K on its biological characteristics at different temperatures in order to obtain the attenuated candidate strain of EV-A71. Methods FY23-K strain was cloned and selected at 33 ℃, and then subcultured at 33 ℃ and 35 ℃ respectively. The virus growth and proliferation, the pathogenicity of the virus and the immunogenicity in mice were detected. Analysis of its biological characteristics of changes. Results Six clones with high infectious titer were selected and their infectious titers were gradually decreased after continuous passage at 33 ℃. Under the same conditions of inoculation, the harvesting time reaching 75% Increased gradually extended to the 26th generation, until the eighth day after vaccination, the degree of cytopathic effect only reached 40%. However, the virus which was continuously passaged at 35 ℃ could proliferate steadily and maintain good growth characteristics. The infectious titer of the strain was> 6.5lg CCID50 / ml. The immunogenicity of the strain after 35 ℃ subculture was good. After immunized mice with 4 inactivated samples, the positive rate of antibody reached 100.0%, the GMT reached 1:128, and the highest neutralizing antibody titer was 1: 512. The rate of positive antibody immunization in mice was up to More than 88.9% neutralizing antibody titers> 1:16. After the passage samples attacked the suckling mice, the suckling mice had no morbidity and mortality, and no significant pathological damage was found in the brain and spinal cord. Only a small amount was found in the intercellular spaces of muscle tissue Inflammatory cell infiltration in the prefrontal lobe appeared several glial nodules. Conclusion The FY-23K clone has stable growth characteristics and good immunogenicity after being continuously passaged at low temperature of 35 ° C. It has no obvious pathogenicity to the suckling mice and can be used as a candidate for further study of live attenuated EV-A71 vaccine Strain.