蚀斑法检测流感病毒滴度方法的建立及初步验证

Development and preliminary verification of plaque assay method for detecting the infectious titers of influenza virus

目的建立流感病毒滴度蚀斑检测方法,研究其适用性,为基于Vero细胞基质的四价流感裂解疫苗原液生产过程提供质控手段。方法通过优化覆盖物琼脂糖含量、病毒吸附时间及培养温度等参数,建立蚀斑法检测流感病毒滴度检测的方法,对其进行初步验证,并与鸡胚法进行比较。结果通过对流感病毒培养条件的优化,确定覆盖物中最佳琼脂糖终浓度为1.0%(P=0.001,P<0.05)、最佳吸附时间为90 min(P=0.001,P<0.05),与对照差异具有统计学意义;不同温度(33℃、35℃和37℃)培养条件对病毒滴度的影响差异无统计学意义(P>0.05)。对病毒液重复性试验结果显示,由同一组试验人员连续测定8次,CV在3.73%~7.04%之间;同一组试验人员在不同工作日内对3批甲型(H3N2)病毒液测定,CV在3.46%~4.12%之间;不同试验人员测定结果的CV在1.77%~5.63%之间。表明蚀斑法重复性好、准确度高。蚀斑法与鸡胚法检测病毒滴度分别为7.84 lgPFU/mL和7.24 lgEID_(50)/mL,CV分别为2.90%(<5%)和10.21%。蚀斑法在流感病毒2017—2018年流行株病毒滴度检测中应用,均获得稳定的检测结果。结论建立的蚀斑法简便、稳定且灵敏度高,能够准确地检测流感病毒的滴度,可用于流感疫苗生产过程的质量控制研究。

Objective To develop a titration assay for the infectious titer of flu virus, and study its applicability, for establishing the quality control method of tetravalent influenza lytic vaccine based on Vero cells matrix. Methods The plaque assay(lgPFU/mL) for the infectious titer was developed and verified by optimizing the parameters of agarose content(%), virus adsorption time(h) and culture temperature(℃), compared with chicken embryo method. Results The final optimized agarose concentration in cover layer was 1.0%, the adsorption time was 90 min, with significant difference in comparison with controls(P=0.001, P<0.05;P=0.001, P<0.05), and there was no significant difference in cultural temperature(33, 35 and 37 ℃, P>0.05).The reproducibility test of virus cultures showed that the CV values were in a rang 3.73%-7.04% with the same group of testers measured 8 times continuously, the CV values were in a rang 3.46%-4.12% in 3 batches of influenza A(H3 N2) in different working days, the CV values were in a rang 1.77%-5.63% by different persons, which showed that the method had better repeatability and accuracy. The virus titers of plaque assay and chick embryo assay were 7.84 lgPFU/mL and 7.24 lgEID_(50)/mL, respectively, the CV values were 2.90%(less than 5%) and 10.21%, respectively. Plaque assay method obtained stable results used in detection the titer of influenza virus 2017—2018 epidemic strains. Conclusion The developed plaque assay used on detection the titers of influenza virus is simple, stable, sensitive and accurate. It could be used for the quality control of influenza vaccine bulk production process.