新型冠状病毒在Vero-E6细胞中的增殖特征分析

Characteristics of 2019-nCoV proliferation on Vero-E6 cells

目的了解新型冠状病毒(2019-nCoV)在Vero-E6细胞中的增殖特征,为病毒的分离培养、抗病毒药物研究以及疫苗研制等工作提供基础。方法将2019-nCoV做半对数系列稀释,测定病毒的半数组织培养感染剂量(TCID 50);以1.74×10^-4 TCID 50/cell的病毒量感染Vero-E6细胞,在病毒吸附后第0~6 d分别收集病毒培养上清,测定上清中病毒感染滴度和病毒核酸拷贝数;测定病毒储存液冻融/非冻融、不同细胞悬液浓度、病毒吸附后洗涤/不洗涤等不同实验条件下的TCID 50。结果2019-nCoV感染Vero-E6细胞后,48 h内培养上清中的病毒TCID 50和病毒核酸拷贝数到达或接近峰值,平均复制周期约为3.29 h;48 h后上清中病毒核酸拷贝数维持在较高水平但病毒感染滴度逐渐下降。病毒储存液的冻融、细胞浓度以及病毒吸附后洗涤等培养条件可轻微影响病毒TCID 50。结论2019-nCoV感染Vero-E6细胞在早期增殖迅速,感染后期病毒感染滴度逐渐下降,但病毒核酸拷贝数在上清中维持在较高水平。

This study aimed to explore the characteristics of 2019-nCoV proliferation on Vero-E6 cells and to determine the necessary experimental conditions for viral isolation and culture,antiviral drug research and vaccine development.The TCID 50 of the 2019-nCoV strain isolated from Fujian province was determined,and virus(1.74×10^-4 TCID 50/cell)was inoculated into the Vero-E6 cell-line.The supernatants of infected cells were collected every 24 hours for subsequent viral titration and determination of the number of genomic copies at 0-6 days post-infection.The effects of different experimental conditions on the TCID 50,such as freeze-thaw of viral stock,cell concentrations for viral inoculation and washing after viral adsorption,were explored.The results showed that the 2019-nCoV virus replicated rapidly on Vero-E6 cells in the first 48 hours,and the average replication period was approximately 3.29 h.The nucleic acid con centration and viral titer peaked at approximately 48 hours and thereafter gradually declined,but the nucleic acid concentration remained high.Freeze-thaw of viral stock,cell concentration and washing of the virus-adsorbed cells did not significantly affect the viral TCID 50.