1. Fujian Center for Disease Control and Prevention, Fujian Provincial Key Laboratory for Zoonoses Research, Fuzhou 350001, China; 2. School of Public Health, Fujian Medical University, Fuzhou 350108, China
Abstract：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 TCID50 of the 2019-nCoV strain isolated from Fujian province was determined, and virus (1.74×10-4TCID50/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 TCID50, 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 concentration 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 TCID50.
[1] 中国疾病预防控制中心新型冠状病毒肺炎应急响应机制流行病学组. 新型冠状病毒肺炎流行病学特征分析[J]. 中华流行病学杂志,2020,41(2):145-151. DOI:10.3760/cma.j.issn.0254-6450.2020.02.003 [2] Zhao S,Lin Q,Ran J,et al.Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV)in China,from 2019 to 2020:a data-driven analysis in the early phase of the outbreak[J]. Int J Infect Dis,2020,92:214-217. DOI:10.1016/j.ijid.2020.01.050 [3] Lu R,Zhao X,Li J,et al.Genomic characterisation and epidemiology of 2019 novel coronavirus:implications for virus origins and receptor binding[J]. Lancet,2020,395(10224):565-574. DOI:10.1016/S0140-6736(20)30251-8 [4] 张炎华, 陈炜, 何文祥,等. 福建省新型冠状病毒的分离和全基因组特征分析[J]. 中国人兽共患病学报, 2020,36(5): 349-353.DOI:10.3969/j.issn.1002-2694.2020.00.059 [5] 国家卫生健康委办公厅. 国家卫生健康委办公厅关于印发新型冠状病毒肺炎防控方案(4版)的通知 [EB/OL].(2020-02-06)[2020-03-22]. http://www.nhc.gov.cn/xcs/zhengcwj/202002/573340613ab243b3a7f61df260551dd4.shtml [6] 鄢心革,冉延超,李晖,等. SARS冠状病毒在Vero-E6细胞中的生活周期分析[J]. 中华微生物学和免疫学杂志,2004,24(9):698-698. DOI:10.3760/j:issn:0254-5101.2004.09.015 [7] Sims A C,Burkett S E,Yount B,et al.SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium[J]. Virus Research,2008,133(1):33-44. DOI:10.1016/j.virusres.2007.03.013 [8] Keyaerts E,Vijgen L,Maes P,et al.Growth kinetics of SARS-coronavirus in Vero E6 cells[J]. Biochem Biophys Res Commun,2005,329(3):1147-1151. DOI:10.1016/j.bbrc.2005.02.085 [9] 卫生部.手足口病预防控制指南(2008年版)[EB/OL].(2008-05-02)[2020-03-22]. http://www.nhc.gov.cn/wjw/gfxwj/201304/72a8f68053d742f392d8ff96bd9692fe.shtml [10] 刘鹏,李佳林,马超,等.流感病毒在Vero细胞系与MDCK细胞系增殖条件的比较[J]. 微生物学免疫学进展,2012,40(5):24-27. DOI:CNKI:SUN:WSMY.0.2012-05-005 [11] 寸怡娜,宋绍辉,周健,等. 乙型流感病毒Vero细胞高产适应株传代稳定性研究[J]. 病毒学报,2019,35(2):220-224.DOI: 10.13242/j.cnki.bingduxuebao.003513
2021, Vol. 37 
