SARS-CoV-2 infection of Vero E6 cell line

The African green monkey kidney Vero E6 cell line was purchased from the Cell Resources Center of Shanghai Institute of Life Science, Chinese Academy of Sciences (China) and cultured in DMEM medium (Cat. No. 12430-054, Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco) at 37 °C with 5% CO₂ atmosphere.

BetaCoV/JS03/human/2020 (EPI_ISL_411953), a SARS-CoV-2 virus strain, was isolated from nasopharyngeal swab of a 40-year-old female confirmed with COVID-19 by reverse transcriptase PCR (RT-PCR) in December 2019. The virus was propagated in Vero E6 cells, and the viral titer was determined by the 50% tissue culture infective dose based on microscopic observation of cytopathic effects. All the in vitro SARS-CoV-2 infection experiments were performed in a bio-safety level-3 laboratory of Jiangsu Provincial Center for Diseases Control and Prevention, Jiangsu, China.

Vero E6 cells were seeded into 12-well plates with a density of 4×10⁴ cells/well for incubation in DMEM medium supplemented with 10% FBS for 16 hours in an incubator with 5% CO₂ at 37 °C for cells to reach 80% confluent. Six wells were infected with the SARS-CoV-2 virus with multiplicity of infection of 0.05 at 37 °C with a 5% CO₂ atmosphere for 48 hours. Then the infected cells of each well were harvested for RNA extraction and quantitation. RNA was extracted from cells using the RNeasy Plus Mini Kit (Cat. No. 74134, Qiagen, Germany) according to the manufacturer's instructions.