SARS-CoV-2 Virus Propagation and Titration

SARS-CoV-2 was isolated and propagated as previously described and used for the infection experiment (50 (link)). The virus used for rechallenge was original virus stock propagated in Vero/TMPRSS2 cells cultured with 2% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C for 3 d. Vero/TMPRSS2 cells were obtained from JCRB Cell Bank and were maintained in 5% FBS DMEM supplemented with 1 mg/mL G418 (Roche). VeroE6/TMPRSS2 (P13) cells were obtained from JCRB Cell Bank and were maintained in 7% FBS DMEM supplemented with 1 mg/mL G418. All cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere. The TCID₅₀ of the virus was measured using VeroE6/TMPRSS2 cells.