SARS-CoV-2 Infection in VeroE6/TMPRSS2 Cells

SARS-CoV-2 (JPN/TY/WK-521 strain) and transmembrane protease serine 2 (TMPRSS2)-expressing VeroE6 (VeroE6/TMPRSS2) cells [25 (link)] were obtained from the National Institute of Infectious Diseases (Tokyo, Japan). For passaging, VeroE6/TMPRSS2 cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum, 2 mM l-glutamine (FUJIFILM Wako Pure Chemical Co., Osaka, Japan), 0.15% NaHCO₃ (FUJIFILM Wako Pure Chemical Co.), 2 µg/mL amphotericin B (Bristol-Myers Squibb Co., New York, NY, USA), 100 µg/mL kanamycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and 100 µg/mL G418 disulfate (Nacalai Tesque Inc., Kyoto, Japan). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2, cultured in viral growth medium (VGM) composed of DMEM supplemented with 1% fetal bovine serum, 20 mM l-glutamine, 0.15% NaHCO₃, 2 µg/mL amphotericin B, and 100 µg/mL kanamycin for 3 d. Culture supernatants were stored at −80 °C as viral stock (viral titer: 7.25 log₁₀ 50% tissue culture infective dose (TCID₅₀)/mL). SARS-CoV-2 was handled in a biosafety level 3 facility.