2.4. CPE assay

The VeroE6/TMPRSS2 cells (Japanese Collection of Research Bioresources Cell Bank, #JCRB1819) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and Penicillin-Streptomycin (Sigma-Aldrich). For isolation of SARS-CoV-2, cells were seeded on a 25-cm 2 cell culture flask (FALCON). The next day, the thawed specimen medium (0.5 mL) was centrifuged at low speed, and the supernatant was mixed with 4.5 mL of isolation medium (DMEM supplemented with 2% FBS, penicillin-streptomycin [Sigma-Aldrich], gentamicin [Sigma-Aldrich], and amphotericin B [Sigma-Aldrich]). The maintenance medium in the flask was then removed, the cells were washed once with isolation medium, and the mixture (5 mL) was added to the flask, followed by incubation at 37 °C. The cells were examined daily for 5 days for cytopathic effect (CPE).