Nafamostat

Vero cells cultured in a 96-well plate at a cell density of 1 × 10⁵ cells/well were treated with 1% dimethyl sulfoxide (Sigma-Aldrich) or 100 µM decanoyl-RVKR-CMK (Cayman Chemical, MN, USA) and 50 µM E64d (Calbiochem, Darmstadt, Germany) or 50 µM nafamostat (Cayman Chemical), or both, for 1 h. The cells were challenged with 15 µL of pseudotype virus or 50 PFU of recombinant virus and incubated for 15 and 20 h, respectively. Luciferase activity was measured using the Luciferase Assay System (Promega) for pseudotype viruses. The infected cells were visualized by an indirect fluorescence assay using an anti-SARS-CoV-2 N protein antibody (a kind gift by Dr. Okamoto) and anti-mouse IgG antibody-CF488 conjugate (Nacalai Tesque).

IEC#17 cells were seeded at a concentration of 1 × 10⁵ per well in a 96-well plate and pretreated with the inhibitors, camostat (Cayman Chemical Company, MI, USA) and nafamostat (Cayman Chemical Company) for 1 h before virus infection. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, MO, USA) was used as a control. After removal of the inhibitors, the cells were washed three times and infected with SARS-CoV-2 at an MOI of 0.1 or 2 × 10⁶ genome equivalents of NoV GII.4 for 1 h at 37 °C. At 48 hpi, the supernatant was collected, and the viral titers of SARS-CoV-2 and NoV GII.4 were measured by the TCID₅₀ method and qRT-PCR, respectively. Each value is representative of at least three independent experiments and is presented as the mean ± SD from three wells of supernatants of each culture group.