Tissue culture infectious dose (TCID50) assay

Vero E6-TMPRSS2 cells were plated in 96-well plates and cultured until 90%–100% confluent. The cells were washed two times in DPBS, then covered with 180 µL of IM. Tenfold serial dilutions of virus at each time point were made in a separate plate. Each dilution was plated in replicates of four. Infection proceeded for 5 days at 37°C, the cells were fixed with 4% formaldehyde, and stained with naphthol blue-black solution. Viral-induced cytopathic effect was scored visually and the titer of infectious virus was calculated based on the Reed-Muench method (37, 44).