Favipiravir and Ivermectin Showed in Vitro Synergistic Antiviral Activity against SARS-CoV-2

Vero E6 cells were seeded in 12-well plates at a density of 2.2×10 5 cells/well, which allowed 100% con uence to be reached within 18 hr. The culture medium was removed, and the cells were inoculated with 100µl of 10-fold serial dilutions of virus supernatants for 1 hr. at 37 o C with 5%CO 2 . After that, the virus supernatants were removed, and the cells were overlaid with 1.56% microcrystalline cellulose (Avicel, RC-591) in 2%FBS-MEM. The cells were further incubated in the standard condition for three days. The overlaid medium was removed, and the cells were xed with 10% (v/v) formalin in phosphatebuffered saline (PBS) for 2 hr. The xed infected cells were washed in tap water, stained with 1% (w/v) crystal violet in 20% (v/v) ethanol for 5 min and washed to remove the excess dyes. The plaques were counted, and the viral titers were calculated in plaque forming units per ml (pfu/ml). 50% cell culture infectious dose (TCID 50 ) endpoint dilution assay Vero E6 cells were seeded in 96-well plates at a density of 3×10 4 cells/well. The culture medium was removed, and the cells were incubated with half-log10 serial dilution of the virus stock for 48 hr. at 37 o C with 5%CO 2 . After that, the cells were xed with 1:1 methanol/acetone for 30 min at 4 o C and the infectivity was detected with an antibody against the SARS-CoV-2 nucleocapsid protein (40143-R001, Sino Biological). The viral TCID 50 titers were calculated using the Reed and Muench method 73 . One-step qRT-PCR for..