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. 2021 Aug 17;65(9):e02680-20. doi: 10.1128/AAC.02680-20

FIG 9.

FIG 9

At equipotent concentrations, simeprevir and grazoprevir but not boceprevir synergized with remdesivir to completely suppress viral infection in Vero E6 cells. Vero E6 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 at an MOI of 0.00002 followed by treatment with 0.4-fold EC50 remdesivir (REM), 1-fold EC50 boceprevir (BOC), 1-fold EC50 simeprevir (SIM), or 1-fold EC50 grazoprevir (GRA) singly or with a combination of remdesivir with either of the PIs, which were administered immediately after infection and subsequently at the indicated time points when cells were split, as described in Materials and Methods. (Left) Percentages of SARS-CoV-2-infected cells on the specified days postinfection were determined by anti-spike protein immunostaining of replicate cultures derived following cell splitting and treatment. (Middle) SARS-CoV-2 RNA titers determined in cell culture supernatants as genome copies per milliliter on the specified days postinfection were determined by RT-qPCR assays. The black lines indicate the LLOQs. In the left and middle panels, to facilitate comparisons, bars are color coded according to the day postinfection, and blue and red dotted lines were inserted to highlight day 1 and 3 values of the nontreated culture, respectively. (Right) Replicate cultures were derived following cell splitting and treatment, immunostained for the SARS-CoV-2 spike protein (green) and counterstained with Hoechst dye (blue), and images were acquired, as described in Materials and Methods. Cultures summarized in this figure are derived from two different experimental setups (REM/BOC/SIM and REM/GRA experiments), each including the respective depicted nontreated control culture. *, culture was terminated, or infection data not recorded, due to virus-induced cell death; #, culture was maintained for a total of 15 days without indication of infection (no observation of single SARS-CoV-2 spike protein-positive cells and RNA titers were around the LLOQ).