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. 2021 Oct 2;564:33–38. doi: 10.1016/j.virol.2021.09.009

Fig. 2.

Fig. 2

Antiviral effects of molnupiravir against HCoV-NL63. (A) Dose-dependent inhibition of HCoV-NL63 replication in Caco2 cell line by molnupiravir treatment. Intracellular viral RNA quantified by qRT-PCR was normalized to housekeeping gene GAPDH and presented relative to the control (CTR) (set as 1) (n = 6). (B) Immunofluorescence microscopy analysis of dsRNA (red), the intermediate of replicating HCoV-NL63 genomic RNA, upon treatment of different concentrations of molnupiravir in Caco2 cell line. Nuclei were visualized by DAPI (blue). (C) Caco2 cells were infected with HCoV-NL63 at an MOI of 0.1 in the treatment of different concentrations of molnupiravir for 48 h. Viral yield in the cell supernatant was quantified by qRT-PCR. Cytotoxicity was determined by MTT assay. The half maximum effective concentration (EC50) and the half maximum cytotoxic concentration (CC50) were calculated based on the model Y = 100/(1 + 10^(LogEC50-X)) using GraphPad Prism 8.0.2 software. The left and right Y-axis of the graphs represent mean % inhibition of virus yield and cytotoxicity of the drugs, respectively. (n = 6–16). (D) Caco2 cells were infected with HCoV-NL63 at the MOI of 0.5, and then untreated or treated with 5 μM molnupiravir for 5 days. Supernatant was collected every day to quantify secreted viruses by qRT-PCR, calculated as genomic copy numbers (n = 6). Standard curve for calculation of genomic copy numbers is included in Supplementary Fig. S3A. (E) Caco2 cells were infected with 0.5 MOI HCoV-NL63, and then untreated or treated with 5 or 50 μM molnupiravir for 48 h. Virus titers from different groups were determined by TCID50 assay (n = 6). (F) HCoV-NL63 was serially passaged in Caco2 cells exposed to no molnupiravir (as control) or increasing concentrations of molnupiravir for 20 passages. 5 μM molnupiravir was used in passage 1–10, which was increased to 10 μM at the subsequent passages. The effect of molnupiravir (5 μM) on HCoV-NL63 harvested at passage 5, 10, 15 and 20 was quantified using qRT-PCR. Data represent as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. HCoV: human coronavirus. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)