Fig. 7.

Evaluation of antiviral activity of remdesivir in SARS-CoV-2-infected tonsil organoids. (A) Quantitative RT-PCR showing inhibition of viral RNA titers on day 2 after treatment of SARS-CoV-2–infected organoids with increasing concentrations of remdesivir (0.1, 1, or 10 μM). Mock: non-infected control. RNA copies are presented as means ± SEM of four independent experiments using tonsil organoids from a single donor (no. 20-16). Statistical analysis was performed by ordinary one-way ANOVA with Dunnett's multiple comparison test. **, p < 0.01. (B) Western blot analysis showing reduction of viral spike and nucleocapsid proteins in two different organoids samples (donors 20-16 and 21–36, respectively) in the presence of remdesivir. Beta-actin was used as a loading control. (C) Reduction of nucleocapsid protein by remdesivir was quantified from Western blot analysis with three different organoid samples (donors 21-16, 21–34 and 21–36). Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparison test. ****, p < 0.0001. (D) Cell viability test. Organoids from three different donors (21–26, 21–27 and 22–33) were mock infected or infected with SARS-CoV-2 alone (MOI, 0.1) or in the presence of 100 μM remdesivir. On day 2, organoids from three different wells were dissociated with trypsin-EDTA and individually stained with trypan blue. Live and dead cells were counted by using an automated microscopic cell counter. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test. n.s., statistically not significant. (E) Organoid samples from three different donors (21–26, 21–27 and 22–33) were infected with SARS-CoV-2 (MOI, 0.1) and treated with increasing concentrations of remdesivir. On day 2 post-infection, RNA samples were harvested from culture supernatants for qRT-PCR against the viral nucleocapsid gene. EC₅₀ values are calculated in three independent experiments and expressed as mean ± SD. (F) Confocal miscopy showing reduction of viral spike protein by remdesivir. Tonsil epithelial organoids (donor 21–36) were mock-infected (upper) or infected with SARS-CoV-2 at an MOI of 0.1. The infected organoids were treated with DMSO (middle) or remdesivir (10 μM; lower) for 2 days. Viral spike protein (blue) and its binding receptor ACE2 (red) were stained with their specific antibodies. Nuclei (blue) were counter-stained with Hoechst 33342. Merged images and their zooms are displayed on the right side. Scale bar is 100 μm.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)