Fig. 5.

Infection and amplification of SARS-CoV-2 in human tonsil epithelial organoids. (A, B) Immunofluorescence staining for viral spike protein (A) and dsRNA (B) (green) and a cellular receptor, ACE2 (red), in tonsil epithelium organoids on day 3 after SARS-CoV-2 infection at MOI of 0 (Mock), 0.1, or 1. Scale bar is 100 μm. Merged images with nuclei (blue) and their zooms are presented in the right panels. (C) Quantitative RT-PCR for detecting viral RNA in the culture supernatants of tonsil organoids infected with SARS-CoV-2 at an MOI of 0.1 or 1 at various time points after infection. (D) Determination of the amount of infectious viral particle in culture supernatants of SARS-CoV-2–infected tonsil organoids from the same donor of (C). Viral titers were measured by infection of fresh Vero CCL-81 cells with organoid culture medium for 2 days and by staining with anti-spike protein antibody, as described in Materials and Methods. (E, F) Both viral RNA copies (E) and infectious SARS-CoV-2 titers (F) in culture supernatants were determined independently from three additional tonsil organoids (donors 20-13, 20-14 and 2016). Two-way ANOVA with Dunnett's multiple comparison test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (G) TEM analysis showing accumulation of SARS-CoV-2 particles on the apical surface of tonsil organoids on day 3 (lower) but not on 3 h (upper) after infection at an MOI of 0.1. Specific in the red rectangle are enlarged at right to show budding-out of viral particles on 72 h post-infection. Mock-infected samples are used as a control for showing pathogen-free samples. Scale bars are 1 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)