Fig. 2: Effect of G12(S) on β coronavirus OC43 replication in infected human cells and mice.

OC43 NP levels in HCT-8 cells infected with OC43 (multicity of infection (MOI) = 0.01, n = 3) in the absence (a) and presence of 2 μM G12(S) in the medium (b). Cell lysates were prepared at 2, 3, 4, and 5 days post-infection (dpi) in the absence (a) and presence of 2 μM G12(S) in the medium (b), and determined by western blotting with anti-OC43 NP and anti-actin antibodies. c Changes in NP/actin ratio (n = 3, normalized with the ratio of (a) at 2 dpi as 1) without (−, from a) and with (+, from b) G12(S). d OC43 RNA levels in OC43-infected (MOI = 0.01) cells (left) and culture medium (right) in the absence (control) and in the presence of 2 μM G12(S). e Effect of G12(S) on OC43 virus titers (pfu) of the medium. f OC43 RNA levels in the culture medium. Statistical analyses of the significance of G12(S) effect were performed by Two-Way ANOVA and data are shown by box-and-whisker plots including all data plots (c–f) (c n = 3, F(3,8) = 25.28, P = 0.0002; d left, n = 3, F(1,4) = 250.8, P < 0.0001; d right, n = 3, F(1,4) = 1800, P < 0.0001; e n = 3, F(1,4) = 5.245, P = 0.0838; f n = 3, F(1,4) = 77.53, P = 0.0009). Sidak’s multiple comparison test was used and the P-values are indicated. g Effect of 1 and 2 μM G12(S) on the CPE of OC43 (MOI = 0.1)-infected MRC5 cells (3 dpi). h OC43 infection in nasal cavities and olfactory bulbs of newborn mice. Immunofluorescence images of OC43 NP (yellow) and nucleus (DAPI, blue) are shown. Scale bars indicate 1 mm (i) and 20 μm (iii, iv) lengths. See Supplementary Fig. 3 for entire images. i, j Prevention of mice toxicity due to OC43 mouse infection by G12(S). OC43 infection and single-dose (i, n = 22) and three-dose (j, n = 13) schedules of G12(S) are shown. The survival rate is shown as Kaplan–Meier curves. P-values were determined by Logrank test.