The 25-HC antiviral tests in vivo and in vitro. Treatment with 25-HC inhibited the proliferation of rPRV HN1201-EGFP-Luc in vitro. PK-15 cells were pretreated with the indicated concentrations of 25-HC for 4 h and then infected with rPRV HN1201-EGFP-Luc (MOI = 0.01) along with the same concentrations of 25-HC for 18 h. Luciferase assays (A), FACS analysis (B) and fluorescence microscopy (C) were performed. Data are shown as the mean ± SD from three independent experiments. Scale bar, 200 μm. ***P < 0.001. D Bioluminescent images of mice pretreated with 25-HC or DMSO at 24 h after rPRV HN1201-EGFP-Luc infection. E Bioluminescent images of mice in which 25-HC or DMSO was injected later upon rPRV HN1201-EGFP-Luc infection. Whole animal bioluminescence imaging was performed 24 h after rPRV HN1201-EGFP-Luc infection.