PK-15 cells were seeded in 12-well plates (2 × 105 cells/well) and incubated at 37 °C with 5% CO2 for 12 h. PK-15 cells were infected with rPRV HN1201-EGFP-Luc (MOI = 0.01) per cell and 25-HC at the corresponding concentration for 18 h after pretreatment with 25-HC (HY-113134, MCE) (0.1 μM, 0.3 μM, 1 μM, 3 μM or 10 μM) for 4 h. DMSO solvent-only control was used in the DMSO group (1 μL of DMSO was added to 1000 μL of cell culture medium). The inhibitory effects of 25-HC on PRV proliferation in PK-15 cells were evaluated with firefly luciferase assays, flow cytometry assays and fluorescence analysis.

Six-week-old mice were randomly divided into a 25-HC group and DMSO group (three mice per group). For the 25-HC group, 25-HC (10 mg/kg) was administered to mice by intraperitoneal injection for 2 consecutive days (once per day). For the DMSO group, mice received the same volume of DMSO. Twelve hours after the last injection of 25-HC, all mice were injected I.M. in the left leg with rPRV HN1201-EGFP-Luc (1 × 104.5 TCID50 per mouse). The rPRV HN1201-EGFP-Luc distributions in mice were measured with whole animal bioluminescence imaging by using an IVIS Lumina III (Perkin Elmer) instrument after intraperitoneal injection of D-luciferin sodium salt (HY-12591, MCE) (3 mg per mouse) at 24 h after viral infection.

To determine the suitability of 25-HC as a curative agent, we administered 25-HC after infection. In the experimental animal groups, the injection dosage and methods for 25-HC and DMSO were the same as above, except that each mouse was first infected with 1 × 104.5 TCID50 rPRV HN1201-EGFP-Luc and then injected with 25-HC or DMSO 6 h and 12 h after virus infection. The rPRV HN1201-EGFP-Luc distributions in mice were measured with whole animal bioluminescence imaging 24 h after viral infection by using an IVIS Lumina III (Perkin Elmer) instrument after intraperitoneal injection of D-luciferin sodium salt (HY-12591, MCE) (3 mg per mouse).

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