CO mediates the anti-PRV activity of HO-1. (A) PK-15 or (B) ST cells were infected with PRV at a MOI of 0.01, prior to harvesting at 12, 24, 36, and 48 hpi to detect total HbCO levels via ELISA. (C) PK-15 or (D) ST cells were incubated with 50 μM of CoPP for 24 h, prior to harvesting to detect total HbCO levels via ELISA. PRV-infected PK-15 cells were treated with 50 μM of CoPP, in the presence or absence of 50 μg/ml of Hb. After 24 h, cells were harvested to assess PRV gB mRNA and protein expression levels via (E) RT-qPCR and (F) western blotting, respectively, while (G) supernatants were harvested to measure virus copies via RT-qPCR. PRV-infected PK-15 or ST cells were incubated with 3% FBS + DMEM containing 0, 10, 25, and 50 μM of CORM-2 or 50 μM of inactive CORM-2 for 36 h. Cells and supernatants were collected for PRV replication analysis via (I,K) western blotting and (H,J) RT-qPCR. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. CO, carbon monoxide; PRV, pseudorabies virus; HO-1, heme oxygenase-1; Hb, hemoglobin; ELISA, enzyme-linked immunosorbent assay; CoPP, cobalt-protoporphyrin; RT-qPCR, reverse transcription-quantitative PCR; CORM, CO-releasing molecules.