Synergistic Antiviral Effects of Cross-Type Bovine Interferons via Enhanced ISG Activation

Antiviral potency, evaluated via the Madin-Darby bovine kidney (MDBK)-vesicular stomatitis virus (VSV) system, showed BoIFN-α with the highest specific activity (1.50 ± 0.98 × 107 U/mg), followed by BoIFN-λ3 (2.39 ± 0.15 × 106 U/mg), BoIFN-γ (8.10 ± 0.21 × 104 U/mg), and BoIFN-β (1.25 ± 0.38 × 104 U/mg). MTT assays revealed minimal cytotoxicity of all BoIFNs (alone or combined) on embryonic bovine kidney (EBK) and MDBK cells. Using TCID50, plaque reduction, and semi-quantitative RT-PCR (sqRT-PCR) assays, BoIFN-λ3 matched type I/II BoIFNs against bovine enterovirus (BEV), cytopathic bovine viral diarrheal virus (cpBVDV), and noncytopathic BVDV (ncpBVDV), with dose- and time-dependent activity and greater potency against BEV than infectious bovine rhinotracheitis virus (IBRV). Notably, BoIFN-λ3 synergized with BoIFN-γ against IBRV and with BoIFN-α/β against BEV in EBK/bovine testicular (BT) cells. Western blotting demonstrated BoIFN-λ3 robustly induced myxovirus resistance protein 1 (Mx1) expression across four bovine cell lines: BT cells, bovine mammary epithelial cells (BMEC), EBK and MDBK cells. Dual-luciferase assays showed differential interferon-stimulated response element (ISRE) activation: BoIFN-α/β triggered rapid peaks at 12 h, while BoIFN-λ3/γ induced sustained activation peaking at 48 h; combinations synergistically enhanced activity.