A High-Potency Bovine Consensus Interferon Delivered by an Oral Lactococcus lactis System Exhibits Antiviral Activity Against VSV and BEV.

Interferon-alpha (IFN-α) mediates innate antiviral immunity. Although it comprises multiple subtypes, their functional divergence and application potential remain incompletely characterized. In this study, we identified 13 BoIFN-α subtypes (A1-A13) in the bovine genome through bioinformatic analysis. Prokaryotic expression and functional comparison revealed substantial differences in antiviral activity among these subtypes (up to 77.7-fold), with BoIFN-A8 being the most potent. Based on these findings, we designed a novel consensus interferon, BoIFN-Acons, by multiple sequence alignment and successfully synthesized it. When expressed in E. coli, BoIFN-Acons exhibited superior antiviral potency (2.36 × 105 U/mg), which was 16.2-fold higher than that of the most active natural subtype. To establish a safe delivery system, we integrated the codon-optimized gene into a food-grade L. lactis expression platform and achieved efficient supernatant production. In vitro assays confirmed that the recombinant BoIFN-Acons effectively suppressed replication of Vesicular stomatitis virus (VSV) and Bovine enterovirus (BEV) and significantly upregulated interferon-stimulated genes (ISGs) such as Mx1 and OAS1. Furthermore, in vivo experiments demonstrated that oral administration of the engineered L. lactis strain alleviated virus-induced clinical symptoms, reduced viral loads in major tissues, and induced upregulation of immune-related genes in the spleen, showing significant prophylactic and therapeutic efficacy. Collectively, this study systematically elucidated the functional diversity of BoIFN-α subtypes and developed a highly active consensus interferon delivered via an oral lactic acid bacteria system, providing a new strategy for the eco-friendly control of animal viral diseases.