Chicken miR-26a-5p modulates MDA5 during highly pathogenic avian influenza virus infection

MicroRNAs play crucial roles in immune-related pathways in host animals. In this study, we aimed to investigate the systemic biological function of gga-miR-26a-5p, a chicken miRNA, in the immune responses to HPAIV H5N1 infection in the Vietnamese Ri chicken line. Our results showed a significant downregulation in gga-miR-26a expression in the lung tissue of Ri chickens during HPAIV H5N1 infection. Overexpression of gga-miR-26a and the reporter construct, either containing the wildtype or mutant melanoma differentiation-associated protein 5 (MDA5) 3' untranslated region (3' UTR)-luciferase, into a chicken fibroblast cell line, revealed that gga-miR-26a can act as a direct translational repressor of MDA5 by targeting the 3' UTRs. Additionally, miR-26a negatively regulated the expression of the signaling molecules related to the MDA5 signaling pathway, including MDA5, mitochondrial antiviral-signaling (MAVS), interferon regulatory factor 7 (IRF7), p38 mitogen-activated protein kinases, and nuclear factor-kappa B (NF-κB). Moreover, downstream of the IRF7 and NF-κB signaling pathway, the proinflammatory cytokines such as IL-1β, IFN-γ, IFN-α, IFN-β, and the interferon-stimulated gene (Mx1) were, likewise, downregulated by the overexpression of gga-miR-26a. These findings suggest that gga-miR-26a-5p serves as an important regulator in the MDA5 signaling pathway and antiviral response. Overall, our results contribute to an improved understanding of the biological functions of gga-miR-26a-5p, alongside the mechanisms underlying the MDA5 signaling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens.