NA-R289K drug-resistant mutant H7N9 avian influenza recombinant virus regulates host cell biology by exosomal miR-335-5p

Background: Drug resistance mutations in influenza virus can significantly alter the host cell immune response and clinical prognosis. Exosomal microRNAs(miRNAs) play a critical role in regulating host cell function during viral infections. This study investigates the role of exosomal miRNAs in modulating host cell functions during infection with oseltamivir-resistant NA-289K H7N9 strains and oseltamivir-sensitive NA-289R H7N9 strains. Methods: Recombinant viruses NA-289R and NA-289K were constructed and used to infect A549 cells. Exosomes generated following infection were analyzed systematically. The impact of hsa-miR-335-5p on cellular function was evaluated using CCK8 and TUNEL assays, and genes related to infection and inflammation were identified via RT-qPCR. Results: Infection with the NA-289K strain significantly upregulated exosomal miRNA levels, particularly hsa-miR-335-5p, in compared to the NA-289R strain. Bioinformatic analysis predicted TGF-β2 and MAPK1 as potential targets of hsa-miR-335-5p. This miRNA modulated the TGF-β/SMAD pathway and MAPK1 gene expression in infected cells. Consequently, hsa-miR-335-5p overexpression reduced cell viability, promoted apoptosis following NA-289K and NA-289R infection, and decreased IL-8 and MCP-1 expression in cells infected with NA-289K. However, only MCP-1 expression was reduced in NA-289R post-infection cells. Conclusion: The NA-289K drug-resistant strain may exploit hsa-miR-335-5p in exosomes to modulate host cell functions, potentially inhibiting proliferation and promoting apoptosis, thereby facilitating influenza virus infection. In contrast, the NA-289R strain may utilizes alternative hsa-miR-335-5p-dependent pathways for host modulation. These findings significantly advance our understanding of pathogenesis in resistant influenza by identifying EV miRNA manipulation as a critical mechanism. Furthermore, they highlight miR-335-5p as a potential therapeutic target to counteract virulence and EV-mediated pathology associated with oseltamivir resistance.