Abstract

Introduction Marek’s disease virus (MDV) is a highly immunosuppressive alphaherpesvirus. However, whether and how MDV exploits extracellular vesicles (EVs) to evade host immunity, particularly the critical type I interferon (IFN-I) response, remains unknown. We hypothesized that MDV reprograms the EV microRNA (miRNA) cargo to facilitate its escape from the IFN-I-mediated antiviral state. Methods Small RNA (sRNA) sequencing was conducted to profile and compare the expression patterns of EV miRNAs in chicken embryo fibroblast (DF-1) cells under four conditions: control, MDV infection, chicken interferon-alpha (chIFN-α) treatment and MDV–chIFN-α co-treatment. Integrative bioinformatic analyzes were employed to identify key differentially expressed miRNAs (DEMs) and predict their target genes within the IFN-I signaling network. Results MDV infection and chIFN-α treatment induced fundamentally distinct EV miRNA profiles. Strikingly, MDV infection counteracted the specific EV miRNA signature triggered by chIFN-α. We identified 65 key DEMs with the potential to cooperatively target multiple nodes of the IFN-I pathway. Among these, gga-miR-20a-5p and gga-miR-148a-3p were experimentally validated to directly target the 3′ untranslated regions of the key innate immune sensors cGAS and TLR3, respectively, leading to a significant suppression of downstream IFN-I signaling activation. Conclusion This study identifies dysregulated EV miRNAs during MDV-interferon antagonism, validating their direct targeting of innate immune sensors. These findings provide new insights into viral pathogenesis and pinpoint specific miRNA-target axes as potential avenues for antiviral intervention.