Marek's disease virus infection alters the composition of exosomes in chicken fibroblasts and promotes viral replication.

Marek's disease (MD) is a highly contagious lymphoproliferative disease caused by Marek's disease virus (MDV), typically characterized by severe immunosuppression. In this study, exosomes were isolated and purified from chicken embryo fibroblasts (CEFs) infected with the virulent MDV strain Md5 (Exo-Md5) and uninfected controls (Exo-NC), respectively. Combining small RNA sequencing, mass spectrometry, and Western blot analysis, we confirmed that MDV infection significantly remodels the exosomal cargo composition. Exo-Md5 exhibited a predominant downregulation of miRNAs. Subsequent GO and KEGG enrichment analyses revealed that these downregulated miRNAs were significantly enriched in critical pathways closely associated with host immunity and viral replication, including T cell receptor signaling, Toll-like receptor signaling, type I interferon response, and cell cycle regulation. Proteomic analysis further demonstrated that MDV infection resulted in 147 upregulated proteins in exosomes. Integrated enrichment analysis using EggNOG, KEGG, and GO databases showed that differentially expressed proteins were predominantly enriched in pathways associated with intracellular transport, metabolic regulation, immune modulation, organismal homeostasis, and tumor-related processes. Additionally, 20 MDV-derived components were identified in Exo-Md5, including the tegument protein VP22 mediating intercellular spread, the envelope glycoprotein gB involved in viral entry, and the phosphoprotein pp38 associated with cell transformation, indicating that MDV-infected cells can package viral proteins into exosomes through sorting mechanisms. Functional assays further demonstrated that exosomes derived from Md5-infected cells (Exo-Md5) were able to reduce the proportion of CD4+ T lymphocytes and promote viral replication, while inhibition of exosome secretion suppressed viral propagation. Collectively, the findings of this study provide novel experimental evidence for elucidating the molecular mechanisms underlying MDV-mediated immunosuppression, immune evasion, and efficient replication.