Objective:The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans.Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the MAPK signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines.Methods: Resistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, their tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing. Results:A gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

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Title of the manuscript: Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14 ABSTRACTObjective: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 μg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by qRT-PCR.Results: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted IFNGR2, MAPK1, MAP3K5, and MAP3K14. Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (ISGs) (MX1, EIF2A, and OASL) and proinflammatory cytokines (IFNG, IL1B, and TNFA) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion:The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against HPAIV H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.