Molecular characterization and functional analysis of interferon regulatory factor 9 (irf9) in common carp Cyprinus carpio: a pivotal molecule in the Ifn response against pathogens

In the present study, interferon (IFN) regulatory factor (IRF) 9 gene (irf9) was identified and characterized in common carp Cyprinus carpio. The predicted protein sequence of Irf9 contains a DNA binding domain (DBD) that possess five tryptophans, an IRF association domain (IAD) and two nuclear localisation signals (NLS). Alignment of Irf9 of C. carpio with the corresponding Irf9 proteins of other species showed that the DBD is more highly conserved than the IAD. The putative Irf9 protein sequence of C. carpio shares higher identities with teleosts (53.8-82.3%) and lower identities with mammals (30.2-31.0%). Phylogenetic studies of the putative amino-acid sequence of IRF9 based on the neighbour-joining method showed that Irf9 of C. carpio has the closest relationship with the grass carp Ctenopharyngodon idella. Tissue distribution analysis showed that irf9 transcripts were detectable in all examined tissues with the highest expression in the skin and the lowest expression in the head kidney. Poly I:C and Aeromonas hydrophila stimulation up-regulated irf9 expression in the spleen, head kidney, foregut and hindgut at different time intervals. In addition, irf9 was induced by Poly I:C and lipopolysaccharides (LPS) in vitro. These results indicate that Irf9 participates in antiviral and antibacterial immunity. Transfection of irf9 up-regulated the expression of cytokines, including type I IFN, protein kinase R (PKR), interferon-stimulated gene (ISG)15 and tumour necrosis factor (TNF)α in epithelioma papulosum cyprini cells (EPC) upon poly I:C and LPS stimulation. A dual-luciferase reporter assay revealed that Irf9 has no effect on NF-κB activation. The present study on Irf9 provides new insights into the IFN system of C. carpio and a valuable experimental platform for future studies on the immune system of fish.