Signaling pathways of duck RIG-I in gene-edited DF1 chicken cells.

Retinoic acid inducible gene I (RIG-I) is an innate immune RNA sensor which can detect viral infection such as influenza viruses. Duck but not chicken has an RIG-I gene. However, the immune responses could be induced in chicken cells by transferring the duck RIG-I transgene. However, effects of other pathogen-recognition receptor (PRR) genes such as Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5) could not be ruled out. In this study, we knocked out TLR3 and MDA5 genes using gene-editing protocol, and stably transferred the duck RIG-I transgene into TLR3/MDA5 double knockout (KO) chicken DF1 cells. We investigated the antiviral responses induced by duck RIG-I in chicken cells. Duck RIG-I induced the expression of interferon-stimulated genes (ISGs) and inflammatory cytokines such as interferon regulatory factor 7 (IRF7), interferon β (IFNβ), Mx1, and protein kinase R1 (PKR1) after treatment with polyinosinic: polycytidylic acid (poly I:C) in TLR3/MDA5 double KO DF1 cells. Additionally, to examine the duck RIG-I signaling cascade, we knocked out mitochondrial antiviral-signaling protein (MAVS), which encodes an antiviral signaling factor in innate immunity. Duck RIG-I in TLR3/MDA5/MAVS triple KO DF1 cells did not activate downstream expression of ISGs. Finally, to analyze the global signaling pathways of duck RIG-I in chicken cells, next-generation sequencing of total mRNAs with and without poly I:C treatment was conducted. In conclusion, duck RIG-I mediated antiviral signaling independently of TLR3 and MDA5, and MAVS induced and stimulated ISGs by duck RIG-I in chicken cells.