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Retinoic acid-inducible gene I (RIG-I) activates mitochondrial antiviral signaling proteins, initiating the antiviral response.RIG-IandRNF135, a ubiquitin ligase regulator, are missing in domestic chickens but conserved in mallard ducks. The chickens’RIG-Iloss was long believed to be linked to increased avian influenza susceptibility. We reinstated both genes in chickens and examined their susceptibility to infection with an H7N1 avian influenza virus. UninfectedRIG-I-expressing chickens exhibited shifts in T and B cells. At the same time, the H7N1 infection led to severe disease, persistent weight loss, and increased viral replication compared to wild-type chickens. The simultaneous expression ofRIG-IandRNF135potentiated theRIG-I activity and was associated with exacerbated inflammatory response and increased mortality without influencing virus replication. Additional animal infection experiments with two other avian influenza viruses validated these findings. They confirmed that the harmful effects triggered byRIG-IorRIG-I-RNF135-expression require a minimum degree of viral virulence. Our data indicate that the loss ofRIG-Iin chickens has likely evolved to counteract deleterious inflammation caused by viral infection and highlight the outcome of restoring evolutionary lost genes in birds.

Significance Statement

The evolutionary loss of a crucial innate immune sensor likeRIG-Iin domestic chickens and its presence in closely related avian species such as ducks has long puzzled researchers. We genetically reinstatedRIG-Iin chickens, alongside its ubiquitination factor,RNF135, to uncover their roles in responding to influenza virus interactions in chickens. Our findings suggest that the loss ofRIG-Iin chickens may have occurred as an adaptive strategy to mitigate harmful inflammation associated with influenza infection. We shed light on the outcome of reinstating evolutionarily lost genes in birds and open new avenues for understanding immune responses in vertebrates.