A chicken lncRNA is identified as a critical regulator that increases influenza virus replication by impairing innate antiviral responses.

Influenza A virus (IAV) poses a significant global health threat to both humans and animals. Increasing evidence highlights long noncoding RNAs (lncRNAs) as critical regulators of various physiological and pathological processes, but their roles in virus‒host interactions in chickens remain elusive. This study identified chicken lncRNAs and further investigated their functional involvement in IAV-host interactions. Transcriptome sequencing of chicken DF-1 cells revealed that 2118 lncRNAs were differentially expressed following H9N2 avian influenza virus infection. Among these, a lncRNA that we named lncRNA-up4 was significantly upregulated by H9N2 infection. LncRNA-up4 was predominantly localized in the nucleus but also detected in the cytoplasm. Furthermore, we found that the virus-induced expression of lncRNA-up4 was regulated by the pattern recognition receptor-dependent NF-κB signalling pathway. Functional analysis demonstrated that silencing lncRNA-up4 impaired IAV replication by upregulating the expression of several critical antiviral molecules, including IFN-β, MX1, and OAS-1. Conversely, overexpression of lncRNA-up4 increased viral replication, as evidenced by increased viral NP protein and virus titers. Moreover, we observed that lncRNA-up4 positively regulated the expression of IL-6, TNF-α, and IL-1β but suppressed the expression of IFN-β at the mRNA level. These results reveal that the newly identified lncRNA-up4 plays an important role in influenza virus replication through the regulation of cytokine production and antiviral gene expression in chicken cells. This study provides valuable insight into the regulatory function of chicken lncRNAs in innate immunity.