In vitro functional characterization of Tadarida brasiliensis interferon-β: promoter responsiveness, antiviral activity, and JAK-dependent transcriptional responses

Introduction Bats display diverse antiviral responses, but interferon-β remains poorly characterized in many bat species. Here, we characterized interferon-β from Tadarida brasiliensis (TBIFN-β) in Tb 1 Lu cells, focusing on its sequence features, promoter responsiveness, antiviral activity, and downstream signaling. Methods TBIFN-β was analyzed by comparative sequence analysis and homology modeling. TBIFNB promoter fragments were evaluated using dual-luciferase assays following batIRF1 expression or poly(I:C) treatment. Antiviral activity was assessed using VSV-GFP and VACV-GFP infection models, together with viral transcript quantification by RT-qPCR. Downstream signaling was examined through interferon-stimulated gene expression, Pyridone 6-mediated JAK inhibition, conditioned-medium stimulation, STAT1 phosphorylation, and exploratory RNA sequencing followed by GO/KEGG enrichment analysis and RT-qPCR validation. Results TBIFN-β retained the principal predicted structural features of mammalian type I interferons. VSV-GFP and VACV-GFP infection induced distinct temporal changes in endogenous TBIFNB and interferon-stimulated gene expression. Both −900/0 and −500/0 TBIFNB promoter fragments responded to batIRF1 and poly(I:C). Transfection with the TBIFN-β expression construct reduced VSV-GFP and VACV-GFP fluorescence and decreased VSV-NP and VACV-C23L transcript copy equivalents. TBIFN-β induced context-dependent expression of PKR, OAS1, and Mx-1, with OAS1 showing the most consistent response. Pyridone 6 attenuated TBIFN-β-associated STAT1 and ISG responses and partially reduced its antiviral effects. Conditioned medium containing Flag-tagged TBIFN-β induced Pyridone 6-sensitive STAT1 phosphorylation in recipient cells. Transcriptomic analysis further revealed coordinated interferon-associated and antiviral transcriptional responses following TBIFN-β overexpression. Discussion These findings provide an integrated in vitro characterization of TBIFN-β promoter responsiveness, antiviral activity, and JAK-dependent transcriptional effects in Tb 1 Lu cells. The results establish a basis for further functional studies of T. brasiliensis interferon biology, while validation in primary bat cells and in vivo models remains necessary.