Identification of interferon-stimulated response elements (ISREs) in canines

Interferon (IFN) responses are vital for antiviral defense, with interferon-stimulated response elements (ISREs) crucial for regulating IFN signaling. While ISREs are well-studied in humans and mice, research on canine ISREs is limited. This study aimed to clarify the role of canine ISREs and create a new method for detecting IFN activity. Canine IFN alpha (CaIFN alpha) was produced using the Pichia pastoris (P. pastoris) system, and an ISRE-based flow cytometry method was developed to measure its activity. ISREs for CaIFN alpha were predicted via bioinformatics analysis. Subsequently, viral suppression assays were conducted using vesicular stomatitis virus, canine influenza virus, and H9N2 to evaluate the antiviral activity of recombinant CaIFN alpha. Fluorescence analysis confirmed that CaIFN alpha activates ISRE2, ISRE8, and ISRE10, thereby enhancing the transcription and expression of the enhanced green fluorescent protein (EGFP) fusion gene. A novel ISRE and EGFP based flow cytometry method enabled precise quantification of CaIFN alpha levels through fluorescence cell counts, with a detection sensitivity reaching 0.1 x 10- 7 mg/mL. Results demonstrate that CaIFN alpha possesses multiple antiviral activity and activates specific ISREs, augmenting gene expression. This approach advances the study of canine ISREs and supports the development and clinical application of CaIFN alpha for diagnosing viral infections and monitoring treatment efficacy.