Establishment of a Dual-Reporter Minigenome System for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) poses a significant global health challenge, particularly affecting infants, the elderly, and immunocompromised individuals. Despite recent progress in the development of vaccines and monoclonal antibodies, effective antiviral therapies remain limited. To advance the discovery of antiviral drugs, we have developed a dual-reporter RSV minigenome system, providing a safe and robust platform for antiviral evaluation. This system incorporates NanoLuc luciferase and superfolder GFP (sfGFP) linked by a self-cleaving P2A peptide, allowing for the simultaneous detection of orthogonal signals. Validation with L polymerase inhibitors confirmed the system's reliability for screening small-molecule inhibitors. The linear correlation observed between the reporter signals enhances the assay's reliability for antiviral assessment. This dual-reporter minigenome system advances targeted therapeutic strategies against RSV.