A high-throughput cell-based platform for Rhinovirus C research and antiviral drug discovery.

Rhinoviruses (RVs) are the most common cause of upper respiratory infections, yet RV-C is particularly associated with severe respiratory disease, including exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Despite its clinical significance, RV-C research and antiviral development have been hindered by the lack of scalable and biologically relevant models for high-throughput screening (HTS). Here, we present the development, optimization, and validation of a scalable high-content imaging-based platform for RV-C research. Our approach combines engineered HeLa cells expressing CDHR3C529Y, a genetically stable mGreenLantern-reporter RV-C15 virus, and a phenotypic selection strategy to identify monoclonal cell lines with maximal and sustained permissiveness. This approach enabled assay optimization in both 96- and 384-well formats, achieving robust performance (Z' > 0.7) and reproducibility across more than 100 runs with reference antiviral compounds. The platform was readily adapted to RV-A16, RV-B14, and additional RV-C types (C11 and C41), demonstrating broad applicability across rhinovirus species. A pilot screen of 10 240 compounds identified confirmed RV-C inhibitors, highlighting the readiness of the platform for integration into drug discovery pipelines. This platform provides a robust and scalable tool for systematic antiviral discovery and a foundation for mechanistic studies of RV-C replication. Significance statementRhinovirus C (RV-C) is strongly associated with severe respiratory disease such as asthma and COPD exacerbations, yet its study has been limited by the absence of scalable cell-based systems. We developed a high-content imaging-based high-throughput screening platform that supports efficient RV-C replication in engineered CDHR3-expressing cells and enables systematic antiviral discovery. The platform integrates a genetically stable fluorescent reporter virus with phenotypically selected permissive monoclonal cell lines, achieving high reproducibility and scalability in both 96- and 384-well formats. Its adaptability across multiple rhinovirus species establishes a unified framework for antiviral testing and mechanistic investigation. A pilot screen of 10K compounds confirmed the applicability of the platform to drug discovery campaigns.