A dual-reporter HCoV-OC43 for coronavirus biology and countermeasure development

A reliable experimental system is essential for advancing coronavirus biology and expediting countermeasure development. In this study, we report the construction of an infectious clone of human coronavirus OC43 (HCoV-OC43) using an in vitro ligation strategy. The clone-derived virus faithfully recapitulates the replication characteristics of the parental isolate in vitro . Leveraging this platform, we have developed a dual-reporter virus, OC43-mNG-Nluc, by replacing the viral accessory gene ns2 with a gene cassette encoding both nanoluciferase and fluorescent protein mNeonGreen. This construct enables dual-mode detection via chemiluminescence and fluorescence within a single assay. OC43-mNG-Nluc maintains replication kinetics identical to wild-type HCoV-OC43 in standard immortalized cell lines but exhibits marked attenuation in primary human airway epithelial cultures, underscoring the critical role of ns2 in viral replication within physiologically relevant systems. The dual-reporter virus also demonstrates excellent genetic and phenotypic stability following fifteen passages in vitro . Furthermore, we validate the proof-of-concept utility of OC43-mNG-Nluc in high-throughput antiviral screening, showcasing its advantages in streamlining and facilitating hit identification, triaging, and prioritization. Collectively, this study provides valuable insights into HCoV-OC43 biology and introduces a versatile and robust tool for coronavirus research and therapeutic development.