An efficient and versatile reverse genetics system for bovine coronavirus facilitates antiviral evaluation

Bovine coronavirus (BCoV), a primary etiological agent of calf diarrhea, exhibits dual tropism for the respiratory and enteric tracts. However, research on BCoV gene function and pathogenesis has been limited by the lack of convenient and flexible reverse genetics tools. Herein, we report a robust reverse genetics system for BCoV based on an optimized circular polymerase extension reaction (CPER) methodology. We successfully rescued recombinant BCoV (rBCoV) in HRT-18G cells and engineered recombinant BCoV expressing ZsGreen fluorescent protein (rBCoV/Zs-T2A-ns2). Phenotypic analysis demonstrated that both unmodified rBCoV and rBCoV/Zs-T2A-ns2 maintained growth kinetics comparable to the wild-type BCoV. Importantly, the reporter virus demonstrated remarkable genetic stability for over ten passages, identifying the ns2 locus as a suitable insertion site for stable foreign gene expression without compromising viral fitness or sequence integrity. As a proof-of-concept application, the rBCoV/Zs-T2A-ns2 was employed for antiviral compound evaluation, successfully identifying four compounds-camostat, nafamostat, niclosamide, and nelfinavir-capable of effectively inhibiting BCoV replication. Further time-of-addition assays revealed that nelfinavir and niclosamide targeted the BCoV replication stage, while camostat and nafamostat interfered with BCoV internalization. This versatile CPER-based reverse genetics system provides a critical platform for advanced BCoV pathogenesis research and accelerated development of novel vaccines and effective antiviral therapeutics, ultimately contributing to enhanced bovine health and sustainable livestock production.