Oya virus reporter systems for high-throughput screening of viral replication inhibitors in mosquito cells

Orthobunyaviruses are a diverse group of arboviruses, yet reporter virus-based inhibitor screening in mosquito-derived cells remains underexplored. Oya virus (OYAV), a Simbu serogroup orthobunyavirus detected in humans, mosquitoes, and midges, currently lacks reverse genetics tools and quantitative compound screening platforms. In this study, we established the first plasmid-based reverse genetics system for OYAV, successfully rescuing recombinant wild-type virus (rOYAV/WT) alongside fluorescent reporter viruses expressing enhanced green fluorescent protein (rOYAV/eGFP/S) or mCherry (rOYAV/mCherry/S). Both reporter viruses maintained stable reporter expression and infectivity across multiple vertebrate and mosquito-derived cell lines, although reporter insertion resulted in attenuated replication compared with the parental virus. Leveraging the robust fluorescence signal and retention of the reporter-containing region during serial passage, we developed a mosquito cell-based high-content screening platform to identify compounds inhibiting OYAV replication in C6/36 cells. The assay demonstrated high robustness and reproducibility, with a Z' factor of 0.755, and showed strong correlation with wild-type virus replication profiles. By screening an 800-compound library, we identified 10 candidates exhibiting >95% inhibitory activity against OYAV replication. Among these, saikosaponin B2, fingolimod (hydrochloride), and fangchinoline displayed micromolar inhibitory effects against both reporter and wild-type OYAV replication, with limited cytotoxicity in C6/36 cells. Collectively, this study establishes the reverse genetics system for OYAV and a quantitative mosquito-cell-based screening platform for orthobunyavirus replication inhibitors, providing a valuable tool for studying OYAV replication and evaluating antiviral compounds in mosquito-derived cells.IMPORTANCEOya virus (OYAV) is an emerging arbovirus circulating in Asia, with documented infection in humans and arthropod vectors. However, the absence of reverse genetics and reporter systems has limited mechanistic studies and inhibitor discovery for OYAV, particularly in arthropod-derived cells. Here, we established a reverse genetics system and generated fluorescent reporter viruses that enable real-time monitoring of OYAV replication. The optimized reporter virus was further used to establish a high-throughput inhibitor screening platform in mosquito C6/36 cells, leading to the identification of three compounds with inhibitory activity and low cytotoxicity in this system. These tools provide a foundation for investigating OYAV biology and facilitate the discovery and evaluation of inhibitors in vector cells and potentially in live vectors.