Engineering an efficient tomato spotted wilt virus mini-replicon system for high-throughput antiviral compound discovery

First establishment of a plant negative-strand RNA virus reverse genetics system in the absence of viral suppressors of RNA silencing (VSRs). Development of a dual-vector-driven mini-replicon system enabling high-throughput screening of antiviral agents for sustainable crop protection. Plant viral diseases pose a persistent threat to global agriculture, requiring efficient platforms for antiviral agent screening to ensure sustainable crop protection. Here, we developed a simplified mini-replicon reverse genetics (RG) system for tomato spotted wilt virus (TSWV) based on co-expression of SR (+)eGFP replicon and L (+)opt genome in the absence of viral suppressors of RNA silencing (VSRs). Using this system, we demonstrated that the movement protein NSm and M (-)opt genome significantly enhance both the replication and cell-to-cell movement of the TSWV mini-replicon. We further constructed a tandem expression vector (SR (+)eGFP -M (-)opt ), and established a novel dual-vector-driven TSWV RG system. Notably, this system achieved 100% systemic infection efficiency without requiring any VSR. This is unprecedented for plant negative-strand RNA virus reverse genetics. This optimized system enabled the screening of antiviral agents, among which ribavirin, ningnanmycin, chitooligosaccharide, cellobiose, azadirachtin, and copper sulfate (CuSO 4 ) potently inhibited TSWV infection. Our study provides a new RG manipulation tool for functional genomics of plant negative-strand RNA viruses (NSVs) and a powerful platform for high-throughput antiviral agents discovery.