Single-cycle Rift Valley fever virus particles from stable replicon cells enable discovery of antiviral CNX-1351 for multiple RNA viruses.

Rift Valley fever virus (RVFV) is a high-containment pathogen that causes severe diseases in humans, with no approved therapeutics available. Its classification as a biosafety level 3 (BSL-3) agent has limited research and therapeutic development due to safety concerns. In this study, we developed a stable replicon cell line maintaining the replication of L and S genomic segments of RVFV. Single-cycle viral replicon particles (VRPs) could be efficiently packaged through trans-complementation of glycoproteins from different strains, recapitulating authentic viral entry and replication while minimizing biosafety risks. Using this system, we conducted high-throughput screening of a small-molecule compound library and identified CNX-1351 as an antiviral agent for multiple RNA viruses. Mechanistic studies revealed that CNX-1351 inhibits viral replication, potentially by targeting the PI3K-Akt signaling pathway. This single-cycle VRP system provides a valuable tool for studying RVFV biology, host interactions, antiviral and vaccine development under reduced biosafety constraints.