The Lassa virus (LASV), a member of the Old World arenaviruses and classified as a risk-group 4 pathogen, causes severe hemorrhagic fever in humans with significant mortality. Here, we developed a robust LASV reverse genetics system designated LASVsg that models the complete LASV life cycle under biosafety level-2 (BSL-2) conditions. Using this system, we screened a library of nucleoside/nucleotide analogs for anti-LASV activity. Gemcitabine, a clinically approved nucleoside analog, was identified to exhibit pronounced antiviral efficacy against LASVsg, which was further confirmed using authentic LASV in a BSL-4 laboratory. Mechanistic analyses revealed that gemcitabine suppresses LASV infection by reducing viral protein levels without directly inhibiting viral RNA replication or transcription. In summary, we developed a robust BSL-2 reverse genetics system for LASV research, enabling the identification of gemcitabine as a potential antiviral candidate.
