Selectivity index of candidate compounds.

Lumpy skin disease (LSD) is an emerging transboundary disease caused by lumpy skin disease virus (LSDV), posing significant threats to global cattle health in the absence of validated drugs. Here, we constructed a recombinant LSDV (rLSDV) expressing both mCherry and luciferase reporters for high-throughput drug screening, and the rLSDV retained virological characteristics phenotypically indistinguishable from the parental strain, with the reporter genes serving as precise and sensitive quantitative indicators for viral replication. Leveraging this platform, we identified six candidates from a library of anti-orthopoxvirus compounds, namely cytarabine (AraC), enrofloxacin (ENR), idoxuridine (IDU), fialuridine (FIAU), ribavirin (RBV), and vidarabine (AraA), demonstrating high antiviral activity concomitant with low cytotoxicity. Time-of-addition experiments revealed that all identified candidates primarily inhibited the viral replication phase. Mechanistical analysis revealed that anti-LSDV agents suppressed synthesis of both viral and host DNA and/or RNA. In particular, AraC markedly blocked viral DNA synthesis and prevented activation of viral late gene promoters, thereby arresting the replication cycle at an early stage. Structural alignment data suggested that AraC may bind to the viral DNA polymerase at residues D554, R639, K666, N670, and D758 to inhibit its activity. Notably, AraC induced only minimal host DNA damage and apoptosis, and host DNA synthesis gradually recovered during treatment, although these residues are conserved in bovine DNA polymerase. Hence, the mechanistic landscape delineated herein, together with the established clinical availability of the anti-orthopoxvirus agents, underscore their potential as repurposable therapeutics for LSDV infection.