<p>Selectivity index of candidate compounds.</p>

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.牛结节性皮肤病（Lumpy skin disease, LSD）是由牛结节性皮肤病病毒（lumpy skin disease virus, LSDV）引发的新发跨边界传染病，在尚无获批验证的治疗药物的情况下，对全球肉牛健康构成严重威胁。本研究构建了同时表达mCherry与荧光素酶（luciferase）报告基因的重组牛结节性皮肤病病毒（recombinant LSDV, rLSDV），用于高通量药物筛选；该重组病毒的病毒学表型特征与亲本毒株无显著差异，且报告基因可作为病毒复制精准且灵敏的定量检测指标。依托该筛选平台，我们从抗正痘病毒（anti-orthopoxvirus）化合物库中筛选得到6种候选药物，分别为阿糖胞苷（cytarabine, AraC）、恩诺沙星（enrofloxacin, ENR）、碘苷（idoxuridine, IDU）、氟阿糖碘胞苷（fialuridine, FIAU）、利巴韦林（ribavirin, RBV）以及阿糖腺苷（vidarabine, AraA），这些化合物均展现出较强的抗病毒活性且细胞毒性较低。加药时间窗实验结果显示，所有筛选得到的候选药物均主要作用于病毒复制阶段。机制研究表明，抗牛结节性皮肤病病毒药物可同时抑制病毒与宿主的DNA和/或RNA合成。其中阿糖胞苷（AraC）可显著阻断病毒DNA合成，并抑制病毒晚期基因启动子的激活，从而将病毒复制周期阻断于早期阶段。结构比对数据显示，阿糖胞苷可能通过结合病毒DNA聚合酶的D554、R639、K666、N670及D758位点抑制其酶活性。值得注意的是，尽管上述结合位点在牛源DNA聚合酶中高度保守，但阿糖胞苷仅会引发极轻微的宿主DNA损伤与细胞凋亡，且宿主DNA合成在给药过程中可逐渐恢复。综上，本研究阐明的抗牛结节性皮肤病病毒作用机制，结合抗正痘病毒药物已具备的临床应用基础，均凸显了这些药物作为老药新用疗法治疗牛结节性皮肤病病毒感染的潜力。