Semi-Synthetic Tylosin A Derivative T3 Inhibits Lumpy Skin Disease Virus Replication at the Viral DNA Replication Stage

Lumpy skin disease virus (LSDV), a capripoxvirus that causes substantial animal-health and economic losses in cattle, remains a major veterinary concern, with few specific antiviral options available. Here, a series of semi-synthetic Tylosin A derivatives were evaluated for anti-LSDV activity using an LSDV-eGFP infection model in Madin–Darby bovine kidney cells. Initial screening identified T3 and T8 as the most active derivatives, reducing infectious virus production by 89.6% and 91.0%, respectively, at 200 ppm. Dose–response analysis based on infectious-virus titration yielded IC50 values of 23.02 ppm (95% CI, 18.48–28.33) for T3 and 19.13 ppm (95% CI, 13.87–25.32) for T8. The corresponding CC50 values were 748.3 and 621.4 ppm, resulting in selectivity indices of 32.50 and 32.48, respectively. Mechanistic studies focusing on T3 showed that antiviral activity was most pronounced when the compound was administered after viral entry and during the early post-infection period. T3 reduced viral genomic DNA accumulation beginning at 8 h post-infection while largely preserving early viral gene transcription and suppressing DNA-replication-dependent intermediate and late gene expression. This phenotype closely resembled that induced by the DNA synthesis inhibitor cytarabine (AraC), supporting an inhibitory effect of T3 at or upstream of the viral DNA replication stage. Proteome-wide molecular docking did not reveal preferential docking of T3 to the selected viral DNA replication-associated proteins, and direct target engagement remains to be established. Collectively, these findings identify T3 and T8 as promising anti-LSDV lead compounds and support further optimization of the Tylosin A scaffold, while mechanistic studies suggest that T3 exerts its antiviral effects primarily during the viral DNA replication phase.