Abstract
The highly pathogenic Micropterus salmoides rhabdovirus (MSRV) poses a serious threat to largemouth bass aquaculture, leading to considerable economic losses. We assessed the anti‐MSRV activity of milbemycin A4 (A4), a macrolide derived from Streptomyces bingchenggensis in both cell‐based and animal models. Cytotoxicity assays revealed that A4 was not toxic to EPC cells at concentrations up to 6.4 mg/L. It significantly reduced viral N gene expression by approximately 90.25%. A4 also suppressed MSRV‐induced apoptosis, decreasing the apoptotic cell population by about 21.88%, and preserved mitochondrial membrane potential. Investigations of the viral life cycle demonstrated that A4 directly targets virions, impairing their binding and internalization, and additionally inhibits early post‐entry replication events. In vivo analyses further revealed that A4 treatment led to significant induction of antiviral‐related genes ( IRF3 , IRF7 and IFN‐γ ), which was accompanied by enhanced survival of infected fish (54%), along with reduced viral accumulation in the liver and spleen and alleviated histopathological damage. These findings suggest that A4 exhibits strong anti‐MSRV activity by targeting early infection stages, protecting mitochondrial integrity and enhancing host innate immunity, highlighting its potential for application in aquaculture disease control.