Milbemycin A4, Derived From Streptomyces bingchenggensis Exhibits Antiviral Activity Against MSRV In Vitro and In Vivo

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.