3-Indoleacetonitrile inhibits Singapore grouper iridovirus via dual mechanisms: direct interference with viral life cycle and activation of IRF3-mediated type I interferon response.

Singapore grouper iridovirus (SGIV) causes severe systemic infections and high mortality in farmed groupers, leading to substantial economic losses. Natural products with immunomodulatory properties have emerged as promising eco-friendly alternatives for antiviral prevention and control in aquaculture. In this study, the antiviral activity and underlying mechanisms of 3-indoleacetonitrile against SGIV were investigated in grouper spleen (GS) cells and in hybrid grouper. The results showed that 3-indoleacetonitrile exhibited no cytotoxicity at concentrations up to 62.5 μg/mL in GS cells, and significantly inhibited SGIV replication in a dose-dependent manner. Further mechanistic studies revealed that 3-indoleacetonitrile targeted multiple steps of SGIV infection by reducing viral particle infectivity, disrupting viral attachment and entry, and inhibiting intracellular viral replication. Furthermore, 3-indoleacetonitrile alone strongly induced the expression of type I interferon (IFN) and interferon-stimulated genes (ISG15 and MXI), and promoted the nuclear translocation of interferon regulatory factor 3 (IRF3). Notably, 3-indoleacetonitrile also enhanced the IFN response triggered by SGIV infection. In vivo, 3-indoleacetonitrile pretreatment significantly reduced SGIV load in grouper liver and spleen. Overall, these findings demonstrate that 3-indoleacetonitrile inhibits SGIV infection through both direct antiviral action and activation of the IRF3-mediated type I IFN pathway, suggesting its potential as a natural candidate for the prevention and control of grouper iridovirus disease. To our knowledge, this is the first report describing the antiviral efficacy of 3-indoleacetonitrile against the aquatic ranavirus SGIV.