Palmatine inhibits largemouth bass ranavirus replication and enhances host antiviral defense.

Largemouth bass ranavirus (LMBV), a member of the family Iridoviridae, is a major viral pathogen in largemouth bass aquaculture. Here, we investigated the antiviral activity of palmatine, a plant-derived isoquinoline alkaloid, against LMBV infection in vitro and in vivo. Palmatine showed no detectable cytotoxicity in epithelioma papulosum cyprini (EPC) cells at concentrations up to 100 μg/mL and inhibited LMBV replication in a dose-dependent manner, with an IC50 of 21.53 μg/mL. At 100 μg/mL, palmatine reduced viral gene expression by more than 90%, markedly attenuated cytopathic effects (CPE), and decreased extracellular and intracellular viral titers. Virucidal, adsorption, internalization, and time-of-addition assays showed that palmatine did not directly inactivate viral particles and had limited effects on viral entry, but most strongly inhibited infection when present during the intracellular replication phase, particularly between 4 and 8 hpi. Consistent with this mode of action, palmatine alleviated LMBV-induced cytoskeletal disruption, preserved mitochondrial membrane potential, and restored mitochondrial elongation. Transcriptomic analysis further showed that palmatine partially reversed LMBV-induced host transcriptional reprogramming, including changes in pathways related to mitophagy, ferroptosis, cytokine signaling, and aminoacyl-tRNA biosynthesis, while reducing stress- and inflammation-associated responses. In vivo, palmatine treatment improved survival of LMBV-infected largemouth bass, reduced viral loads in the liver, spleen, and kidney, and enhanced early induction of ifnγ and irf3. Together, these results indicate that palmatine inhibits LMBV infection primarily through host-directed antiviral and immunomodulatory effects and support the potential of palmatine as a candidate antiviral agent for largemouth bass aquaculture.