A novel host-targeted inhibitor of clathrin-mediated endocytosis limits spring viremia of carp virus infection

ABSTRACT Spring viremia of carp virus (SVCV), an aquatic rhabdovirus, causes lethal disease and major economic losses in carp aquaculture, yet no licensed antivirals are available. In this study, we identify the plant-derived diterpenoid quinone cryptotanshinone (CPT) as a potent inhibitor of SVCV that acts by inhibiting clathrin-mediated endocytosis (CME) and attenuating early mitochondrial apoptosis. CPT was well tolerated by epithelioma papulosum cyprinid cells (50% cytotoxic concentration [CC 50 ] = 303.2 µM) and suppressed SVCV replication in a dose-dependent manner (50% inhibitory concentration [IC 50 ] = 3.2 µM), reducing nucleoprotein (SVCV- N ) gene expression and infectious virion production by >99% at 16 μM. Time-of-addition/removal and virion pre-incubation assays revealed that CPT acts predominantly at early entry or early post-entry steps: CPT did not impair viral attachment but selectively blocked internalization, causing DiO-labeled SVCV particles to remain trapped at the plasma membrane. Mechanistically, CPT inhibited CME, as evidenced by reduced transferrin uptake and redistribution of clathrin heavy chain from the cytosol to the cell surface. CPT also preserved mitochondrial integrity by limiting Bcl-2-associated X protein translocation, cytochrome c release, and caspase-9/3 activation. In vivo , therapeutic bath treatment with 4 μM CPT significantly lowered viral loads, decreased cumulative mortality, and reduced horizontal transmission in juvenile common carp, whereas prophylactic oral CPT primed type I interferon and interferon-stimulated gene expression, and markedly improved survival after SVCV challenge. Together, these findings define CME-dependent internalization as a druggable entry checkpoint for an aquatic rhabdovirus and highlight CPT as a promising antiviral lead for integrated control of SVCV. IMPORTANCE Viral diseases are a major bottleneck to sustainable aquaculture, yet most control strategies still depend on husbandry and culling rather than on mechanism-based antiviral therapy. For spring viremia of carp virus (SVCV), a notifiable rhabdovirus of global concern, it remains unclear whether host entry pathways in fish are realistically “druggable” and how small molecules can be integrated into practical farm interventions. Here, we use cryptotanshinone (CPT) as a chemical probe to demonstrate that clathrin-mediated endocytosis in fish cells can be selectively perturbed to block SVCV internalization, and that this strategy is compatible with immersion and oral delivery in juvenile carp. At the same time, CPT reshapes host responses by stabilizing mitochondrial function and sustaining antiviral signaling, thereby linking a defined entry pathway to cell-death control and innate immunity in vivo . These findings open a conceptual route toward host-targeted, entry-focused antivirals for aquatic rhabdoviruses and provide a translational framework for developing environmentally compatible antivirals in aquaculture.