Zebrafish larvae as an in vivo immersion model to dissect neurotoxicity and innate immune responses induced by nervous necrosis virus

High-content in vivo infection pipelines that integrate phenotypic, molecular, and ultrastructural endpoints can accelerate mechanistic studies and intervention screening for aquatic animal diseases. Zebrafish (Danio rerio) larvae provide a scalable vertebrate system for such applications, particularly at 4 days post-fertilization (dpf) when immunity is exclusively innate and exogenous feeding has not yet begun. Here, we implemented an immersion-based workflow to resolve early host–pathogen dynamics using nervous necrosis virus (NNV), a neurotropic RNA virus of major aquaculture concern. Larvae were challenged by immersion and assessed for neurobehavior, growth, and mortality during an early post-infection window. Viral replication was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR), capsid protein accumulation by Western blotting, and infectious virus was re-isolated from larval tissues. Transmission electron microscopy revealed early neural injury, including mitochondrial swelling and virus-like particles in brain tissue. RNA sequencing further showed pronounced activation of innate antiviral and oxidative stress programs, accompanied by suppression of genes implicated in neuronal maintenance. Pathway enrichment analyses highlighted coordinated perturbation of Janus kinase–signal transducer and activator of transcription (JAK–STAT), Forkhead box O (FoXO), cytokine signaling, and apoptosis-related pathways. Collectively, these results define an early molecular and cellular signature of NNV-associated neurotoxic stress and innate immune dysregulation, and provide a reproducible, information-rich vertebrate workflow with biotechnological utility for mechanistic virology and scalable evaluation of antiviral or host-directed interventions in aquaculture.