Human norovirus (hNoV) is the leading global cause of acute gastroenteritis, imposing a substantial health and economic burden worldwide. Progress in understanding hNoV pathogenesis has been hindered by the lack of tractable small-animal models that recapitulate symptomatic infection. Although zebrafish larvae support hNoV replication, infection remains asymptomatic, limiting their utility for studying disease mechanisms and host-pathogen interactions. In this study, we report that the zebrafish embryo infection model, in which microinjection of hNoV at the early cell stage, resulted in robust systemic viral replication accompanied by overt pathological manifestations, including pericardial and renal edema, yolk and cranial opacity, and mortality by 3 days post-infection. Disease severity displayed marked individual variability and correlated closely with viral burden. Integrated multi-omics analyses, including bulk transcriptomics, untargeted metabolomics, and single-cell RNA sequencing, demonstrated that embryonic infection elicits a stronger and more coordinated antiviral response than larval-stage infection, while enabling widespread viral dissemination across diverse cell lineages. Approximately two-thirds of infected cells were derived from the nervous system or neural crest lineages, providing a potential mechanistic basis for the neurological complications occasionally reported in hNoV-infected patients. Furthermore, we identified a developmental stage-dependent role for extracellular vesicle (EV)-associated hNoV transmission: free virions mediated more efficient infection and higher symptomatic incidence in immunologically immature embryos, whereas EV-associated virions exhibited enhanced infectivity in more immunocompetent larvae. Together, these findings establish the zebrafish embryo as a versatile and accessible in vivo platform for studying symptomatic hNoV infection, reveal host maturity-dependent viral transmission strategies, and provide new opportunities for mechanistic studies and high-throughput evaluation of antiviral and vaccine candidates. ### Competing Interest Statement The authors have declared no competing interest.
Rxiv mechanobio