A standardized microinjection framework with hierarchical quantitative evaluation supports multi-genotype human norovirus replication modeling in zebrafish embryos

AIMS: To establish a reproducible and consistent zebrafish embryo model for multi-genotype human norovirus (HuNoV) replication, by addressing the critical bottleneck of technical variability in microinjection and systematically characterizing the infection dynamics across genotypes. METHODS AND RESULTS: We developed a standardized microinjection framework anchored by a hierarchical quantitative evaluation scheme (based on CLSI EP28-A3c) to minimize operator-dependent variability. This system enabled consistent, low-damage delivery of the viral inoculum. The framework's robustness was confirmed by a strong correlation between net injection efficiency and embryo survival (Pearson r = 0.67, P < 0.05). Leveraging this standardized approach, we successfully constructed stable replication models for five HuNoV genotypes (GII.2[P16], GII.4[P31], GII.4[P16], GII.17[P17], and GII.3[P12]), determining the minimum effective inoculum titer and defining the replication kinetics for each. CONCLUSIONS: Our work provides a comprehensive and standardized methodology that significantly enhances the reproducibility of the zebrafish embryo model for HuNoV research. By effectively decoupling technical variability from genuine biological effects, this framework establishes a robust platform for comparative studies of viral pathogenesis, host interactions, and antiviral efficacy across multiple norovirus genotypes.