Establishment of severe in vitro and in vivo infection models for coxsackievirus B4 and their application in identifying viral virulence determinants.

Coxsackievirus B4 (CVB4) is a highly pathogenic enterovirus associated with severe neurological disease and mortality. To establish research models that recapitulate severe CVB4 infection, two clinical isolates with distinct neurovirulence-the high-virulence strain GZ-HFM01 and low-virulence strain GZ-R6-were used. An in vitro neurocytotoxicity model with human neuroblastoma SH-SY5Y cells showed that GZ-HFM01 produced significantly larger plaques than GZ-R6, reflecting its increased capacity to damage neuronal cells. Concurrently, an optimized in vivo severe infection model was established in 3-day-old ICR mice through intraperitoneal inoculation, which reproduced key clinical features of severe disease. Compared with GZ-R6, GZ-HFM01 infection resulted in significantly reduced survival, progressive neurological impairment, time-dependent viral accumulation in brain tissue, pronounced histopathological injury, and elevated serum pro-inflammatory cytokine levels. To map genomic determinants of neurovirulence, a chimeric virus panel was generated by replacing individual genome segments (5'untranslated region [UTR], P1, P2, P3, or 3'UTR) of GZ-HFM01 with the corresponding regions from GZ-R6. Evaluation with the established models demonstrated that replacement of the P2 region significantly attenuated both the cytopathic effect in SH-SY5Y cells and pathogenicity in 3-day-old ICR mice. Animal studies further indicated that substitution of the 5'UTR or P1 region also reduced virulence-a phenotype absent from the cell-based model-underscoring the multifactorial regulation of CVB4 pathogenesis. In conclusion, this study provides validated in vitro and in vivo models of severe CVB4 infection and identifies key genomic segments that contribute to neurovirulence, offering a foundation for mechanistic research and the development of targeted interventions against severe CVB4-induced disease.