An engineered human hepatitis A virus capable of rapid proliferation in vitro and causing hepatitis in mice

Hepatitis A virus (HAV) remains a significant public health threat. The HM175-mp4-based mouse model has advanced pathogenesis research, but its slow in vitro replication limits reverse genetic studies. We aimed to develop a genetically tractable HAV model through rational mutagenesis.

Two cell-adaptive substitutions, A1052V in the 2B protein and F1163S in the 2C protein, were introduced into HM175-mp4 to generate the recombinant HAV-2m virus. HAV-2m was propagated in Huh7.5.1 cells and intravenously injected into Ifnar1 ^-/- mice to establish the animal model. Virological replication kinetics and shedding, as well as liver histopathology and serum alanine aminotransferase levels were monitored in HAV-2m-infected mice. Single-cell RNA sequencing was employed to characterize liver immune cell dynamics. A K1118M substitution in the HAV 2C protein was introduced to evaluate the biological impact.

HAV-2m replicated effectively in vitro and caused acute hepatitis A symptoms in Ifnar1 ^-/- mice, marked by fecal viral shedding, elevated serum alanine aminotransferase, and inflammatory cell infiltration in the liver. Single-cell RNA sequencing revealed activation of T cells, NK cells, macrophages, neutrophils, and monocytes, but their depletion did not mitigate liver injury. The K1118M mutation enhanced in vitro replication but attenuated in vivo replication and virulence in mice.

The HAV-2m reverse genetic system and its mouse model provide a tractable platform for dissecting HAV pathogenesis and evaluating antiviral strategies.

Hepatitis A virus (HAV) is an ongoing public health concern, yet experimental tools to study recombinant HAV mutants remain limited. We developed a genetically tractable recombinant virus (HAV-2m) that replicates efficiently in cell culture, induces acute hepatitis in mice, and revealed a novel virulence determinant in the viral 2C protein. This platform enables detailed dissection of HAV pathogenesis and the evaluation of antiviral countermeasures.