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

Fig. 1. Recovery and characterization of HAV-2m. (A) Schematic diagram showing amino acid substitutions in recombinant viruses and their rescue in Huh7.5.1 cells. (B) Growth curve of the 3rd passage of HAV-2m in Huh7.5.1 cells. Data are means ± SEM; n = 3. ∗∗∗p <0.001, two-way ANOVA. (C) Immunofluorescence analysis of VP1-positive Huh7.5.1 cells at 4 dpi following HM175-mp4 or HAV-2m infection. Scale bar, 20 μm. (D) Schematic diagram illustrating the genomic structures of repFluc-HAV replicons. (E) Luciferase activity measured at different time points in Huh7.5.1 cells transfected with IVT RNAs of repFluc-HAV replicons. Data are means ± SEM; n = 3. ∗∗p <0.01, ∗∗∗p <0.001, multiple unpaired t tests. dpi, days post-infection; IVT, in vitro transcribed; repFluc-HAV, replicon firefly luciferase hepatitis A virus.

Fig. 2. HAV-2m causes obvious liver injury in Ifnar1-/- mice. (A) Schematic diagram of HAV-2m challenge experiment in Ifnar1-/- mice. (B) Serum ALT levels in Ifnar1-/- mice inoculated with HAV-2m. Data are means ± SEM; n = 3 or 5 (shown by open circles in each bar). ∗∗∗p <0.001, two-way ANOVA. (C) RT-qPCR analysis of viral RNA load in feces. Data are means ± SEM; n = 3 or 5 (shown by open circles in each bar). (D) RT-qPCR analysis of relative viral RNA load in the liver. Data was measured by RT-qPCR and normalized to Gapdh mRNA. Data are means ± SEM; n = 3 (shown by open circles in each bar). (E) H&E staining analysis of liver sections from HAV-2m and MOCK group Ifnar1-/- mice at 14 dpi. Black arrow indicates the hepatitis A foci in the liver. (F) IHC analysis of activated caspase-3 in the liver of HAV-2m and MOCK group Ifnar1-/- mice at 14 dpi. Black arrow indicates caspase-3-positive cells in the liver. The black scale bar represents 20 μm. ALT, alanine aminotransferase; dpi, days post-infection; IHC, immunohistochemistry; RT-qPCR, reverse transcription quantitative PCR.

Fig. 3. Single-cell immune landscape of HAV-2m-infected mice liver. (A) Schematic diagram of single-cell RNA sequencing of liver tissue from mice infected with HAV-2m at 14 dpi. (B) UMAP dimensionality reduction algorithm was used to cluster liver cells from MOCK group (n = 3) and HAV-2m infection group (n = 3) mice. (C) Proportions of each cell type obtained from the liver of MOCK and HAV-2m infection groups. (D) A radar chart illustrates the Log2(fold change) in the proportions of corresponding immune cells in the liver of HAV-2m-infected mice compared to the MOCK group. (E) GO-enrichment analysis of the top 100 upregulated genes in immune cells infiltrating the liver after HAV-2m infection reveals their enrichment in specific pathways. dpi, days post-infection; GO, Gene Ontology; UMAP, uniform manifold approximation and projection.

Fig. 4. Depletion of T cells, NK cells, macrophages, neutrophils, and monocytes in mice fails to alleviate HAV-induced liver injury. (A) Schematic diagram illustrating the experimental design of depleting T cells, NK cells, macrophages, neutrophils, and monocytes in mice before and followed by HAV infection. (B) RT-qPCR quantification of viral RNA in feces of HAV-infected mice. Data are means ± SEM; n = 3 or 4 (shown by open circles in each bar). ∗∗p <0.01, two-way ANOVA. (C) Serum ALT activity levels in HAV-infected mice. Data are means ± SEM; n = 3 or 4 (shown by open circles in each bar).(D) RT-qPCR quantification of viral RNA in liver tissues at 14 dpi. Data are means ± SEM; n = 3 or 4 animals (shown by open circles in each bar). (E) H&E staining and activated caspase-3 IHC of liver tissues from mice at 14 dpi. Black arrows in H&E indicate typical hepatitis A foci; black arrows in IHC indicate caspase-3-positive apoptotic cells. Scale bar represents 50 μm. (F) Immunofluorescence analysis of liver sections at 14 dpi showing caspase-3- and CD45-positive cells. Scale bar represents 20 μm. ALT, alanine aminotransferase; dpi, days post-infection; IHC, immunohistochemistry; NK, natural…

Fig. 5. K1118M substitution in 2C protein leads to attenuation of HAV in Ifnar1-/- mice. (A) Schematic diagram of the amino acid substitution of HAV-2m-K1118M viruses. (B) Growth curves of HAV-2m and HAV-2m-K1118M viruses in Huh7.5.1 cells. Data are means ± SEM; n = 3. ∗p <0.05, ∗∗∗p <0.001, two-way ANOVA. (C) Schematic diagram of the experimental approach for inoculating mice with HAV-2m and HAV-2m-K1118M viruses. (D) RT-qPCR detection of relative viral RNA load in the liver. Data was normalized to Gapdh mRNA. Data are means ± SEM; n = 3. ∗∗∗p <0.001, two-way ANOVA. (E) RT-qPCR quantification of HAV genomic RNA levels in mouse feces. Data are means ± SEM; n = 3 or 6. ∗∗p <0.01, two-way ANOVA. (F) Serum ALT of HAV-2m and HAV-2m-K1118M infected Ifnar1-/- mice. Data are means ± SEM; n = 3 (shown by open circles in each bar). ∗∗p <0.01, two-way ANOVA. (G) H&E staining analysis of liver sections from HAV-infected mice. Black arrows indicate the hepatitis A foci in the liver. Black scale bar represents 50 μm. (H) UMAP dimensionality reduction algorithm was used to cluster liver cells from HAV-2m-K1118M virus infection group mice (n = 3). (I) Proportions of each cell type obtained from the…