A mouse-adapted Yezo virus model for antiviral testing in immunocompetent mice

Figures

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  Fig. 1. Generation and characterization mouse-adapted YEZV (MA-YEZV). a Schematic representation of the viral adaptation process in mice. Three-week-old female C57BL/6 J mice were intraperitoneally inoculated with 104 TCID₅₀ of the T-HLJ01 strain. Every three days, liver tissues from infected mice were collected, homogenized, centrifuged, and filtered before being passaged into naïve mice. From passage 30, the interval between passages was shortened to 2.5 days. At passage 40, the virus was isolated from the liver tissue of infected mice. b Viral titers in mouse liver (n = 1 independent mice per time point) at passages 0, 10, 20, 30, and 40. c, d Comparison of viral RNA copies (c) and infectious titers (d) for T-HLJ01 and MA-YEZV in the supernatants of Vero cells over a 7-day period. Data are presented as mean ± SD (n = 3 biological replicates). Statistical significance between two groups at each time point was determined using a two-sided unpaired Student’s t-test. Exact p-values are indicated in the figure. e Immunofluorescence images of Vero cells infected with T-HLJ01 or MA-YEZV at the indicated multiplicity of infection (MOI). Cells were stained for YEZV nucleoprotein (NP) (green)…

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  Fig. 2. MA-YEZV induces lethal infection in immunocompetent C57BL/6 J mice. a Schematic representation of MA-YEZV infection in C57BL/6 J mice. Three-week-old female and male C57BL/6 J mice were intraperitoneally inoculated with MA-YEZV at different doses (10⁴, 10², or 1 TCID50). Body weight and survival were monitored daily. At designated time points, tissues and serum were collected for viral load analysis. b, c Relative changes in body weight (b) and survival rates (c) of MA-YEZV-infected mice (n = 12 independent mice per group) compared to mock-infected controls (n = 6 independent mice). d Viral loads in tissues and serum of intraperitoneally infected mice (n = 3 biological replicates) measured by RT-qPCR at 3, 7, and 10 days post-infection. Data are presented as mean ± SD (b, d). Source data are provided as a Source Data file.

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  Fig. 3. Liver pathology in C57BL/6 J mice infected with MA-YEZV. Three-week-old male C57BL/6 J mice were intraperitoneally inoculated with 1 TCID50 of MA-YEZV or 104 TCID50 of the T-HLJ01 strain. Ten days post-infection, liver and blood samples were collected for pathological and biochemical analyses. a Liver histopathological assessment, including gross morphology, hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) using an antibody against the YEZV nucleoprotein (NP), was performed to evaluate the pathogenicity of MA-YEZV. Scale bars, 100 μm (H&E) and 50 μm (IHC). b Serum biochemical markers are indicative of liver injury. Data are presented as mean ± SD (n = 3 biological replicates). ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase. Statistical significance was determined by one-way ANOVA (two-sided) followed by Tukey’s multiple-comparison test to adjust for multiple testing. Exact p-values are provided in the figure legends. c Immunofluorescence showing co-localization of YEZV NP (green) with cellular markers: hepatocytes (albumin, ALB; red), liver sinusoidal endothelial cells (LSECs; CD31; red), hepatic stellate cells…

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  Fig. 4. MA-YEZV induces robust inflammatory responses. Three-week-old male C57BL/6 J mice were intraperitoneally inoculated with 1 TCID50 of MA-YEZV or 104 TCID50 of the T-HLJ01 strain. Liver and blood samples were collected at designated time points for cytokine analysis. a Viral loads in the liver and serum of mice infected with MA-YEZV or T-HLJ01 strains (n = 3, biological replicates) were measured by RT-qPCR at 3 and 7 days post-infection (dpi). b Serum cytokine profiles assessed by CBA in mice infected with T-HLJ01 or MA-YEZV (n = 3, biological replicates) at 3 and 7 dpi. Analytes measured include IL-6, IL-10, IL-12p70, IFN-γ, MCP-1, TNF-α. c Hepatic cytokine mRNA expression levels measured by RT-qPCR in MA-YEZV-infected mice (n = 3 biological replicates, relative expression). Data are presented as mean ± SD (a, b). Statistical significance between two groups at each time point was determined using a two-sided unpaired Student’s t-test. For comparisons among multiple groups, one-way ANOVA (two-sided) was performed, followed by Tukey’s multiple-comparison test to adjust for multiple testing. Exact p-values are indicated in the figure. Source data are provided as a Source Data file.

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  Fig. 5. Antiviral efficacy of candidate drugs in MA-YEZV infected C57BL/6 J mice. a Schematic of the antiviral treatment protocol. Three-week-old male C57BL/6 J mice were intraperitoneally inoculated with 1 TCID50 of MA-YEZV. Two hours post-infection, mice were treated with either ribavirin (50 or 100 mg/kg) or remdesivir (10 or 30 mg/kg), while vehicle-treated mice served as controls. Antiviral treatments were administered once daily for a total of 8 consecutive days. Body weight and survival were monitored daily. At designated time points, tissues and serum were collected for viral analysis. b, c Effects of antiviral treatment on body weight changes (b) and survival rates (c) of MA-YEZV-infected mice (n = 12 independent mice per group), compared to vehicle-treated controls (n = 6 independent mice). d Viral load reduction in tissues and serum of infected mice (n = 3 biological replicates) treated with antiviral drugs or vehicle after MA-YEZV infection. e Infectious virus titers in the liver, spleen, and serum of infected mice (n = 3 biological replicates) treated with antiviral drugs or vehicle after MA-YEZV infection. Data are presented as mean ± SD (b, d, e). Statistical significance…

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  Fig. 6. Ribavirin treatment mitigates MA-YEZV-induced hepatic pathology and inflammation. Three-week-old male C57BL/6 J mice were intraperitoneally inoculated with 1 TCID50 of MA-YEZV. Ten days post-infection, liver and blood samples were collected for pathological, biochemical, and cytokine analyses. a Liver histopathology assessment, including gross morphology, hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) staining using an antibody against the YEZV nucleoprotein (NP), comparing mock-infected mice to MA-YEZV-infected mice treated with ribavirin or remdesivir. Scale bars, 100 μm (H&E), 50 μm (IHC). b Serum biochemical markers are indicative of liver injury. Data are presented as mean ± SD (n = 3 biological replicates). ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase. Statistical significance was determined by one-way ANOVA (two-sided) followed by Tukey’s multiple-comparison test to adjust for multiple testing. Exact p-values are provided in the figure legends. c Hepatic cytokine mRNA expression levels measured by RT-qPCR in infected mice treated with ribavirin and remdesivir (n = 3 biological replicates, relative…