Development of a lethal adult mouse model of Coxsackievirus B1 infection for antiviral research

Figures

• 

  Figure 1. Rescue and characterization of rCVB1-XM0108 strain derived from the cDNA clone (A) The viral genomic RNA of CVB1-XM0108 generated by in vitro transcription of the infectious cDNA clone. (B) Characterization of cytopathic effects induced by CVB1-XM0108 and rCVB1-XM0108 in HeLa cells at 12 hpi. (C) Visualization of CVB1 viral particles using transmission electron microscopic analysis. (D) Dose-response cytotoxicity of CVB1-XM0108 and rCVB1-XM0108 in HeLa cells was assessed across a logarithmic MOI range (101 to 10−5). Viability measurements (n = 3 per point) from three independent experiments are expressed as percentages of untreated controls and presented as mean ± SEM. (E) Comparative one-step growth curves of CVB1-XM0108 and rCVB1-XM0108 (MOI = 0.1) in HeLa cells. Viral progeny production was monitored at indicated time points (n = 3 per time point) through three independent experiments, presented as means ± SEM. (F) Immunofluorescence staining for viral VP1 protein (green) and double-stranded RNA (red) in HeLa cells infected with CVB1 strains at an MOI of 1 for 14 h. The cell nuclei were stained blue using DAPI. Scale bars: 200 μm in (B), 50 nm in (C), and 10 μm in (F),…

• 

  Figure 2. Virulence characterization of rCVB1-XM0108 in BALB/c infection model Dose-dependent paralysis and mortality in 7-week-old BALB/c mice infected with serial doses of rCVB1-XM0108 (1 × 105–1 × 108 TCID50). (A) Body weight (mean ± SEM), (B) clinical scores (mean ± SEM), and (C) survival rates were monitored for 21 days post-infection (n = 6 per group). (D) Representative clinical manifestations including progressive emaciation and hunched posture in rCVB1-XM0108-infected mice compared with mock-infected control. Statistical significance between dose groups was annotated on the graphs. For statistical analysis, body weight (A) was analyzed using a mixed-effects model with the Šídák multiple-comparisons test, clinical scores (B) were analyzed using two-way ANOVA, and survival curves (C) were compared using the log rank (Mantel-Cox) test. ∗, p < 0.05; ∗∗∗∗, p < 0.0001. Experiments were independently repeated three times, and representative results are shown.

• 

  Figure 3. Pathological alterations and serum biochemistry in rCVB1-XM0108-infected BALB/c mice Seven-week-old BALB/c mice were intravenously injected with 1 × 107 TCID50/mouse of rCVB1-XM0108 or equal volume of PBS (mock). At 4 days post-infection (dpi), organs (heart, liver, spleen, lung, kidney, brain, pancreas, and limb muscle) were harvested from moribund mice for histopathological analysis. Concurrently, serum biochemical parameters were assessed over 7 dpi. (A) Pancreatic histology showing acute inflammatory lesions (solid black triangles). (B and C) Comparative H&E staining and VP1 immunohistochemistry (IHC) of PBS (B) and rCVB1-XM0108-infected (C) tissues. In mock pancreas (B), dashed line demarcated islet architecture. rCVB1-XM0108-infected pancreas (C) exhibited disrupted islet-acinar interfaces (open triangle) and VP1+ signals in exocrine pancreas (solid triangle). Scale bars: 50 μm. (D–F) Serum biochemistry (n = 5 per time point) revealed elevated α-amylase (α-AMY) (D), alanine aminotransferase (ALT) (E), and aspartate aminotransferase (AST) (F) levels during the acute infection period (1–7 dpi). Data are shown as individual values, with the horizontal line indicating the mean.…

• 

  Figure 4. Tissue tropism of rCVB1-XM0108 in murine pancreas Pancreatic tissues were harvested from 7-week-old BALB/c mice at 4 days post-infection with 1 × 107 TCID50/mouse of rCVB1-XM0108 or PBS (mock). Multiplex immunofluorescence analysis was performed on serial sections co-stained for insulin (red), glucagon (yellow), VP1 protein (green), and DAPI (blue). (A) Mock-infected control showing intact islet-acinar architecture. (B) rCVB1-XM0108-infected pancreas demonstrating viral tropism for exocrine regions. Scale bars: 100 μm. Representative images from three independent experiments are shown.

• 

  Figure 5. Tissue viral load in rCVB1-XM0108-infected mice Seven-week-old BALB/c mice were intravenously inoculated with rCVB1-XM0108 at a dose of 1 × 107 TCID50/mouse. Serum and tissue samples (including heart, liver, spleen, lung, kidney, brain, pancreas, and limb muscle) were collected daily from 1 to 6 days post-infection (dpi). Subsequently, viral RNA copies in these tissues were quantified by RT-qPCR. As shown in the figure, viral loads across different tissues are presented as mean log10 copies/mL or/mg tissue ± SEM (n = 3 per time point). For reproducibility, the experiment was independently repeated three times, with representative results displayed.

• 

  Figure 6. Evaluation of inflammatory responses in vivo Seven-week-old BALB/c mice were intravenously inoculated with 1 × 107 TCID50/mouse of rCVB1-XM0108. Serum was collected at 4 days post-infection (dpi) and analyzed using the Mouse Cytokine Array Panel A (R&D Systems). (A) Displaying a 40-cytokine detection matrix. (B) Representative cytokine expression profiles in rCVB1-XM0108-infected serum compared with PBS controls, with significantly altered cytokines/chemokines boxed in black. (C) Quantified serum cytokine levels (mean ± SEM, n = 3 per group). (D–G) IL-6, IL-10, IFN-γ, and TNF-α levels were measured by ELISA from 1–7 dpi (n = 5 per time point). Data are shown as individual values, with the horizontal line indicating the mean. Data were analyzed by the unpaired two-tailed Student t test in GraphPad Prism 10.0. n.s., not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. Experiments were independently repeated three times, and representative results are shown.

• 

  Figure 7. Protection efficacy of anti-CVB1 sera in BALB/c infection model (A–C) Seven-week-old BALB/c mice (n = 6 per group) were intravenously inoculated with 1 × 107 TCID50/mouse of rCVB1-XM0108. At 2 h post-infection, 200 μL of serially diluted anti-CVB1 sera or PBS were intraperitoneally administered. Mice were monitored for 21 days to assess (A) body weight (mean ± SEM), (B) clinical scores (mean ± SEM), and (C) survival rates. Statistical significance between dose groups was annotated on the graphs. Clinical scores (B) were analyzed using a mixed-effects model with the Šídák multiple-comparisons test. Survival curves (C) were compared using the log rank (Mantel-Cox) test. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. Experiments were independently repeated three times, and representative results are shown.

• 

  Figure 8. Antiserum therapy attenuates rCVB1-XM0108-induced tissue damage and viral dissemination Seven-week-old BALB/c mice were intravenously injected with 1 × 107 TCID50 of rCVB1-XM0108. At 2 h post-infection, 200 μL of 1:10-diluted antiserum or PBS control was administered intraperitoneally. (A) Gross pancreatic morphology at 4 dpi: antiserum-treated mice show intact architecture, while PBS controls exhibit inflammatory foci (triangles). (B and C) H&E and immunohistochemical analysis of multiple organs (heart, liver, spleen, lung, kidney, brain, pancreas, and limb muscle) at 4 dpi: (B) antiserum-treated group, (C) PBS-treated group. Scale bars: 50 μm. (D) Viral RNA loads in tissues and serum from 1–6 dpi (mean ± SEM, n = 3 per group/time point). At each time point, viral RNA loads between the antiserum-treated and PBS control groups were compared using an unpaired two-tailed Student t test. n.s., not significant; ∗, p < 0.05; ∗∗, p < 0.01. Experiments were independently repeated three times, and representative results are shown.