Coxsackievirus A6 Infection Causes Neurogenic Pathogenesis in a Neonatal Murine Model

Figures

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  Figure 1. Body weight, survival rate, and clinical score of suckling mice in inoculation route and F-virus challenge dose experiments. Five-day-old ICR mice were infected with strain F219 at 103, 104, 105, and 106 TCID50 by intracranial injection (A,D,G), intraperitoneal injection (B,E,H), and intramuscular injection (C,F,I), respectively (n = 7 per group); control mice were inoculated with cell culture maintenance medium (MM). Body weight (A–C), survival rate (D–F) and clinical scores (G–I) of 5-day-old mice inoculated via IC, IP, and IM were monitored daily. Clinical scores of CVA6-infected and control mice. 0, no disease; 1, ruffled fur; 2, weight loss; 3, single limb paralysis; 4, hindlimb paralysis; 5, moribund or dead.

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  Figure 2. Virus titers in tissues and histopathological changes of 5-day-old mice. Intramuscular injection of the CVA6 strain F219 was performed in 5-day-old ICR mice. Mice were then sacrificed after 1, 3, and 5 days, and virus titers were measured in the brain and muscle tissue (A,B). Histopathological analysis showing muscle fiber rupture (D,E), cerebral edema, and neuronal cell swelling (G,H); no changes were observed in control tissues (C,F). The magnification of (C,D,F,G) is × 200, and the magnification of (E,H) is ×400. The scale bar for (C,D,F,G) is 100 μm, and the scale bar for (E,H) is 50 μm.

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  Figure 3. Co-localization of viral antigens with astrocyte marker GFAP in infected brain tissues. Suckling mice were injected with CVA6 or cell culture maintenance medium. Brain tissue sections of suckling mice were fixed and immunostained with DAPI (Blue), VP1 (Red), and astrocyte marker GFAP (Green) (A–H); immunostaining with DAPI (Blue), VP1 (Red), and neuron marker NeuN (Green) (I–P); Magnifications: (A,B) ×400. Scale bar = 20 μm.

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  Figure 4. CVA6 infects U251 cells. Cytopathic effect of virus on U251 at 24 h post infection (A); cells were inoculated with CVA6 at 37 °C, and harvested at 2, 6, 12, 24, and 48 h post-inoculation. Virus titers were determined on RD cells (B); U251 cells infected with CVA6 and control were fixed and immunostained with DAPI (Blue), astrocyte marker (GFAP, Yellow), and VP2 (Green). The representative images were acquired using fluorescence confocal microscopy. Magnifications: (A,B), ×200 × 600. Scale bar = 100 μm (C).

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  Figure 5. Transcriptome analysis of the brain tissues and U251 cells infected with CVA6. Total RNA was extracted from U251 cells and brain tissues of suckling mice and analyzed by sequencing (A). Hiplot Enhanced MA v0.1.0 analysis showing the comparison of the RNA-Seq of differential gene expression of infected U251 cells (B) and that of infected suckling mice brain tissue (C). GO and KEGG pathway enrichment analysis of the RNA-Seq of differentially expressed genes in the infected U251 cells (D,E) and that of infected brain tissue (F,G). The dot plot displays the enrichment score values. Visualization of differentially expressed genes in the brain tissue (H).