Spinal Cord Injury in Enterovirus D68 Infection: Mechanisms and Pathophysiology in a Mouse Model

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  Figure 1. Establishment of EV-D68-infected mouse models. Seven-day-old ICR mice were infected with EV-D68 D2-P9 at doses of 105–1011 TCID50 via i.m. injection (n = 6 per group); control mice were inoculated with uninfected culture medium. The survival rates, clinical scores, and body weight changes were monitored daily (A–C). Institute of Cancer Research (ICR) mice were infected with 109 TCID50 of EV-D68 D2-P9 via i.c., i.p., or i.m., with survival rates, clinical scores, and body weight changes recorded daily (D–F). ICR mice aged 5, 7, or 12 days were infected with 109 TCID50 of EV-D68 via the i.m. route, and survival rates, clinical scores, and body weight changes were assessed daily (G–I). The experiment was independently repeated three times.

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  Figure 2. Virus titers in tissues of mice infected with EV-D68 D2-P9. Seven-day-old ICR mice were infected with EV-D68 D2-P9 via intramuscular injection, and the viral titers in different tissues (hindlimb muscle, forelimb muscle, spinal cord, intestine, kidney, lung, spleen, heart, brain, and liver) were measured at 1, 3, 5, and 7 dpi. Data are presented as the mean ± SD from three mice per group. The experiment was independently repeated three times.

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  Figure 3. EV-D68 infection leads to severe histopathological changes in multiple organs of neonatal mice. Seven-day-old ICD mice were inoculated with EV-D68 D2-P9 strain via intramuscular injection or mock infection as controls. Tissues were collected for hematoxylin and eosin (H&E) staining five days post-infection (dpi). The spinal cord of EV-D68-infected mice showed extensive inflammatory cell infiltration (black arrows) (A,B). The spinal cord of mock-infected control mice exhibited normal structure (C,D). Muscle fibers in the forelimb skeletal muscle of infected mice exhibits necrosis, breakage (arrows), and immune cell infiltration (E,F). No pathological changes were observed in the control group (G,H). Muscle fibers in the hind limb skeletal muscle of infected mice exhibits necrosis, breakage (arrows), and immune cell infiltration (I,J). No pathological changes were observed in the control group (K,L). Lung tissue of the infected group showed significant thickening of alveolar walls (interstitial pneumonia) and inflammatory exudate in alveolar cavities (arrows) (M,N). No pathological changes were observed in the control group (O,P). Mild pathological changes were observed in the…

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  Figure 4. Immunohistochemical detection of the distribution of EV-D68 antigen in multiple tissues of infected mice. After intramuscular inoculation with EV-D68 D2-P9 in 7-day-old mice, IHC was employed to analyze the distribution of viral antigens (arrows) in the hindlimb skeletal muscle (A–D), spinal cord (E–H), lung (I–L), and brain (M–P) at 5 dpi. Magnifications: (A,C,E,G,I,K,M,O), Scale bars: 200 µm; other panels, Scale bars: 50 µm.

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  Figure 5. Immune cell infiltration in EV-D68 D2-P9-infected ICR mice. Flow cytometry analysis of CD45+ leukocytes (total leukocytes) in the spleen, spinal cord, and lung tissues from 7-day-old ICR mice infected with the EV-D68 D2-P9 strain, compared to mock-infected controls (n = 9). (A) Proportions of immune cells in the spinal cord, spleen, and lung; (B–D) The absolute counts of immune cells in the spinal cord, spleen, and lung; (E–H). The significance of the data was determined by the two-tailed Student’s t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001. The experiment was independently repeated three times. Data are represented as mean ± SEM (9 mice per group).

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  Figure 6. Transcriptomic analysis of EV-D68 D2-P9 strain infection in ICR mice. Differential gene expression analysis in tissues from 7-day-old ICR mice infected with the EV-D68 D2-P9 strain at 5 dpi. RNA-seq was performed on spinal cord, brain, lung, and hindlimb skeletal muscle tissues. The heatmap shows the gene expression differences in muscle tissue and spinal cord (A,B). Venn diagram illustrating the overlap of upregulated genes across the four tissues (C). Functional enrichment analysis of the 67 commonly upregulated genes using GO and KEGG pathways (D,E). Comparative analysis of spinal cord-specific DEGs highlights unique pathways (F).