Establishment and application of a wild neonatal mouse model infected with an Echovirus 30 isolate

Figures

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  Fig. 1. The major characterizations of the E30/A538 isolate. RD cell monolayers were inoculated with E30/A538 and subsequently covered with an agarose-containing medium overlay. After 5 days, plaques were visualized by staining with neutral red (A). The CPE of E30/A538 on RD cells was observed at 12, 24, 48, and 72 h after infection using an inverted microscope (B). The cell lysate supernatant was analyzed by western blot (C) and qPCR (D).

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  Fig. 2. Susceptibility of different strains of mice to E30/A538. ICR, BALB/c, KM, and C57BL/6 mice were infected through the IC route with the same dose of E30/A538. The body weights (A), clinical scores (B), and survival rates (C) of each group of suckling mice (n = 6) were recorded until 21 days post-infection (dpi). Black arrows marked the quadriplegia (D) in ICR mice on the 5d post-infection, the moribund state (E) of BALB/c mice on the 5d post-infection, the arched back (F) of KM mice on the 5d post-infection, and the weight loss (G) of in C57BL/6 mice on the 12d post-infection. The results displayed the mean ± standard error of the mean (SEM). ****P < 0.0001, ***P < 0.001, “ns” indicates not significant versus ICR

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  Fig. 3. The E30/A538-infected ICR mouse model was developed by optimizing three factors: infection dose, route, and mouse age. Body weights (A, D, and G), clinical scores (B, E, and H), and survival rates (C, F, and I) were calculated for each group (n = 6). The NC was injected with sterile 1× PBS. The results displayed were the mean ± SEM. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. “ns” means “not significant” compared to the NC group

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  Fig. 4. The repeatability of the E30/A538-infected suckling mouse model. The weight changes (A), clinical scores (B), survival rates (C), and clinical manifestations (D) of the mice were recorded until 21 dpi, with a sample size of n = 6. Black arrows indicate the signs of limb paralysis in mice at 5dpi. The results displayed the mean ± SEM. ****P < 0.0001, ***P < 0.001

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  Fig. 5. Histological, immunohistochemical, and viral load analyses in various tissues of ICR suckling mice infected with E30/A538. Tissues were harvested on the 7th day post-infection, at which time the clinical scores for the E30 group and the NC group were 5 and 0, respectively. (A) Tissues from each group were stained with hematoxylin and eosin (H&E). Scale bar represents 100 µm. “6×” represents the image of the small black box has been magnified 6 times. Yellow arrows marked the lesions in E30/A538-infected neonatal mice. (B) Immunohistochemical staining of each group. Scale bar represents 100 µm. “6×” represents the image of the small black box has been magnified 6 times. The brain and heart contained a large number of brownish-yellow E30 antigen particles. (C) The average viral load in tissue of suckling mice infected with E30/A538. The viral loads in the tissues of mice (n = 3) with clinical scores of 4 or higher were measured using qRT-PCR

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  Fig. 6. Anti-E30/A538 effect of IFN-α2a and IFN-γ in vivo. (A) Schematic diagram of antiviral experiments: Twelve one-day-old ICR mice were infected with 5LD50 E30/A538 by IC. IFN-α2a and IFN-γ were injected at 1 hpi, and the same dose was re-administered every 24 hpi for the next 6 days. The NC were injected with sterile 1×PBS. (B) Clinical manifestations: At 11 dpi, all the mice in the virus group died, while those in the IFN-α2a treatment group experienced weight loss. The mice in the IFN-α2a control group and NC group remained healthy. (C-E) The body weights (C), clinical scores (D), and survival rates (E) of each group of suckling mice were recorded until 21 dpi. The results displayed the mean ± S.E.M. ***P < 0.001; “ns” indicates no significant difference compared to the E30/A538 group. (F) IFN-α2a reduced the extent of pathological tissue changes. The brain, heart, spinal cord, limb muscles, fat, and liver of each group were stained with HE. Scale bar represents 100 µm.“6×” represents the image of the small black box has been magnified 6 times. Yellow arrows marked the specific pathological changes are shown in the Table 2