A neonatal mouse model of central nervous system infections caused by Coxsackievirus B5

Figures & data

Fig. 1 CV-B5 susceptible mouse model with CV-B5/JS417.

a The process of selecting a suitable mouse strain. One-day-old mice of various strains (BALB/c, C57BL/6, KM, ICR, and NIH) were i.p. challenged with CV-B5/JS417 (3.16 × 10⁷ CCID₅₀/mouse). b The process of selecting the suitable age. BALB/c mice at 1, 3, 5, 7, and 14 days of age were i.p. challenged with CV-B5/JS417 (3.16 × 10⁷ CCID₅₀/mouse). c The process for selecting the suitable inoculation route. Three-day-old BALB/c mice were challenged with CV-B5/JS417 (3.16 × 10⁷ CCID₅₀/mouse) via the i.p., intracerebral (i.c.), or intragastric (i.g.) route. The control mice were treated with medium via the same corresponding routes. n = 6 to 10 mice for each group. All mice were monitored daily for body weight and clinical symptoms for 21 dpi. One representation of two independent experiments was shown. The Mantel–Cox log-rank test was used to compare the survival of the pups between each group and the medium control group at 21 days post-infection. ***p < 0.001. **p < 0.01.* p < 0.05 (d). A representative image of 3-day-old BALB/c mice infected with CV-B5/JS417 (the left side) or with medium (the right side) at 7 dpi is shown, and the infected mice exhibited the following clinical symptoms: wasting, hair loss, and hindlimb paralysis (arrows)

Fig. 2 CV-B5 infection in 3-day-old mice resulted in dose-dependent disease and mortality.

Three-day-old BALB/c mice (n = 6 to 10, per group) were i.p. inoculated with CV-B5/JS417 at a dose ranging from 3.16 to 3.16 × 10⁵ CCID₅₀/mouse (10-fold serially diluted). The control mice were treated with medium. The CV-B5/JS417-induced mean score of clinical symptoms and CV-B5/JS417-induced mortality were monitored and recorded daily. Representative results of duplicate experiments are shown. The Mantel–Cox log-rank test was used to compare the survival of the pups between each group and the medium control group at 21 days post-infection. ***p < 0.001. *p < 0.05

Reproducibility of the CV-B5-challenged neonatal mouse model

Experiment #	Onset of symptom (d)	Onset of death (d)	Time of death for all mice (d)	Mortality ratio (%)
1	4	8	8	100
2	3	4	8	100
3	5	8	9	100
4	3	4	9	100
5	3	4	10	100
6	3	4	11	100

Six replicate experiments were performed on different days with three-day-old BALB/c mice (n = 6 to 10 per group) that were i.p. challenged by 26 LD50 CV-B5/JS417. CV-B5/JS417-induced clinical symptoms and mortality were monitored and recorded daily

Fig. 3 HE staining of various tissues from the BALB/c neonatal mice that were i.p. challenged with CV-B5/JS417.

a, c, e, g HE staining of cerebral cortex, spinal cord, myocardium, and hindlimb muscle tissues of the control group, respectively. b, d, f, h HE staining of corresponding tissues of the experimental group. b Necrosis of the cerebral cortex associated with tubular infiltration (arrow); d: degeneration and necrosis of spinal cord nerve cells with glial response (arrow); f: eosinophilic necrosis of cardiomyocytes (arrow); h: necrotic myositis of hindlimb muscle (arrow). Magnifications ×100 (a–c, e, g, h), Magnifications ×200 (d, f). n = 6–10 mice for each group. One representative image is shown

Fig. 4 Immunohistochemical staining of various tissues from the BALB/c neonatal mice following the intraperitoneal injection with CV-B5/JS417.

a, c, e, g IHC staining of the cerebral cortex, spinal cord, myocardium, and hindlimb muscle in the control group, respectively. b, d, f, h IHC staining of the cerebral cortex, spinal cord, myocardium, and hindlimb muscle in the experimental group. Positive staining was detected in the cerebral cortex (b, arrow), spinal cord (d, arrow), myocardium (f, arrow) and hindlimb muscle (h, arrow) of the neonatal mice after the intraperitoneal injection of CV-B5/JS417. Magnifications ×40 (a); magnifications ×100 (b–h). n = 6–10 mice per group. One representative image is shown

Fig. 5 The viral load ratios of tissue/serum in CV-B5-challenged mice.

Three-day-old BALB/c mice were i.p. inoculated with CV-B5/JS417 (3.16 × 10³ CCID₅₀/mouse). To exclude the impact of the viral load in the serum, the viral load ratio (tissue/serum) was calculated by dividing the mean viral load of each tissue with the viral load of the serum at 6, 12, 24, 72, 120, and 168 h post-challenge (n = 3, per time point)

Fig. 6 Evaluation of the protective effect of the inactivated CV-B5 vaccine in the mouse model.

Adult female BALB/c mice were vaccinated with formaldehyde-inactivated CV-B5/JS417 (experiment group) or medium (control group) twice with a 2-week interval and allowed to mate 1 h after the first vaccination. The resulting pups were challenged with CV-B5/JS417 (3.16 × 10³ CCID₅₀/mouse) on day 3 after birth. The mortality, clinical symptoms, and body weight were monitored and recorded daily after the infection (n = 6 to 10, per group). One representative result is shown. The Mantel–Cox log-rank test was used to compare the survival of the pups between each vaccine group and the medium control group at 21 days post-infection. ***p < 0.001

Fig. 7 Evaluation of the protective effect of anti-CVB5 serum in the mouse model.

Serially diluted anti-CVB5 serum (neutralizing antibody 1:768) or medium was incubated with an equal volume of 3.16 × 10³ CCID₅₀ CV-B5/JS417 at 37 °C for 1 h. 3-day-old BALB/c mice (n = 6–10, per group) were i.p. inoculated with the diluted serum. The mortality, clinical symptoms, and body weight were monitored and recorded daily after the infection. One representative result of two independent experiments is shown. The Mantel–Cox log-rank test was used to compare the survival of the pups between each anti-serum group and the medium control group at 21 days post-infection. **p < 0.01, *p < 0.05