Pulmonary edema following central nervous system lesions induced by a non- mouse-adapted EV71 strain in neonatal BALB/c mice

Figures

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  Fig. 1. ZZ1350 infection led to high cytotoxicity against Vero cells and RD cells. Viral RNA in ZZ1350-infected RD cells was extracted and identified by qRT-PCR (a). Evolutionary tree based on VP1 genome (b) was performed using MEGA software. CPEs and EV71 distribution (c) in Vero cells induced by different doses of ZZ1350 were imaged via light microscopy (amplification: 100×) or confocal microscopy (amplification: 400×) at 24 hpi. The viabilities of Vero cells (d) following virus infection were determined using the MTT assay. EV71 positive area (%) (e) was evaluated using Image-Pro Plus 6.0 software. Cell pellets of normal (f), infected RD cells (g, h) were fixed and investigated with electron microscopy. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (n = 3)

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  Fig. 2. Viral virulence of ZZ1350 in neonatal mice. 3-day-old BALB/c mice were inoculated with 2 × 106 PFU ZZ1350 strain via intracerebral (n = 7), intramuscular (n = 6) and intraperitoneal injection (n = 6). Survival rates (a) and mean clinical scores (b) of mice were monitored and recorded every 2 days after infection. n = 5 for normal controls; Data are expressed as mean ± SEM

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  Fig. 3. Clinical presentation in neonatal mice following ZZ1350 infection. Different inoculation routes including intraperitoneal (a), intracerebral (a) and intramuscular (b) injection were performed in the present study. Normal mice (c) were regarded as control. Severe infected mice exhibited flaccid paralysis, either in the forelimbs (d), hind limbs (e) or both; and hairless lesions (f) were also investigated at 5 dpi. Black arrows showed haemorrhagic lesions in the hind limbs or forelimbs

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  Fig. 4. Viral replication in tissues of ZZ1350-infected mice. The IHC expression and localization of VP1 represented as brown staining in cytoplasm (a) were shown for tissues from the brains, spinal cords, skeletal muscle of ZZ1350-infected mice. Viral titers in brain, spinal cord, skeletal muscle, lung, spleen, liver, heart (b) was expressed as log10 TCID50 per gram (n = 5 for each inoculation route and normal control)

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  Fig. 5. Representative histology of various tissues of ZZ1350-infected mice. Three-day-old BALB/c mice were intracerebral, intramuscular, intraperitoneal inoculated with ZZ1350 strain (2 × 106 PFU/mouse), and histopathological changes of tissues from mice were examined at 7 dpi as described in Materials and Methods. Solid yellow arrows: severe necrotizing myositis with fragmentation of myofibers and inflammatory cells infiltration; Solid red arrows: myocarditis with inflammatory cells infiltration or cardiomyocytes necrosis; hollow black arrows: inflammatory cells infiltration; hollow yellow arrows: erythrocyte aggregation; hollow red arrows: villous atrophy with edema fluid accumulation in the lumen

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  Fig. 6. Histopathological analysis of CNS and lungs of mice. Histopathological changes of brain (a), spinal cord (b) and lung section (c) were evaluated by H&E staining. Histology score of brains (d), spinal cord (e) and lungs (f) was evaluated by a person blinded to the treatment groups. Data are expressed as mean ± SEM. *** P < 0.001, intracerebral (n = 15), intramuscular (n = 12), or intraperitoneal inoculation (n = 11) vs normal controls (n = 10)

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  Fig. 7. Association of CNS lesions with lung histological severity. Pearson’s correlation test was used to analyze association of brain score and spinal cord score with lung score of mice with intracerebral (a and d), intramuscular (b and e), intraperitoneal inoculation (c and f) and in total experimental animals (g and h)