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. 2016 Jun 30:6:28876.
doi: 10.1038/srep28876.

A clinically authentic mouse model of enterovirus 71 (EV-A71)-induced neurogenic pulmonary oedema

Carla Bianca Luena Victorio  1   2 , Yishi Xu  1   2 , Qimei Ng  1 , Beng Hooi Chua  3 , Sylvie Alonso  2 , Vincent T K Chow  2 , Kaw Bing Chua  1
Affiliations

A clinically authentic mouse model of enterovirus 71 (EV-A71)-induced neurogenic pulmonary oedema

Carla Bianca Luena Victorio et al. Sci Rep. .

Abstract

Enterovirus 71 (EV-A71) is a neurotropic virus that sporadically causes fatal neurologic illness among infected children. Animal models of EV-A71 infection exist, but they do not recapitulate in animals the spectrum of disease and pathology observed in fatal human cases. Specifically, neurogenic pulmonary oedema (NPE)-the main cause of EV-A71 infection-related mortality-is not observed in any of these models. This limits their utility in understanding viral pathogenesis of neurologic infections. We report the development of a mouse model of EV-A71 infection displaying NPE in severely affected animals. We inoculated one-week-old BALB/c mice with an adapted EV-A71 strain and identified clinical signs consistent with observations in human cases and other animal models. We also observed respiratory distress in some mice. At necropsy, we found their lungs to be heavier and incompletely collapsed compared to other mice. Serum levels of catecholamines and histopathology of lung and brain tissues of these mice strongly indicated onset of NPE. The localization of virally-induced brain lesions also suggested a potential pathogenic mechanism for EV-A71-induced NPE. This novel mouse model of virally-induced NPE represents a valuable resource for studying viral mechanisms of neuro-pathogenesis and pre-clinical testing of potential therapeutics and prophylactics against EV-A71-related neurologic complications.

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Figures

Figure 1
Figure 1. Disease severity in 1-week-old mice infected with EV71:BS, EV71:TLLm, or EV71:TLLmv.
Kaplan-Meier survival curves of mice inoculated with EV71:BS, EV71:TLLm, or EV71:TLLmv via I.P. (intraperitoneal) (A) or I.M. (intramuscular) route (B). Deaths recorded were a result of disease or euthanasia at moribund stage. Mean duration of survival were compared using the Mantel-Cox log-rank test. *p < 0.05; **p < 0.005; ***p < 0.0005.
Figure 2
Figure 2. Dependence of EV71:TLLmv infection severity on the dose, age, and inoculation route.
(A) Kaplan-Meier survival curve of 1-week-old mice inoculated I.P. with different doses of EV71:TLLmv. (B) Age and inoculation route dependence of survival of mice inoculated with 106 CCID50 virus. (C,D) Kaplan-Meier survival curves of mice inoculated with 106 CCID50 virus via I.P. (C) or I.M. (D) route. Deaths recorded were a result of disease or euthanasia at moribund stage. Mean duration of survival were compared using Mantel-Cox log-rank test. **p < 0.005; ***p < 0.0005; ****p < 0.0001. Mock. Sham-inoculated controls; wo, mouse age in weeks.
Figure 3
Figure 3. Clinical presentation of EV71:TLLmv infection in 1-week-old mice.
Mice inoculated with 106 CCID50 virus by I.P. (A) or I.M. (B) injection were grouped into four categories based on clinical signs observed throughout the observation period. Severely infected mice (both Class-I and Class-II) exhibited flaccid paralysis (C), either in the forelimbs (yellow arrows), hind limbs (red arrows), or both; and hairless lesions (green arrows) (D) that persisted for several days and typically disappeared within 1 week. A few Class-I mice also exhibited haemorrhagic lesions in the hind feet (E), which appear to be due to vasculitis (blue arrows).
Figure 4
Figure 4. Evidence of EV71:TLLmv-induced neurogenic pulmonary oedema (NPE) in Class-IA mice.
(A) Representative images of lungs (top- and side-views) at necropsy. Note the incomplete collapse of the lungs in Class-IA mice (white arrows). (B) Comparison of wet weights of harvested lungs. Columns represent median values (n = 4), which were compared using Mann-Whitney test. Error bars represent the range of values. *p < 0.05. (C) Representative tissue cross sections (5 μm) of lungs shown in low- and high-power magnifications. Note the presence of pink proteinaceous fluid in the alveolar spaces (red asterisks), some of which were also filled with erythrocytes (yellow arrows), in Class-IA lung sections. (D) Comparison of adrenaline (epinephrine) and noradrenaline (norepinephrine) levels in terminally collected sera. Columns represent mean values, which were compared using t-test with Welch’s correction for unequal variance. Error bars represent SD. *p < 0.05; **p < 0.005.
Figure 5
Figure 5. Localization and distribution of viral antigens and lesions in Class-IA and Class-IB brains.
Representative images of mouse brain coronal sections marked to demonstrate the localization of viral antigens (brown dots) and pathological lesions (blue dots) observed in the brains of infected mice. Larger dots represent more intense signals or greater lesion size. Shown are the cerebellar cortex (CTX) (A–C); hypothalamus (HY) (A,B); hippocampus (HP) (B,C); thalamus (TH) (B); midbrain (MB) and pons (P) (C); and cerebellum (CBX) and medulla oblongata (MY) (D). Templates of coronal sections were downloaded from www.brainstars.org and http://mouse.brain-map.org/static/atlas.
Figure 6
Figure 6. Localization and distribution of viral antigens and lesions in Class-IA and Class-IB mouse spinal cords.
Representative images of spinal cord coronal sections from Class-IA (A) and Class-IB (B) mice marked to show the localization of viral antigens (brown dots) and pathologic lesions (blue dots). Larger dot sizes indicate more intense signals or lesions. V, ventral side; D, dorsal side.
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