Pathological features of African horse sickness virus infection in IFNAR-/- mice

Abstract

African Horse Sickness (AHS) is a vector-borne viral disease of equids. The disease can be highly lethal with mortality rates of up to 90% in non-immune equine populations. The clinical presentation in the equine host varies, but the pathogenesis underlying this variation remains incompletely understood. Various small animal models of AHS have been developed over the years to overcome the financial, bio-safety and logistical constraints of studying the pathology of this disease in the target species. One of the most successful small animal models is based on the use of interferon-alpha gene knock-out (IFNAR^-/-) mice. In order to increase our understanding of African Horse Sickness virus (AHSV) pathogenesis, we characterised the pathology lesions of AHSV infection in IFNAR^-/- mice using a strain of AHSV serotype 4 (AHSV-4). We found AHSV-4 infection was correlated with lesions in various organs; necrosis in the spleen and lymphoid tissues, inflammatory infiltration in the liver and brain, and pneumonia. Significant viral antigen staining was only detected in the spleen and brain, however. Together these results confirm the value of the IFNAR^-/- mouse model for the study of the immuno-biology of AHSV infections in this particular in vivo system, and its usefulness for evaluating protective efficacy of candidate vaccines in preclinical studies.

Keywords: African horse sickness; arbovirus; immunofluorescence; immunohistochemistry; mouse model; pathology.

Figure 1

Representative images of infected spleen displaying both normal (A; mouse 2) and necrotic states (B, arrowhead; mouse 3). Liver tissue displays multifocal hepatitis (C, arrowhead; mouse 2) and inflammatory infiltrate around the portal triad (D; mouse 3). Lung tissue displays some oedema (E, arrowhead, mouse 1; and F; mouse 2) demonstrates interstitial pneumonia throughout tissue. In the gastrointestinal tract, Peyer's patches display lymphoid depletion after 7 days (G; mouse 3) and severe necrosis after 10 days (H; mouse 4). Cerebral tissue presented perivascular cuffing and meningeal inflammatory infiltrates (I, arrowhead; mouse 2 and K; mouse 3). Perivascular cuffing is also shown at higher magnification (J; mouse 3). Cerebellum tissue appeared consistently normal (L; mouse 2). One individual displayed signs of inflammation in the epicardium (M, arrowhead; mouse 3). Lymph nodes frequently displayed focal severe necrosis and lymphoid depletion (N, mouse 4). Mild inflammatory infiltrates were observed in kidney tissue (O; mouse 3). Pancreatic tissue was consistently normal (P; mouse 1).

Figure 2

Splenic (A, E), lung (B, F), hepatic (C, G) and cerebral (D, H) tissue sections were stained with anti-AHSV VP7 mAb (green), phalloidin (red) and DAPI (blue) and analysed by confocal microscopy. Images from mouse 1 (A–D) and uninfected control mouse (E–H) are shown (no viral antigen was observed in uninfected control mouse tissue). High-magnification image of characteristic hexagonal VP7 crystal included as insert in (A). Scale bars indicate 25 μm.

Figure 3

Representative examples of immunostaining within different murine organs (positive staining in red/brown). T cell staining (A), B cell staining (B), neutrophil staining (C) and macrophage staining (D) were all examined in the spleen. Within the brain, T cell staining (E), neutrophil staining (F), B cell staining (G) and macrophage staining (H) were all examined. In the intestine, T cell staining is visible in necrotic Peyer's patch (I) and surrounding villi (J). Sparse macrophage staining is also present within necrotic Peyer's patch (K). B cell staining in Peyer's patch and surrounding villi (L) is also sparse. Within the lung, staining was performed for T cells (M), B cells (N), macrophages (O) and neutrophils (P).

Figure 4

Image analysis was used to determine the % areas positive staining for different leucocytes in spleen (A – AHSV-4 infected and uninfected) and perivascular cuffs in the brain (B – AHSV-4 infected) tissues. The staining in tissue from an uninfected control mouse was compared to the mean staining in AHSV-4 infected mice using a 2-way ANOVA, where **indicates adjusted p-value < 0.01.