Endothelial damage and cellular apoptosis in horses experimentally infected with African horse sickness virus

Abstract

African horse sickness (AHS) is an infectious, non-contagious vector-borne viral disease caused by the African horse sickness virus (AHSV), a non-enveloped, double-stranded RNA Orbivirus belonging to the Sedoreoviridae family (1). Primarily transmitted by Culicoides midges, AHSV infection results in a mortality rate exceeding 90% in susceptible equine populations (2,3). The disease manifests in four clinical forms: pulmonary (“dunkop”), cardiac (“dikkop”), fever, and mixed form, with severity depending on factors such as viral virulence and host immune status (3,4). Clinical signs of severe AHS infection are consistent with vasculitis, characterized by oedema in the supraorbital fossae, ocular conjunctiva, and intermandibular spaces, as well as pulmonary oedema. Hydropericardium and petechial haemorrhages are also commonly observed during clinical examination and necropsy (5). These clinical manifestations suggest significant vascular endothelial damage as a key aspect of AHS pathogenesis. The endothelium, comprising the inner lining of blood vessels, forms a semi-permeable membrane between blood and surrounding tissues. On its luminal surface lies the glycocalyx, a gel-like layer composed of membrane-attached proteoglycans, glycosaminoglycan chains, and glycoproteins. This structure plays a crucial role in maintaining vascular homeostasis, regulating vascular permeability, and exerting anti-coagulant and anti-adhesive effects (6–8). Syndecan-1 (SYND1), a heparan sulphate proteoglycan expressed on endothelial cells, is a key component of the glycocalyx. When endothelial damage occurs, plasma concentrations of SYND1 increase, as reported in human trauma patients, making it a potential biomarker for endothelial injury (9,10). The shedding of glycocalyx components into the circulation may correlate with disease severity in various conditions characterized by endothelial damage. Previous studies have demonstrated AHSV tropism for the heart, lung, and spleen, with the virus successfully identified in these organs using immunohistochemistry (IHC) (11,12). Microscopic evaluation of endothelial cells in these tissues from horses infected with AHSV have reported ultrastructural changes suggestive of apoptosis, as well as loss of intercellular junction integrity, presence of cytoplasmic projections, and nuclear condensation (13). Given the hallmark finding of vascular permeability and tissue oedema in AHSV-infected horses, this study aimed to quantify the extent of endothelial damage using SYND1 concentrations in the blood as a biomarker and to correlate the severity of endothelial damage with apoptosis observed in endothelial cells, using IHC, of horses infected with AHSV. This study retrospectively evaluated stored plasma and tissue samples (spleen, heart, lung) from four Boerperd mix horses that were experimentally infected with AHSV. Syndecan-1 plasma concentrations in horses experimentally infected with AHSV showed no significant change over the course of infection (Skillings-Mack test = 13.516; P = 0.077). Immunohistochemical analysis showed positive staining for AHSV viral protein 7 (VP7) and non-structural protein 4 (NS4) antigens in endothelial cells and some mononuclear cells in spleen, lung, and heart tissues of infected horses. Tissue from control horses were negative for both antigens. Immunolabeling for caspase-3, a marker for cellular apoptosis, in the spleen of AHSV-infected horses showed a marked increase in positive cells (186.0 to 262.4 cells/image) compared to controls (1.0 to 25.0 cells/image). Similarly, in lung tissue, AHSV-positive horses exhibited higher caspase-3 positive cell counts (11.6 to 75.4 cells/image) than controls (0.4 to 7.4 cells/image). However, cardiac tissue showed no significant difference in caspase-3 labelling between infected and control horses. Immunohistochemistry for leucocyte markers in infected horses demonstrated strong labelling for ionized calcium-binding adapter molecule 1 (IBA-1), a marker for mononuclear cells, within splenic lymphoid tissue and lung parenchyma. T lymphocytes observed in splenic periarteriolar lymphoid sheaths and peribronchiolar interstitium stained positive for cluster of differentiation 3 (CD3). CD20-positive B lymphocytes were extensively labelled throughout splenic lymphoid follicles. These findings suggest that AHSV infection does not result in direct damage to endothelial cells; instead, it induces a significant apoptotic response in the spleen and lungs of infected horses, but not in the heart, and is associated with notable changes in mononuclear leucocyte distribution and viability in affected tissues.