Phenotypic characterisation of cell populations in the brains of horses experimentally infected with West Nile virus

Abstract

Background: West Nile virus (WNV), a mosquito borne member of the Flaviviridae, is one of the most commonly diagnosed agents of viral encephalitis in horses and people worldwide.

Objectives: A cassette of markers for formalin-fixed paraffin-embedded tissue and an archive of tissues from experimental infections in the horse were used to investigate the equine neuroimmune response to WNV meningoencephalomyelitis to phenotype the early response to WNV infection in the horse.

Study design: Quantitative analysis using archived tissue from experimentally infected horses.

Methods: The thalamus and hindbrain from 2 groups of 6 horses were compared and consisted of a culture positive tissues from WNV experimentally horses, in the other, normal horses. Formalin-fixed paraffin-embedded tissue from the thalamus and hindbrain were immunolabeled for microglia, astrocytes, B cells, macrophages/neutrophils, CD3⁺ T cells. Fresh frozen tissues were immunolabeled for CD4⁺ and CD8⁺ T lymphocyte cell markers. Cell counts were obtained using a computer software program. Differences, after meeting assumptions of abnormality, were computed using a general linear model with a Tukey test (P<0.05) for pairwise comparisons.

Results: In WNV-challenged horses, Iba-1⁺ microglia, CD3⁺ T lymphocyte and MAC387⁺ macrophage staining were significantly increased. The T cell response for the WNV-challenged horses was mixed, composed of CD4⁺ and CD8⁺ T lymphocytes. A limited astrocyte response was also observed in WNV-challenged horses, and MAC387⁺ and B cells were the least abundant cell populations.

Main limitations: The results of this study were limited by a single collection time post-infection. Furthermore, a comprehensive analysis of cellular phenotypes is needed for naturally infected horses. Unfortunately, in clinical horses, there is high variability of sampling in terms of days post-infection and tissue handling.

Conclusions: The data show that WNV-challenged horses recruit a mixed T cell population at the onset of neurologic disease.

Keywords: West Nile virus; horse; immunohistochemistry; immunopathology.