Sessions

West Nile virus (WNV), belonging to the Orthoflavivirus genus, is a neurotropic virus that occurs in an enzootic cycle between birds and ornithophilic mosquitoes. Humans and horses act as incidental hosts for WNV, with approximately 80% of infections being subclinical. However, if the virus invades the nervous system, West Nile Neuroinvasive Disorder (WNND) can occur presenting with neurological signs. Human and animal studies indicate that the cellular immune response to WNV and other orthoflavivirus infections plays a major role in clinical manifestations and protection. Recent research has shed light on the potential application of cellular assays in orthoflavivirus diagnostics.

The aim of this research is to characterise the cellular response in horses infected with WNV by employing an equine-interferon gamma (IFN𝛾) Enzyme-linked Immunospot (ELISpot) assay. Additionally, the relationship between the ELISpot response and humoral immunity will be investigated.

This study was conducted using 14 client-owned horses all of which were proven to be infected with WNV and showed neurological signs. Blood samples were collected using EDTA tubes and separated into peripheral blood mononuclear cells (PBMCs). Samples with a viability of >90% were included in the study. A WNV capsid protein mix was used to stimulate PBMC samples. WNV IgG ELISA tests and virus neutralisation tests (VNT) were performed to determine the WNV-specific neutralising antibody titer and to exclude other orthoflaviviral infections. DFR was used for statistical analysis.

Samples were taken 290 +/-62.5 days after infection diagnosis. Originally 14 samples were included in the study however only 10 showed positive IFN𝛾 response. The study population ranged in age from 3-16 years including 8 mares and 4 geldings of varying breeds. A pattern was not observed between clinical signs such as ataxia grade and ELISpot results. Nor was a correlation seen between age, gender or breed and the results of the ELISpot. Each subject had a positive WNV IgG ELISA and WNV titres ranged from 1:64–1:1024. Furthermore, no correlation was found between cellular and humoral responses.

In this study we established the equine IFN𝛾 ELISpot assay is a suitable method for detection of WNV-specific IFN𝛾 production in PBMC samples from horses with previous WNND. Cellular assays could hold great potential for diagnostic capabilities in the future. Lack of correlation between humoral and cellular immunity could represent the potential role of memory T cells in the protection against re-infection.

Certain limitations occurred during the study. A lower sample size resulted in reduced statistical sensitivity. It should be noted that IFN𝛾 is produced not only by CD4+ and CD8+ cells but also natural killer cells, dendritic cells and macrophages. Immunophenotyping is necessary in future studies to determine IFN𝛾-secreting cell populations.