Outbreak of Western Equine Encephalitis Virus Infection Associated with Neurological Disease in Horses Following a Nearly 40-Year Intermission Period in Argentina

Abstract

Western equine encephalitis virus (WEEV) is a mosquito-borne arbovirus (genus Alphavirus, family Togaviridae) that has re-emerged in South America in late 2023, causing severe disease in both horses and humans after a nearly 40-year intermission period. We here describe the virological, serological, pathological, and molecular features of WEEV infection in horses during the 2023-2024 outbreak in Argentina. WEEV-infected horses developed neurological signs with mild to severe encephalitis associated with minimal to abundant WEEV-infected cells, as demonstrated by WEEV-specific in situ hybridization. The distribution of viral RNA was multifocal, with predominance within neuronal bodies, neuronal processes, and glial cells in the medulla oblongata and thalamic regions. Phylogenetic analysis of partial nsP4 sequences from three viral isolates obtained from three different provinces of Argentina support grouping with other temporally current WEEV strains from Uruguay and Brazil under a recently proposed novel lineage.

Keywords: Argentina; WEE; WEEV; Western equine encephalitis virus; encephalitis; horse; in situ hybridization; outbreak; viral tropism.

Figure 1

Geographical distribution of WEE cases included in this study. (A) Cases included derived from 8 provinces in Argentina (marked in red). (B) Case distribution by province.

Figure 2

Phylogenetic analysis of partial nsP4 nucleotide sequences of three WEEV isolates from horses from Argentina (marked with a bracket). Current WEEV strains group closely with other reported strains from Uruguay and Brazil (lineage C, red leaves). Maximum likelihood trees were constructed using partial nucleotide sequences and the tree was edited with TreeViewer. A North American variant of EEEV was used as an outgroup. Bootstrap values (>50%) for 1000 replicates are shown as nodes in grades of gray, where black nodes show the highest support (>90%).

Figure 3

Virus-neutralizing antibody dynamics spanning a 3-month period in four horses that recovered from neurologic disease during the 2023–2024 WEEV outbreak. Timepoints 1 and 2 are at a 1-month interval, while timepoints 2 and 3 are at a 2-month interval. Neutralizing antibody titers gradually decline following infection.

Figure 4

Histological lesions in the CNS associated with WEEV infection in horses. (A,B) In the cerebral cortex, there are multiple foci of gliosis within the gray (A, asterisks) and white matter (B) with occasional neuronal necrosis (A, inset and arrow). Inflammatory cells (predominantly lymphocytes, macrophages, and a few neutrophils) infiltrate perivascular spaces (B, arrow) as well as the neuroparenchyma. (C) The molecular layer of the cerebellum has multiple foci of gliosis and inflammatory cells (arrows). (D–F) The neuroparenchyma of the medulla oblongata has frequent foci of gliosis (arrows) and infiltrating neutrophils (E) with occasional blood vessels affected by fibrinoid change (F). Hematoxylin and eosin. (A–C,E,F), 200× total magnification; (D), 40× total magnification.

Figure 5

Intralesional detection of WEEV genomic RNA in the CNS of infected horses via RNAscope^® in situ hybridization. Viral RNA (Fast Red) was detected in all areas of the encephalon examined, including the cerebral cortex (A), thalamus (B), cerebellum (C,D), and brainstem (E,F). Overall, viral RNA was detected within the cytoplasm of neurons (D–F, arrows) as well as within neuronal projections and glial cells within areas of gliosis. In the cerebellum, viral RNA was detected within the molecular and granular cell layer (C), as well as in Purkinje cells (D). (A–D), 200× total magnification; (E), 40× total magnification; (F), 100× total magnification.

Figure 6

Quantitative analysis of WEEV genomic RNA distribution in the CNS of infected horses. Semiquantitative scores (A) correlate with quantitative pathology analysis (B), with the thalamus and brainstem being the sites with more abundant viral RNA. The mean % positive area was 0.166% (±0.363%). (C) Graphical view of the equine encephalon. Regions are classified based on the abundance of WEEV RNA in high (***), medium (**), or low (*) based on the quantitative analysis (B).