Seroprevalence and Passive Clinical Surveillance of West Nile Virus in Horses from Ecological High-Risk Areas in Western Romania: Exploratory Findings from a Cross-Sectional Study

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Abstract
This cross-sectional study evaluated the seroprevalence and clinical impact of West Nile virus (WNV) infection in horses from three ecologically high-risk counties in western Romania (Timiș, Arad, and Bihor) between 2023 and 2025. A total of 306 unvaccinated horses were tested using a commercial ELISA, with 8.17% testing positive for WNV antibodies, indicating prior exposure. Passive surveillance for clinical signs during mosquito seasons identified 16 horses with acute neurological symptoms, four of which were confirmed as clinical cases based on WNV-specific IgM positivity, suggesting probable silent WNV circulation in the region. The overall case fatality rate among confirmed clinical cases was 25.0%. WNV seropositivity was highest in Bihor (8.85%), followed by Arad (8.57%) and Timiș (7.32%). Statistical comparisons using χ2 tests and binary logistic regression indicated no significant differences in seroprevalence between counties, sexes, or age groups, consistent with the overlapping 95% confidence intervals. These findings suggest the continued silent circulation of WNV in the region and support the integration of equine surveillance into the One Health framework as a potential tool for early detection and risk mitigation. However, in the absence of molecular confirmation (e.g., RT-PCR or virus isolation), these results should be interpreted as indicative of prior exposure rather than direct evidence of ongoing viral activity.

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