Standardized reference sera: advancing harmonization of equine viral arteritis serologic testing across national reference laboratories in the European Union

Equine viral arteritis is a contagious disease affecting equids, caused by equine arteritis virus (EAV). Accurate serologic testing is essential for surveillance, breeding management, and international trade. The virus neutralization test (VNT), prescribed by the World Organisation for Animal Health, remains the gold standard, but variability in quantitative results between laboratories limits harmonization. We aimed to produce and validate standardized EAV-positive reference sera to improve VNT consistency across European Union national reference laboratories, and to assess their impact on interlaboratory proficiency testing (IL-PT). Three sera from a vaccinated pony and one from a naturally infected horse were produced and evaluated for reproducibility, homogeneity, and stability. These sera were first distributed during an IL-PT campaign in 2013 to allow participants to characterize them and define expected titers and acceptance ranges. Once established, the sera were reused in subsequent IL-PT campaigns (2016, 2020, 2024) as positive controls. All reference sera had strong reproducibility, homogeneity, and stability. Their use was associated with an improvement in interlaboratory performance, with results within expected ranges increasing from 51.1% in 2013 to 81.7% in 2024. These standardized sera offer a robust tool for harmonizing EAV VNTs, improving test reliability, and supporting surveillance and trade.