West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive
infections in humans and horses. The confirmation of
flavivirus infections is mostly based on rapid serological tests such as
enzyme-linked
immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E)
glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific
epitopes. In order to improve the serological differentiation of
flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified
antigens were covalently bonded to fluorescent beads. The
microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine
immune sera from natural and experimental
flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled
microspheres captured specific
antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.