A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

Abstract

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.

West Nile virus epizootics in the Camargue (France) in 2015 and reinforcement of surveillance and control networks

“…For equine suspect cases reported through either RESPE or local Veterinary Services, sera were first screened for anti-WNV antibodies by competition ELISA (IDScreen ® West Nile competition, IDVet, Montpellier, France) in local veterinary laboratories approved for WNV indirect diagnosis (Bouches-Du-Rhône, Calvados, Gard, Hérault). Competition ELISA-positive sera were further analysed by MAC (M-antibody capture)-ELISA for IgM detection (IDScreen ® West Nile IgM capture, IDVet) in these same laboratories and at ANSES (11). The WNV infection was then confirmed at ANSES by virus neutralisation tests (VNTs), as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual) (12), as well as by flavivirus Luminex assay, as described by Beck et al (11).…”

“…Competition ELISA-positive sera were further analysed by MAC (M-antibody capture)-ELISA for IgM detection (IDScreen ® West Nile IgM capture, IDVet) in these same laboratories and at ANSES (11). The WNV infection was then confirmed at ANSES by virus neutralisation tests (VNTs), as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual) (12), as well as by flavivirus Luminex assay, as described by Beck et al (11). Given the long persistence of anti-WNV IgG antibodies in horses, a horse found positive by competition ELISA but negative by IgM capture ELISA was regarded as an unconfirmed case, but assumed to be carrying markers of previous infection (or vaccination).…”

Exposure of Wild Ungulates to the Usutu and Tick-Borne Encephalitis Viruses in France in 2009–2014: Evidence of Undetected Flavivirus Circulation a Decade Ago

“…Antibodies against flaviviruses were detected using a commercial competitive enzyme-linked immunosorbent assay (cELISA; ID Screen ® West Nile Competition, ID Vet, Montpellier, France) based on purified whole WNV antigen for the detection of antibodies directed against the envelope E protein common to all flaviviruses. Although this ELISA test is designed for WNV detection, the high cross-reactivity observed makes this method suitable for the detection of a broad range of flavivirus antibodies [38,39]. The test was performed according to the manufacturer's instructions and interpreted following their recommendations: samples with a sample-to-negative control (S/N) ratio ≤ 40%, [40-50%] and > 50% were considered positive, doubtful and negative, respectively.…”

Viral Equine Encephalitis, a Growing Threat to the Horse Population in Europe?

“…One recent and striking example of delayed identification of emerging arboviruses due to similarities in clinical presentation and cross-reactive diagnostic tools was given during WNV introduction in the United States, when WNV was initially misdiagnosed with the closely related SLEV [183]. Bearing in mind that three flaviviruses responsible for equine encephalitis are described in Europe, and that serological cross-reactivity is frequently observed in flavivirus indirect diagnosis assays, the development of multiplex approaches that allow the comparison of serological reactions against a wide range of pathogens appear to be valuable options [184,185]. Furthermore, because in about one-half of infectious equine encephalitis, no known pathogen can be evidenced [4], identification of unknown neuropathogenic viruses by classical (electron microscopy) and more recent high-throughput techniques (next generation sequencing for example) is highly desirable [186,187].…”