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Biomed Res Int. 2015;2015:678084. doi: 10.1155/2015/678084. Epub 2015 Sep 17.

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

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UMR 1161 of Virology, ANSES, INRA, ENVA, ANSES Animal Health Laboratory, EU-RL on Equine Diseases, UPE, 94701 Maisons-Alfort, France.
UMR PIMIT (I2T Team), INSERM U1187, CNRS 9192, IRD 249, Technology Platform CYROI, University of Reunion, 97491 Saint-Clotilde, Réunion ; Department of Infections and Epidemiology, Institut Pasteur, 75724 Paris, France.
Department of Infections and Epidemiology, Institut Pasteur, 75724 Paris, France.
Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria ; Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, 123 Al-Khoudh, Oman.
INRA UE 1277, Plate-Forme d'Infectiologie Expérimentale, 37380 Nouzilly, France.
Equine Research Institute, Japan Racing Association, Tochigi 329-0412, Japan.


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.

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