Finally, EDIII IgM antibodies detected soon after infection in model or naturally infected mammals [18] efficiently neutralise the virus in MNT but are not detected by our indirect IgG MIAs. In conclusion, when used in a multiplex MIA format, rEDIIIs were able to specifically identify WNV and TBEV antibodies, particularly if sera were sampled after D20 PI. 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. == 1. Introduction == Many flaviviruses such as West Nile virus (WNV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), or dengue virus are emerging or reemerging diseases threatening humans and/or animals [14]. Most flaviviruses are arboviruses that can be transmitted by ticks,AedesorCulexmosquitoes, or by unknown vectors [5,6]. Mosquito-borne viruses are grouped into serocomplexes such as the FNDC3A dengue or Japanese encephalitis serocomplexes on the basis of their antigenic relationships [7]. WNV and JEVtwo major encephalitic viruses in horses and humans [8,9]belong to the Japanese encephalitis serocomplex which is composed of eight virus species and two subtypes [10]. TBEV, another encephalitic flavivirus known to cause severe infections in humans and dogs, belongs to the group of mammalian tick-borne viruses [2]. Only low levels of viremia are induced after infection with most flaviviruses (apart from dengue), so diagnosis is confirmed mainly through serological tools. Rapid serological tools are widely used in human and veterinarian diagnostic laboratories, in particular enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays (IFAs). ELISAs and IFAs are very sensitive but suffer from a lack of specificity, generating positive reactions for close flaviviruses within a specific serocomplex or even for flaviviruses belonging to other serocomplexes [6]. For example, JEV- or TBEV-infected patients can generate false positive reactions in WNV ELISAs [11,12]. Serological assays should thus be interpreted with care GR148672X taking into account the patient’s history of vaccination and past infection and confirmed by comparative virus neutralisation tests (VNTs) [7,13]. Nevertheless, the VNT is usually less sensitive than ELISA or IFA and some residual cross-reactions can be observed within the Japanese encephalitis serocomplex. This time-consuming immunoassay generally requires the production of infectious virus particles in BSL3 facilities. Rapid flavivirus serological assays have already been misinterpreted in the past, highlighting the need for improved serological tools. During the emergence of WNV GR148672X in the USA in 1999, the first WNV cases were initially thought to be Saint Louis encephalitis cases, a flavivirus prevalent in North America [14]. Consequently, the flavivirus surveillance programmes currently in operation in many countries could detect belatedly, or even fail to detect, the emergence of a new flavivirus. Moreover, the spatial and temporal overlapping in the circulation of flaviviruses makes it difficult to precisely monitor the dynamics of a specific flavivirus. Mosquito-borne flaviviruses like WNV or Usutu virus are widely distributed throughout Europe with high spatial overlapping, for example, in Italy, Austria, and Hungary [1517], while WNV and TBEV are regularly isolated in Central Europe [6]. Most antibodies elicited during flavivirus infections are directed against the highly immunogenic envelope (E) protein, which contains both flavivirus cross-reactive and virus-specific epitopes [18,19]. The E glycoprotein is composed of three domains named EDI, EDII, and EDIII, altogether composing the soluble ectodomain of E (sE). Both EDI and EDIII contain virus-specific epitopes [2022], and EDIII mediates virus attachment to the cell membrane [23,24]. Potent neutralising antibodies have been shown to map to EDIII [2529], and amino acid substitutions within EDIII may influence the pathogenicity of flaviviruses [27,3034]. GR148672X The development of xMAP microsphere immunoassays (MIAs) or Luminex assays for human and veterinary diagnoses offers promising multiplexing approaches to the capture of specific antibodies directed against flavivirus antigens [3538]. To significantly improve the specificity.
Finally, EDIII IgM antibodies detected soon after infection in model or naturally infected mammals [18] efficiently neutralise the virus in MNT but are not detected by our indirect IgG MIAs
