Beck Cécile, Desprès Philippe, Paulous Sylvie, Vanhomwegen Jessica, Lowenski Steeve, Nowotny Norbert, Durand Benoit, Garnier Annabelle, Blaise-Boisseau Sandra, Guitton Edouard, Yamanaka Takashi, Zientara Stéphan, Lecollinet Sylvie
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.
Biomed Res Int. 2015;2015:678084. doi: 10.1155/2015/678084. Epub 2015 Sep 17.
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.
西尼罗河病毒（WNV）、日本脑炎病毒（JEV）和蜱传脑炎病毒（TBEV）是黄病毒，可导致人类和马匹发生严重的神经侵袭性感染。黄病毒感染的确诊主要基于快速血清学检测，如酶联免疫吸附测定（ELISA）。这些检测的特异性较差，主要是由于黄病毒成员之间存在抗原交叉反应。因此，可靠的诊断需要通过病毒中和试验（VNT）进行验证，而病毒中和试验耗时且需要生物安全3级设施。黄病毒包膜（E）糖蛋白胞外域由三个结构域（D）组成，分别命名为DI、DII和DIII，其中EDIII包含病毒特异性表位。为了提高黄病毒感染的血清学鉴别能力，利用果蝇S2表达系统合成了WNV E的重组可溶性胞外域（WNV.sE）以及WNV、JEV和TBEV的EDIIIs（rEDIIIs）。将纯化的抗原共价结合到荧光珠上。用来自自然感染和实验性黄病毒感染的约300份马免疫血清以及172份非免疫马血清作为阴性对照，检测与WNV.sE或rEDIIIs偶联的微球。与rEDIII偶联的微球在阳性马血清中捕获了针对WNV、TBEV或JEV的特异性抗体。这种创新的多重免疫测定法是ELISA和VNT用于兽医诊断黄病毒相关疾病的有力替代方法。
