Schuster Isolde, Mertens Marc, Köllner Bernd, Korytář Tomáš, Keller Markus, Hammerschmidt Bärbel, Müller Thomas, Tordo Noël, Marianneau Philippe, Mroz Claudia, Rissmann Melanie, Stroh Eileen, Dähnert Lisa, Hammerschmidt Felicitas, Ulrich Rainer G, Groschup Martin H
Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
Institute of Immunology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
Antiviral Res. 2016 Oct;134:161-166. doi: 10.1016/j.antiviral.2016.09.004. Epub 2016 Sep 10.
Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans with case-fatality rates of up to 80%. CCHF is considered to be one of the major emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma function as vector as well as natural reservoir of CCHFV. Ticks feed on various domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs). Those animal species play an important role in the life cycle of the ticks as well as in amplification of CCHFV. Here we present a competitive ELISA (cELISA) for the species-independent detection of CCHFV-specific antibodies. For this purpose nucleocapsid (N) protein specific monoclonal antibodies (mAbs) were generated against an Escherichia coli (E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous and heterologous recombinant CCHFV antigens in ELISA and Western blot test and 20 of those 33 mAbs reacted additionally in an immunofluorescence assay with eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a prototype of the cELISA and nine of them competed with positive control sera of bovine origin. The cELISA was established by using the mAb with the strongest competition. For the validation, 833 sera from 12 animal species and from humans were used. The diagnostic sensitivity and specificity of the cELISA was determined to be 95% and 99%, respectively, and 2% of the sera gave inconclusive results. This cELISA offers the possibility for future large-scale screening approaches in various animal species to evaluate their susceptibility to CCHFV infection and to identify and monitor the occurrence of CCHFV.
克里米亚-刚果出血热病毒（CCHFV）在亚洲、非洲和欧洲的许多国家传播。CCHFV可导致人类严重出血热，病死率高达80%。CCHF被认为是传播至欧洲并在欧洲境内传播的主要新兴疾病之一。璃眼蜱属蜱类既是CCHFV的传播媒介，也是其天然宿主。蜱以各种家畜（如牛、绵羊、山羊）和野生动物（如野兔、刺猬）为食。这些动物物种在蜱的生命周期以及CCHFV的扩增中发挥着重要作用。在此，我们展示了一种用于非物种特异性检测CCHFV特异性抗体的竞争性酶联免疫吸附测定（cELISA）。为此，针对大肠杆菌表达的CCHFV核衣壳（N）蛋白制备了特异性单克隆抗体（mAb）。33种mAb在酶联免疫吸附测定和蛋白质免疫印迹试验中与同源和异源重组CCHFV抗原发生反应，其中20种mAb在免疫荧光试验中还与表达N蛋白的真核细胞发生反应。在cELISA原型中对10种mAb进行了进一步表征，其中9种与牛源阳性对照血清发生竞争。通过使用竞争最强的mAb建立了cELISA。为进行验证，使用了来自12个动物物种和人类的833份血清。cELISA的诊断敏感性和特异性分别确定为95%和99%，2%的血清结果不明确。这种cELISA为未来对各种动物物种进行大规模筛查提供了可能，以评估它们对CCHFV感染的易感性，并识别和监测CCHFV的发生情况。
