Development of Multispecies Recombinant Nucleoprotein-Based Indirect ELISA for High-Throughput Screening of Crimean-Congo Hemorrhagic Fever Virus-Specific Antibodies
Development of Multispecies Recombinant Nucleoprotein-Based Indirect ELISA for High-Throughput Screening of Crimean-Congo Hemorrhagic Fever Virus-Specific Antibodies
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DOI：
10.3389/fmicb.2019.01822
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发表时间：
2019-08-23
影响因子：
5.2
通讯作者：
Dash, Paban Kumar
中科院分区：
文献类型：
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作者：
Shrivastava, Neha;Shrivastava, Ambuj;Dash, Paban Kumar
克里米亚-刚果出血热（CCHF）是一种在亚洲、欧洲和非洲许多地区流行的再次出现的人畜共患病毒性疾病。病原体克里米亚-刚果出血热病毒（CCHFV）通过硬蜱传播。蜱病媒介，特别是属玻眼的蜱病媒介，是该疾病的寄主和扩增宿主。包括绵羊、山羊和牛在内的脊椎动物是连接病毒和蜱虫的短暂桥梁。CCHFV在人类中引起致命的出血热。人类通常通过受感染蜱虫的叮咬或与受感染动物的密切接触感染CCHFV。免疫检测，主要是使用全病毒抗原的酶联免疫吸附试验（ELISA），广泛用于动物血清监测。但是，整个病毒抗原具有很高的生物危害风险，只能在生物安全4级实验室中生产。本研究以重组CCHF病毒核蛋白（NP）为抗原，建立了一种安全、灵敏、特异的IgG间接酶联免疫吸附测定法（iELISA）。合成经密码子优化的NP基因序列，克隆并在pET28a+载体上表达。通过亲和层析纯化重组NP，并通过Western blot和MALDI-TOF/MS分析对重组NP进行鉴定。利用动物血清建立间接IgG微孔板酶联免疫吸附试验。将内部ELISA与市售ELISA试剂盒（Vector-Best, Russia）进行比较，其中76份疑似样本的一致性为90%，灵敏度和特异性分别为79.4和100%。精密度分析表明，该方法在不同的条件下具有稳健性和重复性。此外，该测定法用于对来自印度不同地区的反刍动物进行血清监测，结果显示反刍动物血清阳性率为18%，表明病毒在该地区继续传播。研究结果表明，利用重组NP构建的IgG iELISA是一种安全且有价值的工具，可用于多物种cchfv特异性抗体的大规模高通量筛选。
Crimean-Congo hemorrhagic fever (CCHF) is a re-emerging zoonotic viral disease prevalent in many parts of Asia, Europe, and Africa. The causative agent, Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV), is transmitted through hard ticks. Tick vectors especially belonging to the Hyalomma species serve as the reservoir and amplifying host. The vertebrate animals including sheep, goat, and bovine act as a short-lasting bridge linking the virus and ticks. CCHFV causes fatal hemorrhagic fever in humans. Humans are usually infected with CCHFV either through the bite of infected ticks or by close contact with infected animals. Immunological assays, primarily enzyme-linked immunosorbent assay (ELISA) using whole viral antigen, are widely used for serosurveillance in animals. However, the whole virus antigen poses a high biohazard risk and can only be produced in biosafety level 4 laboratories. The present study focuses on the development and evaluation of safe, sensitive, and specific IgG indirect enzyme-linked immunosorbent assay (iELISA) using recombinant nucleoprotein (NP) of CCHF virus as an antigen. The codon-optimized NP gene sequence was synthesized, cloned, and expressed in pET28a+ vector. The recombinant NP was purified to homogeneity by affinity chromatography and characterized through Western blot and MALDI-TOF/MS analysis. The characterized protein was used to develop an indirect IgG microplate ELISA using a panel of animal sera. The in-house ELISA was comparatively evaluated vis-a-vis a commercially available ELISA kit (Vector-Best, Russia) with 76 suspected samples that revealed a concordance of 90% with a sensitivity and specificity of 79.4 and 100%, respectively. The precision analysis revealed that the assay is robust and reproducible in different sets of conditions. Further, the assay was used for serosurveillance in ruminants from different regions of India that revealed 18% seropositivity in ruminants, indicating continued circulation of virus in the region. The findings suggest that the developed IgG iELISA employing recombinant NP is a safe and valuable tool for scalable high-throughput screening of CCHFV-specific antibodies in multiple species.