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-à-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.
Crimean-Congo hemorrhagic fever (CCHF) virus, a highly pathogenic viral agent is responsible for severe fatal hemorrhagic infections in many parts of the world. The early diagnosis of CCHF infection is important for successful clinical management and epidemiological control. The nucleoprotein (NP) of CCHFV being highly conserved and immunogenic is used as early diagnostic marker. In this study, we report a rapid and sensitive double antibody based antigen capture ELISA to detect Crimean-Congo hemorrhagic fever virus (CCHFV). Highly specific polyclonal and monoclonal antibody against NP has been generated and used as capture and detector antibody respectively. The assay was able to detect viral nucleoprotein in different matrices including human serum, ticks and culture supernatant. The detection limit of the developed sandwich ELISA assay was 25 ng of purified antigen. Comparison with a real time RT-PCR revealed its detection limit to be 1000 genome equivalents of CCHFV. Further the assay was comparatively evaluated with a commercial kit employing gamma irradiated CCHFV, revealing a sensitivity and specificity of 100%. This newly developed sandwich ELISA (sELISA) with high sensitivity and specificity could be used as an efficient method for the detection of CCHF virus in humans, ticks and culture supernatant. The assay will be useful as alternate tool for diagnosis of acute infection and is amenable for screening of large scale samples in resource limited settings.
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