Evaluation of Crimean-Congo Hemorrhagic Fever Orthonairovirus AviTagged Nucleoprotein for Potential Application in Diagnosis

Background: Crimean-Congo hemorrhagic fever (CCHF) is an acute viral zoonotic disease, with a mortality rate of 30-50%. There is no approved vaccine or any specific antiviral treatment for CCHF; therefore, the rapid diagnosis seems to be crucial for both efficient supportive therapy and control of infection spread. In this study, the potency of recombinant nucleoprotein of virus expressed in prokaryotic system was investigated for diagnosis of the infection. Methods: The DNA sequence of complete nucleoprotein ORF was codon optimized based on E. coli codon usage and synthesized commercially. The gene was subcloned in pCA4 vector and expressed in E. coli BL21 (DE3). Refolding and simultaneous purification of nucleoprotein were performed using protein folding liquid chromatography method. The recombinant nucleoprotein was analyzed by Western blotting, ELISA, immunofluorescence assay, and circular dichroism. Forty eight human samples, in three IgM positive and three negative control groups, were evaluated using recombinant nucleoprotein in a capture ELISA setting. Serum from healthy individuals, those suspected to viral hemorrhagic fevers, and positive samples of Chikungunya and Dengue were considered as negative controls. Results: The existence and structure of recombinant nucleoprotein were verified and confirmed. Capture IgM ELISA detected all positive samples (sensitivity of 100%), but none of the 25 negative samples was detected as positive (specificity of 100%). The test also detected all the included genotypes of virus. Conclusion: Our recombinant nucleoprotein can be used in IgM capture ELISA for easy and efficient detection of CCHF in any lab in endemic regions.