Image_1_Development of Multispecies Recombinant Nucleoprotein-Based Indirect ELISA for High-Throughput Screening of Crimean-Congo Hemorrhagic Fever Virus-Specific Antibodies.JPEG

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.</p

The percentage of the genome recovered and the reads mapped for the each CCHFV sequences retrieved in this study.

The NGS reads were mapped to the L gene (Accession No: KC867272), Asian M gene (Accession No: MN866207), African M gene (Accession No: MH396671), and S gene (Accession No: KX013446).</p

The percentage of the nucleotide and amino acid divergence for each gene with respect to the reference CCHFV L, M, S segments having accession number MH396640.

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CCHF Viral RNA, anti-CCHFV IgM and anti-CCHFV IgG kinetics of CCHFV cases.

(A) CCHF viral RNA amongst fatal and survivors (B) CCHF viral RNA amongst individual survivors at different time points (C) Anti-CCHFV-IgM antibodies amongst individual survivors at different time points and fatal cases (D) Anti-CCHFV-IgG antibodies amongst individual survivors at different time points and fatal cases.</p

District-wise CCHF positivity in the humans, tick-pools and livestock from Gujarat State, India during the year 2019.

Image attribution: https://d-maps.com/carte.php?num_car=8623&lang=en. License attribution: https://d-maps.com/conditions.php?lang=en. The map was modified showing the district-wise CCHF human cases, tick-pools and livestock positivity in the year 2019. All the details provided in the figure are created by the author themselves using the licensed version of the online software Biorender.com.</p

Clinical presentation of fatal and survived CCHFV cases.

Clinical presentation of fatal and survived CCHFV cases.</p

Details of CCHF positive cases (n = 34), clusters of cases, clinical outcome, along with livestock and tick pool positivity from different districts of Gujarat state.

Details of CCHF positive cases (n = 34), clusters of cases, clinical outcome, along with livestock and tick pool positivity from different districts of Gujarat state.</p

Details of the time points for the follow-up of the CCHF survivors.

(DOCX)</p

Month-wise distribution of CCHF cases and deaths in Gujarat state during 2019.

Month-wise distribution of CCHF cases and deaths in Gujarat state during 2019.</p

Phylogenetic tree for the L M and S genes for the CCHFV (depicted in A, B and C respectively).

Maximum-Likelihood tree for CCHFV sequences retrieved from the clinical samples of the Gujarat state. The tree was constructed using the representative clades downloaded from Genbank using the General-time reversible model with gamma distribution as the rate parameter. The bootstrap replication of 1000 cycles was used to assess the statistical robustness of the generated tree. The scale depicts the number of base substitutions per site. Clades are marked in different colours. * indicates the fatal CCHF cases.</p