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Background: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies. Methods: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen. Results: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation. Conclusions: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment. [ABSTRACT FROM AUTHOR]

• West Nile and Wesselsbron viruses cause outbreaks of disease in southern Africa. • Isothermal assays have been proposed as fieldable nucleic acid amplification tests. • Non infections transcribed RNA was used to optimise and validate an isothermal RT-RPA. • Amplification products were detected using lateral flow devices. • Specific probe design allowed differentiation of West Nile and Wesslesbron viruses. West Nile virus (WNV) and Wesselsbron virus (WSLV) are mosquito-borne viruses belonging to the Flavivirus genus, family Flaviviridae and cause outbreaks in southern Africa after heavy rain. Isothermal assays have been proposed for application in field situations as well as low resource settings and hence we developed a reverse-transcriptase recombinase polymerase amplification (RT-RPA) to detect WNV and WSLV known to occur in South Africa, causing sporadic outbreaks usually associated with good rainfall favouring mosquito breeding. Infectious virus can only be handled within a biosafety level (BSL) 3 facility, hence we opted to validate the assay with transcribed RNA. Specific RT-RPA primers and probes were designed for detection of WNV and WSLV and products detected using a rapid lateral flow device. The assay was performed in 30 min and detected 1.9 × 10¹ copies of WNV and 3.5 × 10° copies WSLV using noninfectious transcribed RNA controls. In addition, the assay was not inhibited by the presence of mosquito extracts in spiked samples. Mismatches between the WNV and WSLV probes and other flaviviruses will likely prevent cross reactivity. The sensitivity, low RPA incubation temperature and rapid processing time makes assay systems based on RPA technology ideally suited for fieldable diagnostics. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe acute human disease with lethal outcome. The knowledge about the immune response for this human health threat is highly limited. In this study, we have screened the glycoprotein of CCHFV for novel linear B-cell epitopic regions using a microarray approach. The peptide library consisted of 168 synthesized 20mer peptides with 10 amino acid overlap covering the entire glycoprotein. Using both pooled and individual human sera from survivors of CCHF disease in Turkey five peptide epitopes situated in the mucin-like region and GP 38 (G15-515) and GN G516-1037 region of the glycoprotein were identified as epitopes for a CCHF immune response. An epitope walk of the five peptides revealed a peptide sequence located in the GN region with high specificity and sensitivity. This peptide sequence, and a sequence downstream, reacted also against sera from survivors of CCHF disease in South Africa. The cross reactivity of these peptides with samples from a geographically distinct region where genetically diverse strains of the virus circulate, enabled the identification of unique peptide epitopes from the CCHF glycoprotein that could have application in development of diagnostic tools. In this study clinical samples from geographically distinct regions were used to identify conserved linear epitopic regions of the glycoprotein of CCHF. [ABSTRACT FROM AUTHOR]