Crimean-Congo haemorrhagic fever virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Handling the virus requires biosafety level-4 facilities, limiting accessibility for many laboratories. Advances in molecular techniques have allowed preparation of safe recombinant antigens that have application in diagnosis and serosurveillance of CCHFV. The aim of this study was to determine genetic diversity in CCHFV based on all available complete sequence data for the S gene encoding CCHFV nucleoprotein (NP) and antibody cross-reactivity between the NP of a South African isolate and the NP of a Greek isolate (AP92), the most genetically diverse CCHFV strain. The nucleotide sequence diversity and amino-acid diversity between genotypes, within genotypes and the pairwise distances were calculated for a dataset of 45 CCHFV isolates retrieved from GenBank. The most diverse virus, AP92, isolated from a tick in Greece, displayed the highest amino-acid difference (8·7%) with SPU415/85, isolated from a human infection in South Africa. Recombinant NP encoded for by codon-optimized S genes of SPU415/85 and AP92 were expressed in a bacterial host system and used to develop an in-house ELISA to detect IgG antibody against CCHFV in South African patients who survived infection. A total of 14/14 sera reacted with the South African recombinant NP and 13/14 reacted with the Greek recombinant NP. The serological cross-reactivity of the two NP antigens suggests that recombinant antigens prepared from geographically distinct CCHFV will have diagnostic and epidemiological applications worldwide.
Background: Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus belonging to the family Bunyaviridae, which consists of diverse RNA viruses. CCHFV has the propensity to cause nosocomial infections with a high fatality rate and is endemic in South Africa. Handling of the virus requires biosafety level 4 (BSL-4) conditions, which limits diagnostic capacity. Advances in molecular techniques have allowed preparation of safe recombinant antigens that are useful in diagnosis and serosurveillance of CCHFV. The purpose of this study was to examine the global nucleic acid and amino acid diversity between isolates worldwide; clone and express a recombinant CCHFV nucleoprotein (NP) from a southern African CCHFV and distantly related Greek CCHFV strain and determine the antigenic cross-reactivity between the two isolates.Methods & Materials: Phylogenetic analyses based on NP gene of 45 isolates was performed. Nucleotide sequence diversity and amino acid diversity between groups, within groups and pairwise distances were calculated. A previously expressed codon optimized NP from a South African isolate, SPU 415/85 was subcloned into pColdTF vector and was expressed in a bacterial system. Similarly, the gene encoding the NP of a Greek isolate AP92 was codon optimized and expressed in Escherichia coli host cells. Recombinant NP were used to develop in house ELISA to detect IgG antibody against CCHFV in South African patients who survived infection Results: Phylogenetic analyses using nucleotide and amino acid sequences of the NP revealed six different groups. The most diverse strain, AP92, displayed the greatest amino acid difference with SPU415/85 (8.7%). Both proteins were expressed with the aid of a chaperone and were purified from the soluble phase. A total of 14/14 sera reacted with the South African recombinant NP and 13/14 reacted with the Greek recombinant NP. The cross reactivity suggests the presence of highly conserved epitopes.Conclusion: Phylogenetic analyses reveal a high genetic diversity and lower amino acid diversity which suggested synonymous changes in nucleotides, resulting in fewer differences at protein level. The serological cross-reactivity of the two NP antigens suggests that recombinant antigens prepared from geographically specific CCHFV strains will have diagnostic and epidemiological applications worldwide.
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