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Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis distributed widely in Africa, Asia, Russia and the Balkans. The emergence and re-emergence of CCHFV emphasize the importance of increasing both human and veterinary surveillance and developing diagnostic capacity. Recombinant CCHFV nucleocapsid protein (NP) has been expressed using insect cells and mammalian cells and used as a diagnostic tool but bacterial expression has not been described previously. The S gene of CCHFV was codon optimized and the NP expressed in Escherichia coli from the synthetic gene. The protein was reacted against serum samples collected from confirmed CCHFV patients at varying intervals after the onset of illness from acute to convalescent stages using both an ELISA and a Western blot. To confirm that the protein was able to induce a humoral antibody response that could be detected using CCHFV antigen derived from live virus, mice were immunized and serum samples were tested using IF slides prepared from CCHFV infected Vero cells. The recombinant antigen was able to detect IgG antibody in acute and convalescent sera. In addition, a detectable IgG antibody response was induced in mice immunized using NP. The results suggest that proteins expressed in a bacterial system lacking post-translational modifications can be used in ELISA to detect IgG antibody against CCHFV in human sera which may be used for routine diagnosis and seroepidemiology. [Copyright &y& Elsevier]

Abstract: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis distributed widely in Africa, Asia, Russia and the Balkans. Occurrence of segment reassortment has been established and may be associated with pathogenicity. The ability to distinguish between an infection with a reassortant and a non-reassortant variant may have prognostic value. In this study the use of Simple-Probe technology and real-time PCR was investigated to detect rapidly CCHF viral nucleic acid and to distinguish between reassorted and non-reassorted variants of southern African CCHF virus (CCHFV) isolates. A Simple-Probe® was designed based on multiple alignments of 19 CCHFV partial M segment sequences that included reassorted and non-reassorted isolates from two phylogenies. Real-time PCR followed by probe-specific melting-curve analysis allowed differentiation of three selected isolates representative of two phylogenetically distinct groups, SPU 431/85 (group IV), 383/87 (group IV) and 103/87 (group III) based on melting temperatures. Within group IV there are two distinct lineages which were represented in the optimization. A further 16 clinical isolates could be genotyped into groups III and IV using the Simple-Probe real-time PCR assay developed in this study. This assay provides a reliable and sensitive tool for the differentiation between reassorted and non-reassorted variants of CCHFV which finds application for diagnosis and epidemiologic and surveillance studies. [ABSTRACT FROM AUTHOR]