Non-structural proteins of crimean congo hemorrhagic fever virus kelkit strain and their roles in humoral immunity

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widely distributed arbovirus and causative agent of a severe febrile hemorrhagic disease that have a mortality rate between 5-30%. Currently there is no approved vaccine or treatment against this agent which has been classified as a biosafety level 4 (BSL 4) pathogen. BSL 4 facility requirement directs researchers to alternative methods to gather as much information about this pathogen as possible and recombinant protein technology is one of these methods. Recombinantly expressed viral proteins are powerful tools to dissect different stages of life cycle of such viruses. Among other proteins,viral non-structural proteins stand out because of their ability to interact with vector/ host immun system and contribution to viral pathogenesis. Roles of viral non structural proteins at viral assembly, possible activities as a scaffolding proteins, apoptotic/anti-apoptotic activities, inhibition of interferon based reactions and virus attenuation have been shown in many species including arthropod-borne bunyaviruses. Besides their various biological roles at virus life cycle, it has also been shown that these proteins could have potential roles at diagnosis, treatment and vaccine development. CCHFV has one and possibly two non structural proteins originated from M segment (NSm) and S segments (NSs). In this study bacterial expression of nonstructural proteins M and S of CCHFV Kelkit06 strain is aimed. For this purpose, NSm (369 base pair) and NSs (477 base pair) optimized sequences are cloned to various expression vectors (pETSUMO, pET28b and pET30b) and Escherichia coli BL21(DE3) was utilized as an expression host for N or C-terminally His tagged proteins. Recombinant proteins were used in Western Blotting and enzyme immunoassay (EİA) tests against CCHFV antibody positive and negative human serum to detect the antibodies against these proteins. Both proteins reacted with CCHFV antibody positive human sera when tested with Western Blotting, 66 % of all tested CCHFV antibody positive human sera were found to be positive for NSm and NSs specific IgG. Also high titer NSm and NSs specific antibodies obtained from mice immunized with recombinant proteins. Further studies are warranted to specify exact roles played by these proteins.