RESEARCH ARTICLE Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by

Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, se-vere thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombi-nant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was opti-mized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFVmutants. The M segment en-codes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for produc-ing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We stud-ied the role of furin cleavage by rescuing a recombinant CCHFV encoding a viru