The Single Amino Acid Change of R516K Enables Efficient Generation of Vesicular Stomatitis Virus‐Based Crimean–Congo Hemorrhagic Fever Reporter Virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a medically important tick-borne virus, causing severe hemorrhagic diseases in humans. There are no approved vaccines and therapeutics for CCHFV infection. The study of CCHFV authentic virus requires biosafety level 3 facilities, hindering the research and development of antivirals. Here we report the generation of a recombinant vesicular stomatitis virus (VSV) bearing both CCHFV glycoprotein precursor (GPC) and EGFP reporter (rVSV-CCHFV-GFP). We also find that the acquisition of an unexpected single R516K mutation in the GPC protein enables the packaging of high-titer pseudotyped particles. The replication-competent rVSV-CCHFV-GFP reporter virus resembles the entry properties of the authentic virus and allows for rapid assessment of susceptible cell lines, neutralizing antibodies, and host entry factors such as heparan sulfate in fluorescence-based assays. This study provides a valuable strategy for packaging of high-titer CCHFV pseudovirus, and the tool generated here can be served for the identification and evaluation of countermeasures against the cell entry of CCHFV.