Assessment of Inhibitors of Pathogenic Crimean-Congo Hemorrhagic Fever Virus Strains Using Virus-Like Particles

Abstract

Crimean-Congo hemorrhagic fever (CCHF) is an often lethal, acute inflammatory illness that affects a large geographic area. The disease is caused by infection with CCHF virus (CCHFV), a nairovirus from the Bunyaviridae family. Basic research on CCHFV has been severely hampered by biosafety requirements and lack of available strains and molecular tools. We report the development of a CCHF transcription- and entry-competent virus-like particle (tecVLP) system that can be used to study cell entry and viral transcription/replication over a broad dynamic range (~4 orders of magnitude). The tecVLPs are morphologically similar to authentic CCHFV. Incubation of immortalized and primary human cells with tecVLPs results in a strong reporter signal that is sensitive to treatment with neutralizing monoclonal antibodies and by small molecule inhibitors of CCHFV. We used glycoproteins and minigenomes from divergent CCHFV strains to generate tecVLPs, and in doing so, we identified a monoclonal antibody that can prevent cell entry of tecVLPs containing glycoproteins from 3 pathogenic CCHFV strains. In addition, our data suggest that different glycoprotein moieties confer different cellular entry efficiencies, and that glycoproteins from the commonly used strain IbAr10200 have up to 100-fold lower ability to enter primary human cells compared to glycoproteins from pathogenic CCHFV strains.

Author Summary

The tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a frequently life-threatening disease. CCHFV is present in a wide geographic area with potential for expansion. Moreover, CCHFV segmented genome reassortment leads to new strains with potentially different virulence. Studying CCHFV is highly necessary, but requires dedicated, resource-intensive, high biosafety and security laboratories. In part due to the need for high containment, CCHFV studies have been limited, and developing tools to study CCHFV has been difficult. We report the development of a system that mimics the CCHFV life cycle and produces virus-like particles (VLPs) that are similar to CCHFV in cell culture, but do not form infectious CCHFV and therefore do not require the use of special laboratories. We generated VLPs representing several pathogenic CCHFV strains with robust reporter signal activity. This allows VLPs to be used in testing cell entry inhibitors against a wide array of CCHFV strains. In addition, VLPs can be used in a variety of cell lines and in cells directly isolated from humans. Our results also suggest that the CCHFV strain IbAr10200, which is commonly used in the laboratory, may not accurately reflect the activity of circulating pathogenic CCHFV strains, as the surface glycoproteins of IbAr10200 confer reduced entry efficiency of VLP into cells derived directly from humans. In addition, we show that drugs with proven anti-CCHFV properties inhibit VLP activity, and identify a monoclonal antibody that prevents cell entry of VLP made using glycoprotein genes from different, pathogenic CCHFV strains.