Development of a Novel Plasmid-based Eukaryotic Model to Investigate Crimean-Congo Hemorrhagic Fever Virus

Objective: Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne viral disease, caused by the Crimean-Congo Hemorrhagic Fever virus (CCHFV). The global expansion of CCHF and high mortality rates underline the critical need for research and development of effective treatments and vaccines. However, the high risk of transmission and requirement for highcontainment facilities hinder investigations involving live virus. In this study, we aimed to address these challenges by employing a plasmid-based virus -like particle (VLP) system and a minigenome model to investigate the biology and immunology of CCHFV. Methods: The plasmids encoding CCHFV structural genes of CCHFV were transfected into Huh-7 cells. Viral protein expression was confirmed using fluorescence imaging, immunological and molecular methods. A minigenome system was developed, eliminating the need for T7 polymerase, T7-expressing cellular lines, or viral ribonuclear protein complexes, allowing autonomous virus replication without a helper virus or transfections using plasmids in trans. Results: Fluorescence microscopy showed successful production of NP-EGFP and GC-EGFP proteins with various subcellular localizations. Western blot analysis demonstrated the presence of pre-Gc, Gc, pre-Gn, Gn, and Np proteins in cell lysates and supernatants. ELISA analysis suggested that transfection of Np alone, in combination with Gc, or all three proteins might cause distinct VLP formations. Huh -7 cells successfully expressed reporter genes after transfection of minigenome RNA transcripts. Conclusion: The study advances CCHFV research by using novel tools for virus biology and immunology. The findings may provide new avenues for research that promise better public health preparation against this neglected viral disease.