Stably expression of proteins of Crimean-Congo Hemorrhagic Aever virus kelkit strain in eukaryotic cells

Crimean-Congo Hemorrhagic Fever (CCHF) is a zoonotic infectious disease that causes severe illness in humans. This disease is particularly characterized by a high mortality rate in countries where vector ticks are endemic. Viral proteins play a crucial role in the biology and immunology of CCHFV, similar to other viral systems. The significance of CCHFV proteins in various processes has been addressed in numerous studies, and in these investigations, CCHFV proteins are typically produced using transient expression systems. However, stable protein expression proves to be a consistent and reliable model for such studies over time. In this study, the aim was to continuously produce CCHFV glycoproteins and nucleoproteins by using eukaryotic cell lines that mimic glycosylation processes in human cells. Human hepatocellular cell lines (Huh-7) were employed for the stable expression of CCHFV proteins. The resistance of cells to selective antibiotics was evaluated through MTT analysis, followed by transfection using transfection agents such as Lipofectamine® 3000 and polyethyleneimine (PEI). Cells expressing CCHFV nucleoproteins and glycoproteins in a stable manner were generated through the selection of antibiotic-resistant colonies. The detection of the transfected gene in the cells was performed using real-time PCR specific to the transfected CCHFV genes. Additionally, the expression of proteins was tested through immunofluorescence microscopy, Western blot, and enzyme immunoassays. The experiments demonstrated the successful expression of CCHFV proteins in stable cells. Western blot analysis confirmed that the produced proteins in the stable cell lines were of the expected sizes, while enzyme immunoassay (EIA) analysis confirmed their antigenic properties. Furthermore, immunofluorescence microscopic analysis revealed the localization of the produced proteins within the cells. The successful expression of CCHFV proteins in stable cells was demonstrated. These stable cell lines are expected to serve as important tools in further detailed investigations on CCHFV, contributing to the understanding of the virus's biology, immunology, and pathogenesis, as it is considered one of the priority pathogens for research.