Authors:

Sarah Jimenez Rojas

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About Paper:

Hepatitis C Virus (HCV) is a bloodborne pathogen causing chronic liver disease, cirrhosis, and hepatocellular carcinoma, with over 50 million people infected worldwide and 1 million new infections each year. Direct acting antivirals cure up to 95% of chronic HCV infections; however, reinfection can occur after successful treatment, and cases continue to rise across the United States, leading to the urgent need for an HCV vaccine. Rational vaccine design is limited by a lack of structural information. Notably, there is no resolved structure of HCV due to the heterogeneity, lipophilicity, and low yields of cell-cultured virus. To overcome this, an alternative purification approach is to use affinity-tag particles generated by transfection of in vitro transcribed viral RNA. In this research, the transfection of HCV with a polyhistidine and One-Strep-Tag will be optimized in hepatic cell line Huh 7.5. The transfection efficiency of different treatments, such as transfection technique (lipofectamine 3000, PEI-MAX, and electroporation), ratios of DNA to transfection reagent (1:1, 1:2, 1:3, 1:4, 1:5), and harvesting time aim to be evaluated. An immunofluorescence assay, utilizing an E2 antibody, and a focus-forming assay will be used to test transfection efficiency and infectious titer, respectively. All this with the purpose of designing a comprehensive protocol for transfection and quantification, critical aspects to future purification and characterization of viral structure through cryo-EM and other techniques to inform vaccine design. Keywords: Hepatitis C Virus (HCV); Structural Virology; Transfection; Cell-Cultured Virus; Rational Vaccine Design

Source:

Purdue University / 2024

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Co-authors:

Sarah Jimenez Rojas