Generation of HBV DNA Plasmids of Genotype E for In Vitro Infection Model

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Background The African specific HBV genotype E (HBVgtE) is associated to a relative high viral replication and greater risk of hepatocellular carcinoma. However, studies concerning this genotype are still under-represented. This project aims at generating replication competent HBV DNA plasmid of genotype E to better understand biomarker production and pathogenesis by this genotype in vitro. Method The fast cloning was realised following the well-established protocol from Li et al. in 2011. In brief, the primers for insert amplification were designed with insert-specific sequences and additional 15-17 bases overlapping with the vector-ends. The 1.5mer was generated from the 1.1mer template and for the 1.1merG1896A, the mutation G1896A was directly inserted in the primer sequences for 1.1mer vector amplification resulting in the knockdown of HBeAg expression. Then, the recombinant 1.1 and 1.5merHBV-E DNA plasmids were transfected into HepG2-hNTCPcells and their HBsAg/HBeAg Level were measured in the supernatant. Results Transfection of HBV-E Plasmids resulted in a successful production of HBeAg and HBsAg with a higher expression of both biomarkers in 1.5mer. We found similar meanlevels of HBV DNA, RNA and total HBsAg after transfecting the 1.5mer during 8days at ratio 2:1; 3:1; 4:1. Interestingly, mean LHBs were slightly higher using a transfection ratio 3:1 (2.7ng/mL) compared to 2:1 (0.79ng/mL) and 4:1 (0.72ng/mL) suggesting a better viral fitness of HBVgtE. Conclusion The newly engineered HBV-Eplasmids can be transfected in cell cultures hereby showing stable levels of biomarkers. Our model is ready-for-use in further in vitro studies on HBV replication and drug efficacy of HBVgtE. Publication History Article published online: 23 January 2024 © 2024. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

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