Establishment of an In Vitro HBV Infection System and Investigation of a Recently Emerged HBV Genotype

Hepatitis B virus (HBV) research has been hampered due to the lack of experimental infection systems to study the virus. With the discovery of NTCP as a cellular receptor for HBV, transgenic HepG2-NTCP cells offered promise as an infection model, but several limitations have hindered their usefulness for studying all aspects of the HBV life cycle, such as viral spread. Further, most research systems are based on HBV genotype D, with little regard to the other genotypes (A-J) even though the other genotypes have different clinical manifestations and response to treatment. The emergence of HBV genotype E (GTE) is particularly alarming due to its rapid spread throughout a large region in Africa suggesting possible antibody escape in vaccinated individuals. In this thesis, we improved the HepG2-NTCP cell-based infection system allowing for more physiologically relevant infection conditions that allow for HBV spread, and hopefully the study of all HBV genotypes, such as the emerging genotype, GTE. By interrogating the impact of different variables on HBV infection and spread in vitro, optimal virus handling to preserve infectivity and avoiding supraphysiological cellular NTCP levels were found to be key factors for achieving robust viral infection and spread. Additionally, antibody-resistant, NTCP-independent HBV spread was observed, suggesting a novel HBV cell-to-cell spread mechanism. Importantly, we show that HBV GTE has increased expression and altered antigenicity of the viral surface proteins which could contribute to vaccine/immune escape. Overall, this optimized, more physiological relevant HBV infection system allows for study of all aspects of the viral life cycle and should facilitate expansion of research to different HBV genotypes, both of which will ultimately aid in antiviral drug discovery.