Alternative tools to investigate HBV entry pathway and intra-host evolution

Hepatitis B virus (HBV) infects liver cells resulting in hepatitis B infection. In approximately 90% of childhood infections and 10% of adult cases the virus establishes a chronic, lifelong infection. In 2015, it was estimated that approximately 3.5% of the global population were living with chronic HBV infection. Long-term complications of chronic HBV infection such as cirrhosis and hepatocellular carcinoma cause a significant disease burden. The World Health Organisation has set a goal to eliminate the infection by 2030. However, current treatment for HBV does not cure the infection, and most people require the treatment for life. Therefore, a better understanding of the virus lifecycle and viral intra-host evolution is crucial in the development of antiviral drugs. The error-prone nature of the HBV polymerase and combined with high replication rate result in genetic diversity manifesting as a quasispecies in a chronically HBV- infected host. However, the impact of genetic diversity in HBV infections is still poorly understood; while antigenic differences and drug resistant forms of HBV have been assigned to specific viral polymorphisms, the consequence of the majority of genetic variants have not been interrogated. In this study, genetic diversity of HBV infections was investigated using a nanopore sequencing platform to determine longitudinal HBV evolution within hosts. The data extends previous knowledge that greater viral diversity is observed in HBV e-antigen (HBeAg)-negative infection and the rate of evolution in the nonoverlapping region is much higher than that of the overlapping region. The HBV surface proteins (HBsAg) comprise the Large (L), Middle (M) and Small (S). The HBsAg gene is completely overlapped by the polymerase gene in a -1 frameshift. Mutations occurring in one open reading frame (ORF) can also impact on the other; therefore, it is challenging to investigate the phenotype of HBsAg mutations without altering phenotypic behaviour of the polymerase. Moreover, HBV research requires Biosafety Level (BSL)-3 containment, which hinders research progress. To address these limitations, retroviral pseudotyped viruses (PVs) were utilised in this study to investigate HBV entry and the effect of viral polymorphisms. Retroviruses pseudotyped with the HBV glycoproteins, possess the structural proteins of retrovirus and HBV surface glycoproteins and only require BSL-2 laboratory facilities, which offers an alternative to overcome the drawbacks of handling HBV in vitro. This study developed novel pseudotyping techniques to investigate the HBV entry pathway. A sodium taurocholate co-transporting polypeptide (NTCP) overexpressing hepatocellular carcinoma cell line was developed to determine the impact of receptor expression on HBV entry. A new cell line (293T.gtD4.S) constitutively expressing the S protein of a genotype D isolate has also been developed. Using an in-house engineered lentiviral vector pNL4.3.Nluc.R-E- and a plasmid expressing L co- transfecting 293T.gtD4.S cells, the infectivity of HBV PVs was quantified and monitored. The results revealed the importance of L/S ratio in HBV infectivity