Hepatitis B core evolution and cellular localisation

HBV core gene has attracted a resurgence of interest recently because it influences the outcome of hepatitis B infection. The aims of this study were: to characterise the core gene variability in diverse geographic regions and ethnic groups; to determine subtype/genotype-specific variants; to specify country/ethnic-specific variants; and to identify any relationship between intracellular HBcAg and chronic HB disease. The current work can be divided into three main parts. In chapter 3, in the first and second sections, HBV DNA in 139 sera samples from 12 diverse geographic origins was analyzed by sequencing the core gene. We found that certain residues allow definition of subtypes and genotypes. Further, specific nucleotide motifs were defined for particular countries. We also found, intriguingly, a set of amino acid variants in a majority of sequences from South-east Asian patients and also for Western populations. In the second section of chapter 3, core gene variability in the Pacific region, the nucleotide diversity of the genotype C samples was significantly greater than that of genotype D samples, which is consistent with a longer history of HBV infection in genotype C predominant islands. Results obtained from this chapter confirm that HBV strains spread within constrained ethnic groups and that selection pressure defined the sequence variability within each genotype (and subtype). It may be that specific T cell epitopes are specific for particular geographic regions, and thus ethnic groups, which may affect the design of immunomodulatory therapies, including therapeutic vaccines. The third section of this thesis (chapter 4) deals with in vitro biological effects of HBV C gene variability. Confocal microscopy was used to visualise HBcAg cellular expression in 40 clones (including mutagenised samples) from various stages of HB disease frompatients with different HBeAg/anti-HBe status.. This was correlated with sequence variation within B cell epitopes and at the C-terminus. Using site-directed mutagenesis to revert C-terminus and B-cell epitope mutated sequences with cytoplasmic expression to the original (wild type) sequences led to a shift back to predominantly nuclear and both nuclear and cytoplasmic distribution, respectively. In conclusion, we showed that HBcAg reflects the genotype of the whole genome and there are certain residues that are distinct for the HBV genotypes. Moreover, we demonstrate that the pattern of HBcAg localisation in vitro is dependent on sequence and correlates with the serology of chronic HBV infection. It supports the hypotheses that such variants may play a biological role including antigenic expression. This study not only has an impact on the design of the therapeutic and prophylactic vaccines, but on our understanding of the immune response to different HBVs. Chapter 5 is composed of a preliminary study on FT09.1 cell line infectivity by HBV. This cell line which is transfected with a construct containing human-Annexin V, showed propagation of virus in primary and transfer experiments. Due to some difficulties using the Cat III laboratory, this project stopped after the first six months of the research course. The results obtained during that period have therefore been included in a separate chapter (chapter 5)