Genetic variability of hepatitis B virus in chronic infection

Sammanfattning: Chronic hepatitis B is a major health problem, and may lead to cirrhosis and hepatocellular carcinoma. The liver damage in hepatitis B virus (HBV) infection is considered to be a result of the immune response, and not a cytopathic effect of the virus itself. Thus, the course of infection depends on several factors which influence the immune response, such as age at infection, genetic host factors, and probably the genetic variability of the virus, which is observed as the evolution of genotypes in the carrier population and as the emergence of mutations in each infected subject. Eight HBV genotypes (A-H) have been identified. However genotype-related differences in the pathogenicity of HBV have not yet been described. Mutations in certain regions, in particular the core promoter, the precore and core regions have been more thoroughly studied. It is however, not clear to what extent and in what manner these mutations interfere with the course of infection and the liver injury. Because studies of mutations in the entire genome are rare, the frequency and importance of mutations in all coding regions are not known. The aim of our research was to study genetic variability in HBV and its effect on virus replication, course of infection, and outcome of therapy. In this work, we analyzed genotypes and core promoter mutations in 43 chronic HBV carriers of East Asian origin. Patients with genotype C showed higher grade of liver inflammation, and higher frequency of the double mutation at nucleotide 1762-1764 in the core promoter. These findings suggest that pathogenic differences between genotypes may exist and that the core promoter mutation may be useful as a marker for progressive liver damage. In most studies, mutations have mainly been assessed by comparison with a consensus sequence representing the genotype of the strain analyzed, but it is not known to what extent this approach reflects changes taking place during infection in a patient. In this work, the entire viral genome, from patients infected through vertical transmission, was studied. The emergence of mutations was assessed by comparing the individual sequences with the sequence of the strain assumed to have been transmitted. During the HBeAg-positive phase, the HBV genome appeared to be extremely stable despite the high replication level of virus. After loss of HBeAg, an average of 20 mutations was detected, without association to clinical outcome, with the exception of the TGA mutation in the core promoter. Because cytosine at position 1858 (C1858) is characteristic for genotype A we sequenced the complete genome of T1858 variants which we had previously classified as genotype A. Phylogenetic tree and similarity plot analysis of these strains revealed recombination between genotype C and a putative new genotype most similar to genotype A. Our findings encourage further study of genotypes and recombination in HBV. It is still unclear whether viral genetic variability influences response rate, and no specific mutation has reliably been associated with better or poorer response to interferon (IFN). The frequency of mutations in the entire X region, before and after IFN, was analyzed in 26 HBeAg-positive patients with various response patterns. No association between IFN response and any particular mutation or the number of mutations was identified, partly as a consequence of the strikingly low mutation frequency detected. By the genotype-specific core PCR and RFLP assay, designed by us, genotype mixtures were found in 20 of 30 patients treated with interferon. Mixtures were detected in samples collected before and after treatment, and these were easier to detect when IFN dramatically changed the levels of viremia. This could indicate that IFN influences the genotypes differently or that IFN may enhance the immune response toward the major strain more than the minor one. In summary, this study contributes to the understanding of the pathogenesis of chronic hepatitis B and describes how genetic variability of HBV may influence the course of infection and outcome of IFN therapy.

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