Emergence and selection of HBV variants in an anti-HBe positive patient persistently infected with quasi-species

Abstract

Background/Aims: Infection with the hepatitis B e antigen (HBeAg) negative variant of hepatitis B virus (HBV) causes chronic liver disease characterised by occasional acute exacerbations. This virus exhibits a high prevalence of mutations in the core region. Our aim was to study the changes in the pre-core/core region of the virus in relation to exacerbations of the disease. Methods/Results: We performed direct sequencing on DNA amplified from 7 sequential sera taken over a 5-year period from a hepatitis B surface antigen (HBsAg) and anti-HBe positive Greek patient infected with the HBeAg negative variant. The patient had chronic hepatitis with several acute exacerbation episodes and underwent interferon therapy twice. We found significant variability in the core region at different time points. To determine whether these variants were present in the initial serum sample and subsequently selected under immune pressure or whether they arose de novo during the course of the disease, we cloned the pre-core/core region from 4 sera before and after episodes of acute exacerbation. Fifteen clones from each time point were sequenced. Fourteen nucleotide mutations in the pre-core/core region were recorded, 7 (50%) of which led to amino-acid substitutions. All the amino-acid changes occurred at recognised B− and CD4+ epitopes. The cloning results indicate the presence of quasi-species in all the samples investigated. Some of the variants present as a minor population in the first sample appear to have been selected and become dominant in subsequent sera. However, the emergence of novel variants, not present at a detectable level in earlier samples, during the course of the disease, was also established. The quasi-species nature of HBV only became apparent after the cloning experiments and was not obvious from the direct sequencing results. Conclusions: New dominant variants occurring during the course of the disease arose either by the selection of pre-existing mutants that were not detected by direct sequencing or by mutation of existing strains. All changes were located within B− and CD4+ epitopes. The continuous production and selection of variants may enable virus to evade elimination by the immune system, resulting in presistent infection.