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There are two hypotheses explaining a fulminant outcome after hepatitis B virus (HBV) infection, both of which may be applicable at the same time: (i) basal core promoter (BCP) mutations increase viral replication, allowing rapid spread of the virus through the liver, and (ii) pre-core (pre-C) mutations abrogating hepatitis B e antigen (HBeAg) synthesis remove its tolerogenic effect, leading to a vigorous immune response. This study investigated the effect of these mutations on virus replication efficiency and HBeAg production. Substitutions A1762T/G1764A and T1753C, C1766T and T1768A in the BCP region, and G1896A and G1899A in the pre-C region, were examined either alone or in combination, using a common genetic background. Huh7 cells were transfected with these constructs and real-time PCR was used to quantify released virion-associated and intracellular HBV DNA, pregenomic RNA and pre-C mRNA. In addition, culture supernatants were tested for hepatitis B surface antigen (HBsAg) and HBeAg. The double BCP mutation (A1762T/G1764A) and the pre-C mutations (G1896A, G1899A), either alone or in combination, had no appreciable effect on the replication capacity of the virus. In contrast, clones with mutations at positions 1766/1768, 1762/1764/1766 and 1753/1762/1764 exhibited increased-replication phenotypes. HBeAg was undetectable in all cultures transfected with constructs bearing the G1896A stop-codon mutation, as expected. In contrast, constructs with additional mutations in the BCP region had appreciably lower levels of HBeAg expression than the wild type. Thus, core promoter mutations other than those at 1762/1764 appear to upregulate viral DNA replication and, at the same time, greatly reduce HBeAg production.

Variants of hepatitis B surface antigen have been described in different clinical settings, but their replicative capacity in vitro has remained unexplored. Point mutations leading to sT131I, sK141E, and sG145R amino-acid substitutions were engineered by site-directed mutagenesis into an infectious plasmid clone of the virus. The mutated constructs were transfected into Huh7 cells, and their replication capacity was documented by LightCycler (Roche Diagnostics) measurements of virion-associated hepatitis B virus (HBV) DNA, intracellular relaxed circular double-stranded DNA, and pregenomic RNA. The sT131I and sG145R variants replicated with efficiency equal to that of the wild type, whereas the sK141E variant was replication impaired. Hepatitis B virus (HBV) particles have an outer lipid bilayer envelope into which are inserted the large, middle, and small hepatitis B surface antigen (HBsAg) proteins. All 3 proteins share the group-specific “a” antigenic determinant, a hydrophilic region with a conformational structure that constitutes the main neutralization epitope of the virus [1]. Variants of the virus that have amino acid substitutions in this region have been described in different epidemiological and clinical settings. These variants exhibit altered antigenicity, which causes assays either to fail to detect them or to have greatly reduced sensitivity. Such variants have been described in vaccinees, livertransplant patients who have received monoclonal or polyclonal anti-HBs treatment after transplant, cases of occult infection

A patient with agammaglobulinemia developed acute hepatitis that progressed to chronic liver disease with high levels of hepatitis B virus (HBV) DNA in the absence of detectable HBsAg. Sequencing of the a determinant region of HBsAg revealed multiple amino acid substitutions that, unusually, also included a substitution at position 122 that defines subtype specificity. All of these mutations had a profound effect on the antigenicity of this region, which led to the complete failure of variant detection by commercially available routine diagnostic assays or laboratory-based monoclonal antibody assays.

AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection. METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals post-inoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2354-2359, numbering from the EcoRI site). The amplicons were cloned into PCR-ScriptTM and a minimum of 15 clones per time point were sequenced in both directions. RESULTS: Both genotypes persisted for the entire follow-up period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5% level of testing. CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.