Low frequency of mutations in the core promoter and precore regions of hepatitis B virus in anti-HBe positive Brazilian carriers

Abstract

Background: Mutations in the core promoter and precore regions of the hepatitis B virus (HBV) genome, notably the double substitution (AGG to TGA) at nt positions 1762-1764 in the core promoter, and the precore stop codon mutation G to A at nt 1896, can often explain the anti-HBe phenotype in chronic carriers. However, the A1896 mutation is restricted to HBV isolates that have T at nt 1858. The double substitution at positions 1762-1764 has been described to occur preferentially in patients infected with strains showing C instead of T at nt 1858.

Results: HBV DNAs from 29 anti-HBe Brazilian samples were characterized by nucleotide sequencing of PCR products from precore region. Among them, 18 isolates presented C at nt 1858 (mostly genotype A strains). The 11 remaining isolates (genotypes D and F) had T1858. The stop codon mutation at nt 1896 was found in seven isolates (24% of the total and 63% of the isolates that had T1858). The frequency of the double substitution at positions 1762-1764 was surprisingly low (20%) among C1858 isolates. An association between A1896 and TGA 1762-1764 mutations was observed among genotype D isolates: these showed either none of the two mutations or both. Furthermore, strains mutated at positions 1896 and/or 1762-1764 also presented an elevated number of other, less common substitutions in the core promoter and precore regions.

Conclusions: The data reported here are not in accordance with some reports from other parts of the world. In half of the isolates, none of the mutations previously described could explain the anti-HBe phenotype.

Figure 1

Phylogenetic tree of 37 HBV isolates, constructed with the neighbor-joining method, and based on 200 nucleotides (positions 1814-2013). Capital letters A, D, and C designate the respective genotype groupings. Bootstrap values were calculated from 100 replicates and the numbers above 60 are shown. Isolates A1 to A17, D1 to D9, and F1 to F3 are from this work. Their nucleotide sequences have been deposited in the Genbank database under accession numbers AF389988 to AF390016. The other isolates are designated by their Genbank accession numbers.

Figure 2

Alignment of partial nucleotide sequences of HBV isolates derived from anti-HBe carriers. The consensus sequence (based on wild-type genotype A strains) is indicated at the top. Dots represent the same nucleotides as in the consensus sequence. Letters at the left represent the genotypes of the strains. The AT-rich region (nt 1789-1795) and the direct repeat DR1 (nt 1824-1834) are underlined. The two common point mutations, i.e. the double substitution in the core promoter (nt 1762-1764) and the precore stop codon mutation (nt 1896), are indicated by arrows. The other substitutions constitute the uncommon point mutations. The initiation codons for precore and core proteins are also shown. HVR: Hypervariable region (nt 1751-1755).