Molecular characterization of full genome hepatitis B virus sequences from an urban hospital cohort in Pretoria, South Africa

Hepatitis B Virus (HBV) is a DNA virus and belongs to the genus Orthohepadnavirus of the Hepadnaviridae family which represents one of two animal viruses with a DNA genome which replicates by reverse transcription of a viral RNA intermediate. Nucleotide variation led to further sub-classification into 8 genotypes (A to H). The reverse transcription step within its life cycle is prone to the introduction of errors and recombination when dually infected. This leads to a viral quasispecies which forms during the course of infection with many minor population variants; such variants can however only be detected by means of ultra-deep sequencing. A recent study in the Department of Medical Virology (UP) by Mayaphi et al. identified a number of the specimens that partitioned away from the typical subgenotype A1 clades with high bootstrap values and longer branch lengths. Thus, the main objective of the current study was to characterize the full genome of all variants for the outliers observed in the aforementioned study, inclusive of potential recombination, dual infection and minor populations. Twenty samples were selected from a previous cohort for purposes of the present study. The viral DNA was extracted and amplified by PCR according to the methods described by Gunther et al. with modified primer sets. Nineteen of the samples were successfully amplified and 15 of these were sequenced. Specimens were sequenced by NGS on the Illumina MiSeq(TM) sequencer and sequence data used to reconstruct the viral quasispecies of each specimen. Further analyses of the reconstructed variants included molecular characterization as well as phylogenetic analysis and screening for recombination and drug resistance mutations. Full genome coverage was obtained for twelve of the fifteen samples and full genome variants reconstructed, generating nearly 40 full genomes. Phylogenetic analysis showed that the majority of the samples are of genotype A, more specifically of subgenotype A1, differing by less than 4% from known sequences. The phylogenetic analysis revealed a similar clade of outliers, where four samples clustered together with significant bootstrap support (75%) and a fifth sample partitioned separate from, yet close to, this clade, away from the typical African A1 clade. This clade was assigned to genogroup III. Three samples were of the Asian A1 clade (genogroup I) with remaining specimens grouping within genotype D and E. The variants showed low diversity within each specimen with some differing at but a few positions across the genome while even the most diverse quasispecies differed by less than a percentage (32 positions). Several unique and atypical positional variations were observed amongst study samples of which some were present in but one of the variants for that sample. Twenty-six lead to shared amino acid changes. Some observed changes, such as A1762T/G1764A and G1896A, could explain the serological patterns such as HBeAg negativity while others, such as C2002T, were previously implicated in disease progression and severity. Sample N199 presented a longer branch length and revealed short regions within the genome that display evidence of recombination between HBV/A1 and HBV/A2. The results illustrate the utility of NGS technology in characterizing viral variants.