Quasispecies structure, cornerstone of hepatitis B virus infection: mass sequencing approach

Abstract

Hepatitis B virus (HBV) is a DNA virus with complex replication, and high replication and mutation rates, leading to a heterogeneous viral population. The population is comprised of genomes that are closely related, but not identical; hence, HBV is considered a viral quasispecies. Quasispecies variability may be somewhat limited by the high degree of overlapping between the HBV coding regions, which is especially important in the P and S gene overlapping regions, but is less significant in the X and preCore/Core genes. Despite this restriction, several clinically and pathologically relevant variants have been characterized along the viral genome. Next-generation sequencing (NGS) approaches enable high-throughput analysis of thousands of clonally amplified regions and are powerful tools for characterizing genetic diversity in viral strains. In the present review, we update the information regarding HBV variability and present a summary of the various NGS approaches available for research in this virus. In addition, we provide an analysis of the clinical implications of HBV variants and their study by NGS.

Keywords: Gene overlapping; Hepatitis B virus; Linkage analysis; Next generation sequencing; Quasispecies.

Figure 1

Overlapping region of the polymerase and surface genes (A) and its corresponding sequence (B). Translation into amino acids is depicted in blue in the P open reading frame (ORF) (above) and red in the S ORF (below). Most of the main codons related to nucleos(t)ide resistance (framed in blue), and the overlapping codons in the S ORF that may give rise to immune escape or stop codons (framed in red) are located within the fragment analyzed in a previous study by our group[58].

Figure 2

Changes in percentages of reverse transcriptase variants during follow-up of a patient included in one of our studies. Reproduced from Rodriguez-Frías et al[58]. BC: Baseline combination.

Figure 3

Quasispecies of the X gene obtained by unpublished result. The four TA-like boxes are highlighted in red and the eight nucleotides detected are indicated in green.

Figure 4

Secondary structures adopted at the 5’ and 3’ ends of hepatitis B virus pgRNA reported by our group. A: Stem-loop structure of the 5’ with main structural motifs (loops and stems), main codons (1, 14, 15, 28, and 29 of preCore, and 1 of Core gene), and the location of 4-nt primer annealing in the 5’; B: Stem-loop structure of the 3’. The acceptor site (AS), direct repeat region 1 (DR1), and preCore and Core start codons are indicated in the figure. Reproduced from Homs et al[56].