Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus

Abstract

This study assesses the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions of the HBV genome in patients with chronic hepatitis B virus (HBV) infection. Thirty samples from ten chronic hepatitis B patients were included. The polymerase/surface and X/preCore regions were analyzed by deep sequencing (UDPS) in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment. HBV genotype was determined by phylogenesis. Quasispecies complexity was evaluated by mutation frequency and nucleotide diversity. The polymerase/surface and X/preCore regions were validated for genotyping from 113 GenBank reference sequences. UDPS yielded a median of 10,960 sequences per sample (IQR 16,645) in the polymerase/surface region and 11,595 sequences per sample (IQR 14,682) in X/preCore. Genotype mixtures were more common in X/preCore (90%) than in polymerase/surface (30%) (p<0.001). On X/preCore genotyping, all samples were genotype A, whereas polymerase/surface yielded genotypes A (80%), D (16.7%), and F (3.3%) (p = 0.036). Genotype changes in polymerase/surface were observed in four patients during natural quasispecies dynamics and in two patients during treatment. There were no genotype changes in X/preCore. Quasispecies complexity was higher in X/preCore than in polymerase/surface (p = 0.004). The results provide evidence of genotype mixtures and differential genotype proportions in the polymerase/surface and X/preCore regions. The genotype dynamics in HBV infection and the different patterns of quasispecies complexity in the HBV genome suggest a new paradigm for HBV genotype classification.

Fig 1. Regions of the HBV genome amplified for the present study: P/S is indicated in green and X/preCore in pink.

Fig 2. UPGMA tree obtained from the polymerase/surface (a) and the X/preCore (b) regions of the three samples from Pt 4.

The main haplotype of the first sample (First.0.0.0001) was genotyped as A, whereas the main haplotype of the second and third sample (Second.0.0.0001 and Third.0.0.0001) was genotype as D. At the X/preCore region, the main genotype was A, but Gen/DE and F haplotypes were also observed. Genotype reference sequences for the two regions: A2_X02763; B1_AB362933; C1_AB112066; D1_X59795 (for P/S region); DE_ D1_X59795 (for X/preCore region); E1_X75664 (for P/S region); DE_E1_X75664 (for X/preCore region); F1_HM590471; G0_HE981172; H0_AB275308; I0_FJ023664; J0_AB486012.

Fig 3. Distribution of quasispecies complexity in all the samples analyzed according to mutation frequency (a) and nucleotide diversity (b) in the polymerase/surface (P/S) and X/preCore regions.