Evaluation of intra-host variants of the entire hepatitis B virus genome

Abstract

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.

Figure 1. Flowchart of the HBV WG amplification process.

Figure 2. Repeat amplification of the S-gene fragment from first round PCR products.

Two WG first-round products (C2 and C21) containing two nucleotide changes (indicated in red) were subjected to 40 independent nested PCR amplifications; a sample of 5 identical S-gene sequences amplified from each clone is shown.

Figure 3. Reproducibility of EPLD protocol for WG-HBV quasispecies amplification.

Phylogenetic analysis of HBV-WG quasispecies amplified in two independent batches (red and blue colored nodes) of the same high-titer (a) and low- titer (b) sample.

Figure 4. WG-HBV quasispecies amplification of different genotypes.

HBV WG was amplified from samples belonging to genotypes A–G using a universal set of primers. Maximum likelihood tree shows WG quasispecies of genotypes A– E and G. Each cluster in the tree is the WG quasispecies of an individual patient belonging to one genotype.

Figure 5. Detection of co-infections, subpopulations and recombination.

(a) Maximum likelihood trees of HBV-WG quasispecies (blue) and consensus WG sequences from a single patient (viral titer of 10⁶ IU/ml). HBV quasispecies belong to genotypes A and G, while consensus WG sequences (red) obtained at two time-points belong to genotype A. (b) Maximum likelihood tree of HBV-WG quasispecies from a genotype B infected patient (viral titer of 10⁵ IU/ml). Consensus sequence shown in red belongs to a single HBV subpopulation, while the WG quasispecies shown in blue segregate into 2 subpopulations. (c) Maximum likelihood tree of WG quasispecies (blue) sampled from a patient (viral titer of >10⁶ IU/ml) infected with genotype G and recombinant genotype G/A variants. Consensus sequence (red node) belongs to genotype G. Two HBV WG clones, that are genetically distant from the main cluster of genotype G variants, contain genomic regions belonging to genotype A.

Figure 6. Distribution of mutations along the HBV genome.

(a) Polymorphic positions along the HBV WG quasispecies obtained from 4 patients infected with different HBV genotypes (viral titer of 10³–10⁶ IU/ml). (b) Sliding window analysis of polymorphic regions identified among genotype D variants (each shown with different color) implicated into a horizontal transmission among 4 patients (samples 1–4; viral titer of 10⁴–10⁶ IU/ml).

Figure 7. Diversity in naive and treated individuals.

Phylogenetic trees of HBV-WG quasispecies (blue) and consensus sequences (red) from: (a) a treatment-naïve patient (viral titer −10³ IU/ml) and (b) a lamivudine-treated patient (viral titer −10⁶ IU/ml).