Figure 1.
Flowchart of the HBV WG amplification process.
Table 1.
Primers used for HBV whole genome quasispecies amplification.
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).