Temporal variations in the hepatitis C virus intrahost population during chronic infection

Abstract

The intrahost evolution of hepatitis C virus (HCV) holds keys to understanding mechanisms responsible for the establishment of chronic infections and to development of a vaccine and therapeutics. In this study, intrahost variants of two variable HCV genomic regions, HVR1 and NS5A, were sequenced from four treatment-naïve chronically infected patients who were followed up from the acute stage of infection for 9 to 18 years. Median-joining network analysis indicated that the majority of the HCV intrahost variants were observed only at certain time points, but some variants were detectable at more than one time point. In all patients, these variants were found organized into communities or subpopulations. We hypothesize that HCV intrahost evolution is defined by two processes: incremental changes within communities through random mutation and alternations between coexisting communities. The HCV population was observed to incrementally evolve within a single community during approximately the first 3 years of infection, followed by dispersion into several subpopulations. Two patients demonstrated this pattern of dispersion for the rest of the observation period, while HCV variants in the other two patients converged into another single subpopulation after ∼9 to 12 years of dispersion. The final subpopulation in these two patients was under purifying selection. Intrahost HCV evolution in all four patients was characterized by a consistent increase in negative selection over time, suggesting the increasing HCV adaptation to the host late in infection. The data suggest specific staging of HCV intrahost evolution.

Fig. 1.

Divergence (a) and diversity (b) of HVR1 and NS5A quasispecies at different time points in 4 patients. Divergence is calculated as the average distance between variants from different time points and the first time point and is expressed as the percentage of nucleotides that differed over the total length of a region, i.e., 291 bp for HVR1 and 343 bp for NS5A. (c) Distribution of dN/dS values for HVR1 and NS5A quasispecies over different time points.

Fig. 2.

Phylogenetic analysis of the unique HVR1 (a) and NS5A (b) variants obtained from patients A to D. The color bar scale shows the time line of the follow-up since acute infection (in years). Black triangles beside the color bar mark the exact sampling time points. The blue diamonds identify acute-phase HCV variants.

Fig. 3.

MJN communities in HVR1 and NS5A in each patient. (a and c) Area graphs of MJN communities in HVR1 and NS5A. Each area represents the percentage of quasispecies that belong to a given community at each time point (in years since acute infection [x axes]); vertical dotted lines delimit time points. (b and d) MJN divided into communities (HVR1 and NS5A) using network topological parameters. Each node corresponds to a unique nucleotide sequence. Areas representing communities (a and c) and MJN nodes (b and d) that belong to these communities are shown with the same colors.

Fig. 4.

Presence or absence of a given sequence at different time points. Most of the individual sequences are replaced at every time point for both the HVR1 (top panel) and NS5A (bottom panel) regions in patients A to D. More sequences show persistence in time in the NS5A region than in the HVR1 region. The y axis shows the number of unique variants.

Fig. 5.

Temporal distribution of HVR1 quasispecies in an MJN for patients C (a) and D (b). Each node corresponds to a unique nucleotide sequence, and the radius of each circle is proportional to the frequency of the sequence. Sequences found at a given time point are shown in red; all sequences found before and after the given time point are shown in blue and yellow, respectively. The horizontal bar identifies the exact time points (years since acute infection) marked with black triangles along the bar.

Fig. 6.

Schematic representation of the probable stages of HCV infection over the years in four chronically infected naïve patients. The arrows indicate sampling points from acute infection to 9 to 18 years. The color coding of the arrows corresponds to the percentage of the major variant at the indicated time point of sampling. The green color is for variants whose percentage was >30%, indicating a selective sweep around the third year and purifying selection during the later years of chronic infection. The black and gray bar at the top indicates the dispersion of quasispecies in the MJN, where black indicates single communities (local) and gray is for more than one community (dispersed). The second bar indicates selection pressure, where red indicates positive selection, blue indicates negative selection, and green indicates neutral selection pressure. The last bar indicates the probable stages of HCV infection.