Sequential bottlenecks drive viral evolution in early acute hepatitis C virus infection

Abstract

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. RNA level, Shannon entropy, and antibody titers over time for the four early infection subjects.

Panels A and B show two subjects who developed chronic infection (240_Ch, 23_Ch) followed from pre-seroconversion timepoints. Panels C and D show the infection dynamics for two subjects who ultimately cleared the infection (360_Cl, 686_Cl). Red dots represent viremic time points analyzed via next generation sequencing (NGS). The solid line represents the RNA level. The dashed and dotted lines represent the interpolation of the Shannon entropy calculated across the genome at each time point using NGS data. Entropy was calculated using all mutated sites (dotted line), or with only non-synonymous sites (dashed line). The shaded area represents semi-quantitative estimates of the anti-HCV antibody titer (OD: cut-off). Note the varied ranges in the x- and y-axes.

Figure 2. Distribution of the non-synonymous substitutions detected across the genome over the course of the infection.

Panels A and B show the distributions of non-synonymous substitutions in two subjects who developed chronic infection (240_Ch, 23_Ch). Panels C and D show two subjects who ultimately cleared the infection (360_Cl, 686_Cl). Each panel shows the longitudinal analysis of the distribution of non-synonymous substitutions for each subject. In subjects who cleared the infection, substitutions sporadically emerged at low frequency (99%). Colors represent the time course post-infection (see legend).

Figure 3. Founder virus analysis based on partial E2 region of the viral genome.

Panels A and B show the analyses for two subjects who developed chronic infection (240_Ch, 23_Ch) followed from pre-seroconversion timepoints. Panels C and D show the analysis for two subjects who cleared the infection (360_Cl, 686_Cl). Phylogenetic reconstructions and highlighter plots are shown, illustrating the genetic relatedness between HCV variant sequences. Names of each sequence are labeled with a letter (H for haplotype, and C for clone), with the first number representing the sampling timepoint and with the second number representing either the prevalence of the haplotype or the clone number. The phylogenetic trees of subjects 686_Cl, 360_Cl and 240_Ch (panels A, C, D) are consistent with an infection arising from a single founder. The fit with a Poisson model is also consistent with a single founder (p-value > 0.1, see text). As shown by the highlighter plots, founder viruses are identified as the consensus sequence and coincided with the most prevalent variant reconstructed from NGS data, (e.g. for subject 686_Cl H1_0.60 is identical to the consensus sequence and to clone C_12b). The highlighter plots also show the random distribution of mutated sites with respect to the founder sequence (master), which is consistent with a star-like phylogeny. The phylogenetic analysis in 23_Ch (panel B) is consistent with an infection originated from two founder viruses (indicated with an asterisk in the highlighter plot) giving rise to two major clusters, 23A and 23B. This is consistent with the rejection of the Poisson model (p-value = 0). Phylogenetic trees were obtained using PhyML, with Maximum Likelihood methods using a GTR model of substitution as suggested by model testing.

Figure 4. Evolutionary dynamics of HCV variants over the partial E2 region of the genome.

Sequence analyses of the two subjects who developed chronic infection, 240_Ch (A), and 23_Ch (B) revealed the presence of selective sweeps. These sweeps led to the emergence of new variants that replaced the founder viruses (identified with an asterisk). Phylogenetic trees (left panels in A and B) display nucleotide sequences of reconstructed haplotypes derived from NGS data and clonal sequences. Names of each sequence are labeled with a letter (H for haplotype and C for clone), with the first number representing the sampling timepoint and with the second number representing either the prevalence of the haplotype or the clone number. Colors are also used to portray the sampling timepoint (see legend). Infection dynamics for subject 240_Ch are consistent with a single founder, identified with the most prevalent strain of cluster 240A (H2_0.85 and H3_0.97 at time-points 2 and 3 respectively), with clone C2_6, and with the consensus of the sequences from time-point 1. The pre-chronic phase (corresponding with the color-coded time ranges 5 and 6) of infection shows the emergence and dominance of a new subgroup of viruses, designated 240B. 23_Ch has at least two founder viruses that successfully initiated the infection (H1_0.26 and H1_0.16 within the two clusters 23AF and 23BF, respectively), A new cluster 23AC, termed after the dominant variant H5_0.54, emerged in the pre-chronic phase and replaced cluster 23AF. Trees are calculated using Maximum Likelihood method (implemented in PhyML).

Figure 5. Evolution of the partial E2 region of individual HCV variants at the amino acid level.

The plots in A (subject 240_Ch) and B (23_Ch) show the dynamics of the individual viral variants over time. In 240_Ch, infection was initiated with one founder variant, 240AF, which was then replaced sequentially by two related variants, 240AC1 and 240AC2, respectively. In 23_Ch, at least two founders initiated infection, 23AF and 23BF, which both dominated the early phase of infection before being replaced by a new variant in the pre-chronic phase of infection (23AC). The y-axis shows the contribution of each variant with respect to the RNA level. Below each graph is an amino acid alignment indicating the distinguishing residues for the different variants. The location of putative CTL (pink shading) and B cell (green shading) epitopes, and mutations with previously recorded viral fitness costs (light blue shading) are indicated. All the identified epitopes within this region carried at least one amino acid change. Two of these mutations (G483D for 240_Ch and T542I for 23_Ch) generated CTL epitopes with reduced binding CTL affinity, and both subjects showed a substitution at position Y443, known to be within a B cell epitope - all of which is suggestive of immune escape. In addition, in 240_Ch a potential fitness cost associated mutation was observed at T543A .

Figure 6. Demographic reconstruction of the viral populations.

Demographic reconstruction from E1/HVR1 (A,B) and E2 (C,D) sequences for subjects who developed chronic infection, 23_Ch (A,C) and 240_Ch (B,D). In both subjects, and in both genomic regions, the founder effect and sequential bottleneck events are evident. The estimated effective population size (Nτ, the product of the effective population size and generation length in days) had a peak value of the order of 103, and then decreased to values of the order of 102. The longer estimate of tMRCA for 23_Ch when compared to those in Table 1 is likely to be due to the presence of two founder viruses in this subject.