Analysis of the intrahost evolution of hepatitis C virus (HCV) is important for understanding mechanisms responsible for establishment of chronic infections and development of efficient preventive and therapeutic measures. However, analysis of HCV's longitudinal evolution is significantly hindered by difficulties in identifying patients and in their long-term follow-up, starting from the acquisition of infection. In the current study, intrahost variants of two variable HCV genomic regions, HVR1 and NS5A, were sequenced from four treatment-naïve chronically infected patients who were followed from the acute stage of infection for 9 to 18 years. The HCV quasispecies from each region were sampled at 5 to 14 time points from each patient using the endpoint limiting dilution PCR, and ~3,400 clones were obtained (an average of 43 clones per time point per region).
Temporal patterns of communities.
Analysis conducted in this study showed that HVR1 variants in these 4 patients are organized into communities or subpopulations rising to dominance at different time points (). The intrahost HCV population was predominantly found evolving as a single community during approximately the first 3 years of infection, followed by dispersion into several communities or subpopulations. As was shown for all 4 patients, the time order of phylogeny was usually obscured during dispersion into communities. Patients A and D demonstrated the pattern of continuous dispersion for the rest of the observation period. HCV variants in patients B and C converged into another single subpopulation after ~9 to 12 years of dispersion. The final subpopulation in the last two patients was under negative or purifying selection.
Observation of HCV variants detectable at more than one time point, with certain variants even increasing in frequency over time, as in patient C ( and ), indicates that at least some HCV variants are capable of long-term persistence during chronic infection. Most of the individual sequences are replaced at every time point for both the HVR1 and NS5A regions (). More sequences show persistence in time at the NS5A region than at the HVR1 region. Interestingly, the sequences with the highest persistence are usually the most frequent too.
HVR1 and NS5A variants from the final MJN community in patient C were sampled only 0.3 year and NS5A variants in patient B for ~3 years after acute infection (). In patient D, some NS5A variants sampled at the first time point were found in distant regions of the MJN. All these observations suggest that these 3 patients were infected with an assortment of HCV variants belonging to different MJN communities. These communities seemed to vary in their dominance during infection, allowing the sampling of their members only at some time points. HCV variants from the final MJN community in patient C, and most probably in patient B, were present early during infection and were possibly transmitted to these patients from the source. Later in the infection, these variants grew in frequency and were extensively sampled in our experiments. Patient A was infected through blood transfusion (), which also implies transmission of numerous HCV variants to this patient.
A low risk of HCV persistence was suggested to be related to transmission of a relatively homogeneous HCV population to a new host (37
). In the present study, all 4 patients have indications of transmission of HCV variants representing more than one MJN community. The fact that all these patients established chronic infection lends additional support to the importance of the diverse HCV populations at the initiation of infection for escaping clearance.
Varying rate of evolution and complexity of the founder population.
The observed rate of HCV intrahost evolution varied between time points. It ranged from a significant slowdown during late infection, as in patient C (, panels 5 to 10), to a rapid jump from one region of the MJN to another during early infection, as in patient D (, panels 1 to 2). This finding implies various degrees of evolutionary changes at different stages of infection in these 4 patients. However, measures of the evolutionary rate based on calculating differences between nucleotide sequences of viral populations at consecutive time points can be inaccurate in defining the extent of evolution and should be interpreted cautiously. The presence of more than one MJN community, with some evolving in the background, as suggested by the aforementioned observations of the temporal appearance of HCV variants, creates an opportunity for variation in dominance for these communities at different stages of infection, thus confounding the estimation of evolutionary rates calculated using small viral samples. As a result, HCV variants from two consecutive time points may have no direct links to each other in the MJN (, panels 1 to 2, and , panels 7 to 8), which suggests that the succeeding viral population is not always immediately derived from the preceding population.
If viral communities preexist, from where do they originate? We hypothesize that many, if not all, communities are originally established within the expanding founder population, which experiences a bottleneck evolution upon transmission followed by a rapid increase in population size. In all 4 patients studied here, the HCV titer was in the range of 6 × 103
to 9 × 104
IU/ml at acute-phase time points (data not shown), suggesting that within a very short period of time after transmission, the HCV population reached ~107
viral particles in the bloodstream, starting from a limited number of founder particles. Owing to the large number of progeny genomes generated during such a population expansion, the probability of retaining newly arising mutations in progeny increases. Additionally, changes in selection pressures in a new host can lead to adjustment in epistatic connectivity between sites within the viral genome. This process is analogous to the founder-flush speciation model for a population exploring a new ecological niche (51
). In this model, new selectively favorable mutations have a higher probability of survival as a result of the flush increase in population size.
Thus, the first molecular events occurring immediately after HCV transmission may result in the generation of a highly heterogeneous viral population despite the bottleneck. As was shown above, the members of more than one MJN community were sampled at early stages of HCV infection in patients B, C, and D, suggesting transmission of more than one HCV variant, with patient A most probably being infected with many HCV variants through blood transfusion. The transmitted HCV population may acquire, in addition to its original heterogeneity, an extensive genetic variability through the founder-flush process during the initial steps of infection. However, the HCV-specific adaptive immunity likely shapes this founder population into a complex pool of closely related communities, each of which may become dominant at different stages of the HCV infection. HCV's intrahost evolution is, therefore, defined by two distinct processes: incremental changes within viral communities through random mutation and alternations between coexisting communities, reflecting a complex intrahost population dynamic. This consideration implies that succeeding dominant subpopulations may not be directly derived from each other but may rather share an ancestor.
The founder-flush process, although being a powerful tool for producing intrahost heterogeneity, should, however, have limits in its capacity to generate viral variants. Thus, seeding with sequence variants representing different viral communities upon transmission seems to ensure the establishment of the extensive sequence diversity and broad community structure of the HCV population in a new host. It is interesting that HIV infection is usually initiated by a single variant (1
), thus limiting the extent of the initial intrahost heterogeneity generated through the founder-flush process and suggesting that HCV and HIV have different requirements for the initial genetic heterogeneity needed to establish chronic infections.
Contributions of the HVR1 and NS5A regions to intrahost evolution.
Chronic HCV infection is frequently associated with a significant reduction in T-cell immune responses (10
), thus making humoral immunoresponses the dominant force of selection during long-term infection. HCV HVR1 contains a neutralizing antigenic epitope (49
) and is a target for such immune pressure, while evolution of the NS5A region, which harbors T-cell epitopes (32
), is not directly affected by neutralization (27
). Therefore, it is HVR1 that is under the frequently changing selection pressure of sequence-specific neutralizing antibodies elicited at different stages of chronic infection (53
). The similarity observed between the NS5A and HVR1 temporal dispersion patterns among the MJN communities () seemingly suggests that NS5A evolution merely reflects HVR1 evolution. However, analysis shows that NS5A changes may have different characteristics. In patient B, for example, NS5A quasispecies evolved into the final MJN community ~1 year earlier than HVR1 quasispecies (). The disparity between HVR1 and NS5A quasispecies evolution was even more pronounced for patient D. A significant correlation of genetic distances among all time points for HVR1 and NS5A genomic variants was observed for patients A, B, and C. However, such similarity was not observed for the distribution of genetic distances among time points for these two genomic regions in patient D. These observations, together with the finding that NS5A evolved into a single community at the end of the observation period in this patient, while HVR1 variants remained distributed among more than one community (), also suggest an independent role for NS5A in HCV evolution in this patient.
It is important to note that sites across the HCV genome are physically and epistatically linked. Thus, selection on one site may affect the intensity and direction of selection on other sites within the genome. We have shown earlier that such a linkage is globally organized into a scale-free network (8
). Both subgenomic regions, HVR1 and NS5A, are significantly separated in the HCV genome and were treated here as independent entities. However, evolutionary changes analyzed in these two subgenomic regions may reflect strong selection pressures acting somewhere else in the HCV genome.
Identification of the selective sweep immediately before dispersal into the MJN communities in all 4 patients ( and ) suggests a role for frequency-dependent selection in intrahost evolution. The most frequent HVR1 variant likely elicits a strong neutralizing immune response against itself, which should result in reduction in fitness and suppression of the community containing that variant. HCV may undergo more than one round of such frequency-dependent selection, as exemplified in the early infection of patient D (, panels 2 to 8). This period of infection associated with the decrease in HCV fitness seems to be a particularly vulnerable phase in the development of chronic infection and provides a valuable opportunity for initiation of antiviral therapeutic intervention.
In general, HCV's intrahost evolution was characterized by a consistent increase in negative selection during chronic infection in the 4 patients. The final subpopulation in patients B and C was under negative or purifying selection. A significant reduction in HVR1 heterogeneity (, panels 5 to 10) at later stages of infection in these patients most probably resulted from background selection (5
). The increase in viral load observed at this stage likely reflects decline in the effectiveness of neutralizing immunoresponses and, therefore, could be associated with inefficient HCV clearance rather than with improvement in viral replicative fitness.
Adaptation to late stages of infection.
An important indication of improved viral adaptation to the host at later stages of chronic infection for all 4 patients is identification of a positive correlation between viral load and length of infection (r
= 0.585, P
= 0.0001) and a negative correlation between viral load and dN/dS
= −0.383, P
= 0.012). The existence of the negatively selected HVR1 variants in patients B and C suggests a deep adaptation of these populations to their host. The mechanism of this adaptation is not known. HCV may exploit the preexisting host deficiency in controlling this infection. Although immune escape via exploitation of “holes” in HCV-specific immunity has been observed (54
), this mechanism of adaptation should require the frequent availability of various immunological deficiencies in the host population that can be exploited by HVR1 via mutations. Alternatively, as with evasion of innate immunity, HCV may actively affect adaptive immunity, making the host environment conducive to the stable coexistence of the virus with the host. The second mechanism of HVR1 adaptation is consistent with the observation of an HVR1 variant that persisted in patient C for almost the entire observation period but became predominant only during approximately the last 7 years ( and ).
In contrast to HIV, HCV does not cause systemic immunodeficiency (47
). However, the reduction in selection intensity over the course of HCV infection is clearly attributable to the decline in specific immune responses. Although molecular mechanisms responsible for the decline in the immune pressure on HCV HVR1 at late stages of infection are not known, it can be speculated that such an intrahost environment is caused by B-cell dysfunctions, such as those leading to the hyperactivity and exhaustion observed during chronic HIV (29
) and HCV (43
) infections; may be related to or associated with competition between antigenic sites (55
), a paucity of high-affinity immune cell receptor recognition (54
), the “original-antigenic-sin” response (11
), or the enhanced antibody-dependent uptake of some HCV variants (28
); or may be the result of a combination of these conditions.
Irrespective of the mechanisms responsible for reduction of immune pressure on HVR1, the increase of negative selection in all 4 patients during chronic infections suggests that HCV not merely escapes neutralizing immune responses but also promotes intrahost conditions beneficial for stable viral reproduction through temporal cooperation between viral subpopulations at different stages of chronic infection. Such cooperation may assume different forms (15
). In the scenario of “original antigenic sin,″ for example, the succeeding HCV populations may experience a reduction in immune pressure through stimulating moderately cross-immunoreactive memory cells raised against previous populations rather than through eliciting new antibody-producing cells. Thus, we suggest a modification of the continuous-escape (53
) or diversification-stabilization (48
) model for HCV which implies that a viral population at a given stage has a specific effect on the host environment and reduces host selection pressures for the succeeding population until the final population achieves a state of stable coexistence with the host.
HCV as a noncytopathic virus seems to exploit host mechanisms for reducing immune-related liver damage (6
) and for controlling excessive immune responses (42
). It adversely affects many functions of the host immune system but does not incapacitate it completely, suggesting a long history of HCV adaptation to humans. Additionally, HCV-host coevolution, besides having immunological effects, may involve mutual virus-host cell adaptation, as has been observed in vitro
Stages of HCV intrahost evolution.
The model presented here suggests that HCV's intrahost evolution starts in the absence of specific immune responses and proceeds toward inefficient neutralizing immune responses. We hypothesize that in order to achieve this goal, HCV evolves through 4 stages (). In stage 1 (implied, but not studied here), HCV establishes itself in a new host before the surge of adaptive immune responses. This stage is the arena for the founder-flush process. Stage 2 covers a period of incremental evolution of viral variants within predominant communities, reflecting the minimal genetic changes actually required for an effective immune escape. Stage 3 is a period of diversification into a set of subpopulations that become prominent following the decline of the previously dominant population. In stage 4, HCV achieves final settlement under strong negative selection. Patients B and C exhibit distinct settlement phases. Patients A and D show neutral selection pressure in the last time points, and probably a few more years of follow-up would likely show settlement under negative selection.
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 (more ...)
At the final stage, HCV is posited to achieve a stable adaptation to the host, the ultimate goal of intrahost evolution. However, this goal may not always be achieved. As exemplified in patient D, the course of HCV's intrahost evolution seems distressed by strong selection pressure early in infection. Nevertheless, judging from the declining dN/dS values during the last years of infection, HCV evolution in this patient was redirected toward the goal programmed into the genetic composition of the late-stage HCV variants.
The duration and intensity of evolutionary processes at different stages likely play an important role in defining clinical outcomes of chronic HCV infections. Staging a chronic HCV infection should assist in determining the optimal timing for the successful application of therapy, since HCV may be differentially sensitive to interventions at particular stages. For example, it is known that patients with acute HCV infection achieve a complete virological response to interferon therapy more frequently than patients with chronic infections (16
), suggesting that stages 1 and 2 are most sensitive to therapy. Additionally, the finding that HVR1 in baseline samples of nonresponders and transient responders tends to be negatively selected, whereas HVR1 in samples of sustained responders tends to be positively selected (14
), may indicate that HCV at stage 4 is more resistant to combined interferon and ribavirin therapy than at stages 2 and 3.
Our results suggest that the predominant viral subpopulation at the last stage loses its advantages upon transmission, implying that different HCV generations may exhibit variations in transmissibility and, potentially, in virulence. Observation of the reduction in quasispecies diversity associated with severity of liver disease (38
) may be interpreted in terms of variation in virulence during stages 3 and 4 of HCV infection. It is also conceivable that sequence variants prevalent at different stages of infection may have somewhat specific immunological properties, which may be explored for the development of hepatitis C prophylactic or therapeutic vaccines.
Although limited to 4 HCV strains because of significant difficulties in obtaining serial specimens from chronically infected treatment-naïve patients over an extended period of time, the findings made in the present work offer a new framework for studying and exploiting HCV's intrahost evolution for clinical and public health interventions.