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The treatment of HCV infection has seen significant progress, particularly since the approval of new direct-acting antiviral drugs. However these clinical achievements have been made despite an incomplete understanding of HCV replication and within-host evolution, especially compared with HIV-1. Here, we undertake a comprehensive analysis of HCV within-host evolution during chronic infection by investigating over 4000 viral sequences sampled longitudinally from 15 HCV-infected patients. We compare our HCV results to those from a well-studied HIV-1 cohort, revealing key differences in the evolutionary behaviour of these two chronic-infecting pathogens. Notably, we find an exceptional level of heterogeneity in the molecular evolution of HCV, both within and among infected individuals. Furthermore, these patterns are associated with the long-term maintenance of viral lineages within patients, which fluctuate in relative frequency in peripheral blood. Together, our findings demonstrate that HCV replication behavior is complex and likely comprises multiple viral subpopulations with distinct evolutionary dynamics. The presence of a structured viral population can explain apparent paradoxes in chronic HCV infection, such as rapid fluctuations in viral diversity and the reappearance of viral strains years after their initial detection., Competing Interests: RR is employed by BioInfExperts as bioinformatics consultant.

The hepatitis C virus (HCV), which currently infects an estimated 3% of people worldwide, has been present in some human populations for several centuries, notably HCV genotypes 1 and 2 in West Africa and genotype 6 in Southeast Asia. Here we use newly developed methods of sequence analysis to conduct the first comprehensive investigation of the epidemic and evolutionary history of HCV in Asia. Our analysis includes new HCV core (n = 16) and NS5B (n = 14) gene sequences, obtained from serum samples of jaundiced patients from Laos. These exceptionally diverse isolates were analyzed in conjunction with all available reference strains using phylogenetic and Bayesian coalescent methods. We performed statistical tests of phylogeographic structure and applied a recently developed "relaxed molecular clock" approach to HCV for the first time, which indicated an unexpectedly high degree of rate variation. Our results reveal a >1,000-year-long development of genotype 6 in Asia, characterized by substantial phylogeographic structure and two distinct phases of epidemic history, before and during the 20th century. We conclude that HCV lineages representing preexisting and spatially restricted strains were involved in multiple, independent local epidemics during the 20th century. Our analysis explains the generation and maintenance of HCV diversity in Asia and could provide a template for further investigations of HCV spread in other regions.

Virus-specific CD8+ T cell responses play an important role in the natural course of infection; however, the impact of certain CD8+ T cell responses in determining clinical outcome has not been fully defined. A well-defined cohort of women inoculated with HCV from a single source showed that HLA-B27 has a strong association with spontaneous clearance. The immunological basis for this association is unknown. However, the finding is especially significant because HLA-B27 has also been shown to have a protective role in HIV infection. We report the identification of an HLA-B27 restricted hepatitis C virus (HCV)-specific CD8+ T cell epitope that is recognized in the majority of recovered HLA-B27 positive women. In chronically HCV-infected individuals, analysis of the corresponding viral sequence showed a strong association between sequence variations within this epitope and expression of HLA-B27, indicating allele-specific selection pressure at the population level. Functional analysis in 3 chronically HCV-infected patients showed that the emerging variant viral epitopes represent escape mutations. In conclusion, our results suggest a dominant role of HLA-B27 in mediating spontaneous viral clearance as well as viral evolution in HCV infection and mechanistically link both associations to a dominant novel CD8+ T cell epitope. These results support the central role of virus-specific CD8+ T cells and the genetically determined restriction of the virus-specific T cell repertoire in HCV infection.

Multispecific CD8(+) T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8(+) T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8(+) T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8(+) T-cell responses, as well as CD4(+) T-cell responses. Rapid recrudescence also occurred despite broad CD8(+) T-cell responses. Importantly, in vivo suppression of CD3(+) T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8(+) T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.

In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.

Cellular immunity plays an important role in the control of persistent virus infections such as hepatitis C virus (HCV). Antiviral CD4(+) T cell responses have been shown to accompany resolution of acute disease and there is also a consistent association between HLA Class II genes, notably HLADRB1*1101 (and the closely linked HLADQB1*0301) and disease resolution. We initially mapped longitudinal CD4(+) T cell responses in an individual after spontaneous resolution of acute HCV, and identified three HLA-DR11-restricted responses which vary in immunodominance over time. Functional assays and HLA Class II tetramer staining revealed one to be a response to a commonly recognized epitope, NS3(1248-1261), although cytokine capture assays showed these specific cells to be at a very low frequency. In this patient, and in others reported, this most frequently recognized HLA-DR11 restricted epitope is not immunodominant. We analysed whether sequence variability within and between genotypes might account for differences in recognition of HLA-DR11 restricted epitopes. We found that a limited number, including NS3(1248-1261), showed extreme sequence conservation. Within NS3, the ability of peptides to accept amino acid substitutions was clearly related to the structure of the protein. Overall the data provide a deeper understanding of the relationship between protein structure and variability of HLA-DR11 restricted peptides and may explain the apparent dominance of responses to NS3(1248-1261) across studies but not within an individual immune response.

Hepatitis C virus (HCV) persists in the majority of those infected despite host immune responses. Evidence has accrued that selectively fixed mutations in the envelope genes (E1 and E2) are associated with viral persistence, particularly those that occur within the first hypervariable region of E2 (HVR1). However, the individual amino acid residues under selection have not been identified, nor have their selection pressures been measured, despite the importance of this information for understanding disease pathogenesis and for vaccine design. We performed a high-resolution analysis of published gene sequence data from individuals undergoing acute HCV infection, employing two phylogenetic methods to determine site-specific selection pressures. Strikingly, we found a statistically significant association between the number of sites selected and disease outcome, with the fewest selected sites in fulminant HCV cases and the greatest number of selected sites in rapid progressors, reflecting the duration and intensity of the arms race between host and virus. Moreover, sites outside the HVR1 appear to play a major role in viral evolution and pathogenesis, although there was no association between viral persistence and specific mutations in E1 and E2. Our analysis therefore allows fine dissection of immune selection pressures, which may be more diverse than previously thought. Such analyses could play a similarly informative role in studies of other persistent virus infections, such as human immunodeficiency virus.