The results of the present study highlight the diversity and dynamics of acute HCV infection in HVR1 in a small, wellsampled cohort. The anticipated patterns of immune selection were completely masked by the far greater effects of viral diversity, with genotypes 1, 2, and 3 all present in the cohort. We make 2 general observations: first, an extremely high level of within-host diversity, with multiple subtypes coexisting both at single time points and throughout acute infection; and second, the unstable dynamics of acute HCV infection with the apparent disappearance and reemergence of variants over a very short time scale.
We minimized the effects of any methodological shortfalls where possible. The short length of the fragment analyzed limited the robustness of the phylogenetic trees when distinguishing within-subtype variants, but there was sufficient diversity to accurately assess differences between subtypes. This was an unavoidable limitation due to the difficulties in amplifying longer sequences or other genomic regions. PCR is not the ideal tool for assessing clonal diversity because the limited number of clones results in underestimation of the variation present. However, all samples were cross-checked with multiple genotype- specific primers for genotypes 1a, 2, and 1b/3a in order to identify the broadest range at all time points, and all positive samples were cloned. In cases with only 1 dominant viral variant, this affects the resulting clonal proportions very little (subject 6 on days 53 and 93 and subject 7 on day 204); however, where a mixture of genotypes were retrieved the overall proportions do not accurately reflect the viral variants present in the sample (subject 6 on day 81 and subject 7 on days 17 and 199). PCR is also susceptible to random effects and systematic bias. Performing limiting dilutions would annul bias but we were unable to amplify samples by this method, possibly because of poor sample quality. PCR may also overestimate diversity due to incorporation of errors during viral sequence copying. The PCR error rate for this protocol was previously estimated as 5.86×10−6
errors/bp/cycle for 35 cycles. Combined with the estimated rate of RT error (3.0×10−5
errors/bp), we would expect 1 error every 2110 bp, or once every 9.4 sequences [23
]. Finally, samples were obtained from peripheral blood and may not be a true representation of quasispecies diversity in the liver [24
]. Despite potential quantitative errors inherent in the methods used, the qualitative observations of dynamic HCV subtype coinfection are robust, and if anything the true diversity may be underestimated.
It has been reported previously that greater quasispecies diversity may be associated with progression to chronicity [25
], but this relationship was not observed in this (albeit small) cohort. Instead, we observed the coexistence of numerous distinct clades leading to a range of clinical outcomes. Previous studies have noted intra- and intergenotypic superinfection in chronic HCV infection, where bulk sequencing shows the HCV strain to be replaced by an unrelated variant [26
]. Many have produced conflicting results, reporting prevalences of mixed genotypes in chronic infection ranging from 0% to 56% ([31
]; reviewed in [33
]). No coinfection of different HCV subtypes was observed in a study of 12 subjects with acute HCV infection after transfusion [9
], yet levels of diversity similar to our results were found in 12 non-IDU subjects with chronic infection [13
]; this was postulated to have arisen through the role of HVR1 as an immunological “decoy,” but coinfection was not considered. Herring et al [26
] reported a 20% prevalence of HCV coinfection in an IDU cohort in San Francisco, California, and suggested that this was the result of little immunological cross-protection against alternative quasispecies by the adaptive immune response. They later detected heterogeneous quasispecies, analogous to our within-subtype clades, in one-third of a recently infected cohort that included IDUs, transfusion recipients, and plasma donors [34
]. HVR1 is the most variable region of the HCV genome, so it is possible that strong within-host antibody-driven selection here is driving the apparent divergence between within-subtype variants. However, the additional differences between these variants in the more conserved E1/E2 region surrounding HVR1 support the hypothesis that they diverged before entering the host.
The apparent dominance of genotype 1 over genotype 3 infection when both infect the same host was an intriguing finding. This may result from differences in adaptive immune responses or in the response to innate cytokines or from viral interference between genotypes. This is consistent with the higher treatment response rates for genotype 3 than genotype 1, with sustained virologic response rates of 75%–80% versus 40%–45%, respectively, for combination pegylated interferon and ribavirin treatment [35
]. However, studies of patients who experience spontaneous resolution of acute HCV infection have shown no consistent link to the infecting genotype [37
], and the variance in follow-up times in these 6 subjects (range, 41–473 days; standard deviation, 184 days) limits the validity of the comparison.
We observe a general pattern where subjects infected with the highest number of variants also have a fluctuating course of viral replication (subjects 2 and 4–7). In particular, subjects 4 and 5, who were followed up for long periods without treatment, showed highly complex viral diversity and dynamics. A similar pattern of viremia has been described previously in other subjects with acute infection, in health care workers exposed to needlestick accidents [40
] and other IDU cohorts [42
], and in the original cohort from which the present subgroup was obtained [15
]. The combination of subtype coinfection, changing quasispecies profile, and fluctuating viral load points to an unstable system, with changes driven by both virus and host factors. The most intuitive explanation of the multiple subtypes in these subjects is their epidemiology; they are mostly IDUs and therefore are likely to be highly exposed to HCV through contact with contaminated needles and other injection equipment. It is possible that repeated exposure could have occurred before diagnosis or that coinfection of multiple virions within 1 infectious dose led to the existence of multiple strains in the subject at the time the first sample was obtained. Ongoing exposure is unlikely, because the variants that dominate later during acute infection are not newly acquired; they are quasispecies from clusters detected in previous samples, sometimes at low levels, but in many cases forming a dominant earlier clade. The location of this cohort may have facilitated the detection of multiple genotypes, given that a range of genotypes are known to be circulating in central Europe [43
]. In other IDU cohorts in areas with a limited range of genotypes, this phenomenon may be less apparent. Furthermore, these results may not be representative of HCV infections in general; the cohort is enriched for subjects with fluctuating viremia, in whom it may be easier to detect mixed infections compared with the stable high-level replication observed during chronic infection, where the dominant variant may mask others present at low frequency.
In conclusion, this cohort provides a rich within-host data set and highlights the complex diversity and dynamics of acute HCV infection. Most subjects were coinfected with multiple HCV subtypes, which is associated with high levels of exposure. This raises important questions for vaccine design, with a monovalent HCV vaccine unlikely to be effective in western European populations.