# Genetically distinct within-host subpopulations of hepatitis C virus persist after Direct-Acting Antiviral treatment failure

## TL;DR

This study shows that hepatitis C virus maintains distinct genetic subpopulations in patients even after treatment failure, which could impact drug resistance and transmission.

## Contribution

The study demonstrates that structured viral populations persist after DAA treatment failure, challenging assumptions about viral bottlenecks.

## Key findings

- Distinct viral subpopulations persist in most patients after DAA treatment failure.
- Evolutionary rates differ significantly between viral lineages within individuals.
- Lineage-aware analysis is needed to avoid conflating distinct subpopulation traits.

## Abstract

Analysis of viral genetic data has previously revealed distinct within-host population structures in both untreated and interferon-treated chronic hepatitis C virus (HCV) infections. While multiple subpopulations persisted during the infection, each subpopulation was observed only intermittently. However, it was unknown whether similar patterns were also present after Direct-Acting Antiviral (DAA) treatment, where viral populations were often assumed to go through narrow bottlenecks. Here we tested for the maintenance of population structure after DAA treatment failure, and whether there were different evolutionary rates along distinct lineages where they were observed. We analysed whole-genome next-generation sequencing data generated from a randomised study using DAAs (the BOSON study). We focused on samples collected from patients (N=84) who did not achieve sustained virological response (i.e., treatment failure) and had sequenced virus from multiple timepoints. Given the short-read nature of the data, we used a number of methods to identify distinct within-host lineages including tracking concordance in intra-host nucleotide variant (iSNV) frequencies, applying sequenced-based and tree-based clustering algorithms to sliding windows along the genome, and haplotype reconstruction. Distinct viral subpopulations were maintained among a high proportion of individuals post DAA treatment failure. Using maximum likelihood modelling and model comparison, we found an overdispersion of viral evolutionary rates among individuals, and significant differences in evolutionary rates between lineages within individuals. These results suggest the virus is compartmentalised within individuals, with the varying evolutionary rates due to different viral replication rates and/or different selection pressures. We endorse lineage awareness in future analyses of HCV evolution and infections to avoid conflating patterns from distinct lineages, and to recognise the likely existence of unsampled subpopulations.

Hepatitis C virus (HCV) remains prevalent globally, particularly straining healthcare systems in low- and middle-income countries. Despite huge efforts to develop an effective HCV vaccine, deployment is at best several years away. In the meantime, Direct-Acting Antivirals (DAAs), which can cure HCV in up to 95% of cases for some but not all HCV genotypes, are the gold-standard treatment. With the widespread use of DAAs in HCV-infected individuals, the emergence and spread of DAA resistance is imminent. HCV has been observed to maintain a structured population within hosts, where distinct subpopulations coexist and fluctuate in frequency. Using novel genomic methods, we demonstrate the continued presence of the unique within-host viral population structure of HCV after DAA treatment failure, meaning resistance-associated mutations may be present but not observed either before or after treatment failure. The distinct lineages exhibited significantly varying evolutionary rates, suggesting either differential selective pressures acting upon the subpopulations, or the infection of different cell types. In light of these findings, we advocate a lineage-aware approach that recognises the complexity of within-host dynamics of HCV since fluctuating subpopulations may harbour distinct genotypic features that could confer different phenotypes, such as drug resistance or high transmissibility. Unless the lineages are teased apart, within-host evolutionary studies risk reporting averaged and inaccurate properties of distinct sub-populations that may be evolutionarily divergent.