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.

Author Summary

Primary hepatitis C (HCV) infection is typically asymptomatic and commonly results in persistent infection. The characteristics of early infection remain undefined. Four subjects were studied longitudinally from within a few weeks of transmission until resolution of outcome, via a full genome analysis of viral evolution. In the acute phase (<100 days post-infection) there were two periods with a major reduction in genetic diversity (i.e. a bottleneck) irrespective of subsequent clearance (n = 2) or chronic infection (n = 2). The first bottleneck was associated with transmission, with generally only one ‘founder’ virus successfully establishing infection. The second occurred following the primary peak in viraemia, concomitant with seroconversion, approximately 100 days post-infection. In the subjects who became chronically infected, the second bottleneck was followed by emergence of a new cluster of variants, which evolved from the founder(s), and carried only a small number of mutated residues that reached fixation. Some fixations occurred in known targets of CD8 cytotoxic T cell and neutralizing antibody responses. These results indicate a common evolutionary pattern, independent of disease outcome in the acute phase of HCV infection, with strong signatures of selective pressures driving the transition into chronic infection. These novel data will inform preventative vaccine strategies.