Intra-host analysis of hepaciviral glycoprotein evolution reveals signatures associated with viral persistence and clearance

Even 30 years after the discovery of hepatitis C virus (HCV) in humans there is still no prophylactic vaccine available. Reasons for this include the high mutation rate of HCV, which allows the virus to escape immune recognition, in addition to the absence of an immunocompetent animal model for vaccine development. Phylogenetically distinct hepaciviruses (genus Hepacivirus, family Flaviviridae) have been isolated from diverse species, each with a narrow host range: the equine hepacivirus (EqHV) is the closest known relative of HCV. Comparing patterns of intra-host hepaciviral evolution in different species can potentially identify mutational signatures associated with transmission, immune evasion, resolution of infection, progression to chronicity and pathogenesis. In this study, we analyzed the viral intra-host population composition of the surface glycoproteins E1 and E2 in longitudinally sampled sera via amplicon-based deep-sequencing in a cohort of naturally as well as experimentally EqHV-infected horses and compared them to HCV-infected patients. Differences in frequency and number of sites exhibiting intra-host variation were detected between chronic and acute infection in horses. Experimentally infected horses developed selflimiting acute EqHV infections with no pronounced transmission bottleneck and displayed some degree of adaptation to the new host. The overall glycoprotein variability was higher in HCV patients compared to EqHV-infected horses. Additionally, selection pressure in HCV patients was higher, especially within the hypervariable region 1 (HVR1), while only one horse showed elevated selection pressure in the N-terminal region of E2. Furthermore, alignment of glycoprotein sequences from diverse hepaciviruses identified HVR1 as unique characteristic of the HCV E2 N-terminus: hepaciviruses from non-human species lacked this region. The in vivo evolution of hepaciviral glycoproteins showed distinct mutational patterns in acutely and chronically infected horses, as well as differences between HCV and EqHV. In conclusion, experimental EqHV infections of naive horses showed no reduction in population diversity due to a transmission bottleneck but exhibited signs of host-adaptation. Together, these data indicate EqHV infection of horses could represent a powerful surrogate animal model for HCV vaccine development by providing insights into HCVs HVR1-mediated immune evasion strategy.