hepatotropic virus that can cause both transient and persistent infection in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A and its roles in immune evasion and persistence are unknown. To address these knowledge gaps we evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals) and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than observed in horses that spontaneously cleared infection (P = 0.0002) or in SCID foals (P < 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P < 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcome and could be dependent on strain specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank and defined three novel hypervariable regions in E2 at residues 391-402 (HVR1), 450-461 (HVR2) and 550- 562 (HVR3). In the experimentally infected horses entropy analysis focused on the HVRs demonstrated that these regions were under increased selective pressure during persistent 43 infection. Increased diversity in the HVRs was also temporally associated with seroconversion in 44 some horses, suggesting that they may be targets of neutralizing antibody and play a role in 45 immune evasion.