Long-Term Persistence of Infection in Chimpanzees Inoculated with an Infectious Hepatitis C Virus Clone Is Associated with a Decrease in the Viral Amino Acid Substitution Rate and Low Levels of Heterogeneity

Hepatitis C virus (HCV) was first identified in 1989 (6) and is the major causative agent of parenterally transmitted non-A, non-B hepatitis. In general, chronic infections in chimpanzees exhibit only very mild hepatitis, while a wide spectrum of disease is observed in humans, ranging from nonapparent to mild to severe chronic active hepatitis or end-stage cirrhosis and, potentially, hepatocellular carcinoma (25). However, the more serious forms of chronic liver disease in association with HCV in humans are usually not seen until at least the third decade after infection. Very few chimpanzees have been monitored for that length of time; therefore, similar long-range illnesses in this animal model cannot be excluded. The mechanisms leading to viral persistence, which is associated with the more severe forms of liver disease, are as yet undefined. Any single HCV isolate exists as a quasispecies with sequence variability throughout the RNA genome (3, 27). This variation could lead to evasion of the host immune response through the selection of neutralizing antibody or cytotoxic T-lymphocyte escape mutants and thereby the establishment of persistent infection. Evidence for both types of escape mutants have been reported in HCV infections (4, 7, 13, 31). Reports have indicated that hypervariable region 1 (HVR1), located in the N terminus of the E2 protein, evolves more rapidly in vivo than the rest of the viral genome (15, 18) and that it plays a major role in the maintenance of persistent infections (reviewed in reference 17). Previous studies have hypothesized that a higher complexity of virus species provide an indicator of progression to chronicity, particularly in HVR1 (8, 24). However, RNA transcribed from an infectious cDNA clone lacking HVR1 caused a persistent infection in a chimpanzee, indicating that this region is not essential for infection or persistence (10). The quasispecies nature of natural isolates makes it impossible to distinguish true de novo mutations. In this study, we have examined the molecular evolution of HCV over a 4-year period in two chimpanzees infected with a virus consisting of a single sequence as the starting population. The predominant circulating virus at different times after infection was analyzed by direct sequencing of PCR amplicons. We have been able to monitor the true accumulation of mutations in the HCV genome in chimpanzees over time relative to host responses and viral kinetics. We propose that the majority of variant sequences observed during infections with quasispecies isolates, particularly multiple substitutions observed in HVR1, arise primarily through selection of sequence variants already present in the pool of quasispecies rather than by mutation during the time period most humans and chimpanzees have been observed. Our studies indicate that even over several years only single-amino-acid mutations become fixed at distinct sites and that many more years of infection would be required before significant sequence changes would be achieved.