| An estimated 170 million people worldwide are infected with hepatitis C virus (HCV). More than 80% of infections result in persistent viremia, which may be associated with chronic hepatitis, cirrhosis, liver failure, and hepatocellular cancer.Because of limitations of in vitro replication systems, studies of HCV pathogenesis have been limited to observation of natural infection of humans and experimental infection of chimpanzees. The chimpanzee is the best experimental model of HCV infection. However, the data from the experimental infection of chimpanzees may be different from humans. Existing data do not conclusively explain HCV pathogenesis. What is the difference of immune selection pressure between chimpanzees and humans? And there is no experimental immunodeficiency model as a control for HCV infection.Nucleotide substitutions that do not affect the protein structure (synonymous substitutions, or ds) accumulate in a quasispecies over time, reflecting the rate of viral replication among other factors. In contrast, changes in the amino acid composition of the HCV quasispecies (nonsynonymous substitutions, or dN) typically reflect immunologic pressure and are not predicted by replication alone.Using a combination of single-stranded conformational polymorphism (SSCP) and heteroduplex analysis (HDA) on a single gel (SSCP+HDA) for detection of distinct variants, we determined the complexity of the quasispecies in each specimen and selected representative cloned cDNAs for sequenceanalysis. We studied the HCV quasispecies in a cohort of animals through which HCV was serially passed and in two animals with persistent infection. The serial passage experiment was initiated using a well-characterized strain of HCV (H77). In addition, Because HIV infection is broadly immunosuppressive, we also examined the evolution of envelope HCV sequences in persons with chronic hepatitis C before and after HIV seroconversion, including subjects with rapid progression to AIDS.Results were presented as follows:1. The quasispecies complexity was examined by assessing 33 cDNA clones from each specimen using SSCP+HDA. The median clonotype ratio (range) of the acute phase of the serial eight passage lineage chimps, 0.27(0.18 to 0.39), was similar (P>0.5) to that found in the first RNA-positive specimen from acutely infected humans, 0.39(0.09 to 0.67).2. SSCP+HDA analysis indicated the persistence of clonotype a through chimpanzee passages P1 to P8, with the exception of animal P2, which was the animal that developed persistent viremia. In contrast to the persistence of clonotypes during serial passage, acute and chronic specimens from animals P2 and x304 shared no clonotype.3. In each pair of specimens from the same chimpanzee, the complexity of the quasispecies assessed using the clonotype ratio increased with time from inoculation or seroconversion, both during the acute phase and also during the chronic phase. This was also true of four of the six humans who had clonotype ratio increases. However, decreasing complexity was observed in subjects AT and AZ. Of note, the infecting subtype of subjects AT and AZ (1b) differed from that of the other hosts (1a).4. By using SSCP+HDA to select representative cloned cDNAs, 72 distinct cloned cDNAs were identified for sequencing. The most striking aspect of the chimpanzee HCV acute-phase sequences was the low frequencyof substitutions. In contrast, chronic-phase sequences formed monophyletic clusters.5. The mean pairwise genetic distance between acute- and chronic-phase sequences was consistently lower in chimpanzees than in humans. Substitution rates calculated from these data were lower for chronic-phase sequences(P2 and x304) than for humans. The substitutions observed during chronic infection of chimpanzees were predominantly synonymous, resulting in no change in the amino acid sequence, whereas they were predominantly nonsynonymous (resulting in changes in amino acid sequence) in humans.6. It is notable for the persisten...
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