Viral Sequence Evolution in Acute Hepatitis C Virus Infection

Abstract

CD8(+)-T-cell responses play an important role in the containment and clearance of hepatitis C virus (HCV) infection, and an association between viral persistence and development of viral escape mutations has been postulated. While escape from CD8+ -T-cell responses has been identified as a major driving force for the evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), a broader characterization of this relationship is needed in HCV infection. To determine the extent, kinetics, and driving forces of HCV sequence evolution, we sequenced the entire HCV genome longitudinally in four subjects monitored for up to 30 months after acute infection. For two subjects the transmission sources were also available. Of 53 total non-envelope amino acid substitutions detected, a majority represented forward mutations away from the consensus sequence. In contrast to studies in HIV and SIV, however, only 11% of these were associated with detectable CD8+ T-cell responses. Interestingly, 19% of non-envelope mutations represented changes toward the consensus sequence, suggesting reversion in the absence of immune pressure upon transmission. Notably, the rate of evolution of forward and reverse mutations correlated with the conservation of each residue, which is indicative of structural constraints influencing the kinetics of viral evolution. Finally, the rate of sequence evolution was observed to decline over the course of infection, possibly reflective of diminishing selection pressure by dysfunctional CD8+ T cells. Taken together, these data provide insight into the extent to which HCV is capable of evading early CD8+ T-cell responses and support the hypothesis that dysfunction of CD8+ T cells may be associated with failure to resolve HCV infections.

Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity

“…For autologous virus sequencing, viral RNA was extracted from plasma samples using the vRNA extraction kit (Qiagen). A nested PCR with 12 external and 36 internal primer pairs spanning the entire HCV coding sequence amplified overlapping fragments of approximately 500-1.2 kb, as previously described (48). PCR fragments were then purified (PCR Purification Kit; Qiagen) and population sequenced bidirectionally on an ABI 3730 PRISM automated sequencer (48).…”

Hepatitis C Virus Transmission Bottlenecks Analyzed by Deep Sequencing

“…In an infected individual, serum HCV RNA levels can reach 10 to 100 million IU/ml (40). The viral RNA polymerase is estimated to make 1 error per 10,000 to 100,000 bp copied (22), but the viral genome is only 9,600 bases, resulting in diversification of the viral population, so that most viral genomes differ in sequence from the population consensus (16,20,21). Thus, when antiviral pressure is exerted on a viral population, sequence variants with reduced sensitivity may expand in the presence of the selective pressure (30, 41) and cause resistance (37).…”

Automated Low Cost Method of Quantification of Hepatitis C Virus

“…RT-PCR was performed using the RNA reaction mixture (18 µl) containing 4 U of RNAsin, 0.27 µM of each forward and reverse primers (HCV 5UT F and R, Cha et al, 1992) [13][14] , and 15 µl of viral RNA, was prepared. The RNA mix was incubated for 5 minutes at 65 0 C and 10 minutes at room temperature.…”