Effect of bottlenecking on evolution of the nonstructural protein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection

Abstract

Sexual partners of patients infected with the hepatitis C virus (HCV) often have detectable HCV-specific T-cell responses in the absence of seroconversion, suggesting unapparent, spontaneously resolving infection. To determine whether differences in the evolutionary potential of bottlenecked inoculum may explain the low rate of HCV persistence after sexual exposure, we have investigated changes in the entire HCV nonstructural 3 (NS3) gene over time in a chronic carrier and compared his viral quasispecies with that of the acute-phase isolate of his sexual partner, who developed acute resolving hepatitis C. The overall rate of accumulation of mutations, estimated by regression analysis of six consecutive consensus NS3 sequences over 8 years, was 1.5 x 10(-3) mutations per site per year, with small intersample fluctuations related to changes in environmental conditions. Comparison of quasispecies parameters in one isolate of the chronic carrier with those of the acute-phase isolate of the infected partner revealed a higher heterogeneity and lower proportion of nonsynonymous mutations in the former. All NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic isolate, despite complete HLA mismatch between the patients, suggesting bottlenecking during transmission. The low risk of viral persistence after sexual exposure to HCV may be related to the selection of a limited number of viral particles carrying a particular combination of mutations which may further limit the potential of a relatively homogeneous quasispecies to rapidly diversify and overcome the immune response of the exposed host.

FIG. 1.

Representative changes in ALT (solid line) and HCV viral load (dashed line) in patients A and B. Arrows indicate samples obtained for sequencing. The gray area indicates the antiviral treatment period for patient A (February 1996 to March 1997). IFN+Rbv, interferon plus ribavirin.

FIG. 2.

Fragment of the HCV genome investigated in the study and locations of the PCR primers (arrows) used for direct sequencing of PCR products or clones. UTR, untranslated region.

FIG. 3.

(a) Nucleotide mutations per nucleotide position for each consecutive sample plotted against the year the sample was collected and used for regression analysis. (b) Representation of genetic distances between consensus nucleotide sequences from consecutive samples in patient A and an acute-phase sample from patient B. IFN+Rb, interferon plus Ribavirin.

FIG. 4.

Sequence alignment of the complete 631 amino acids of NS3 in 12 clones of patient A close to the time of transmission to patient B (sample A-10/1998, clones A1 to A12) and in 12 clones from patient B (B-9/1998, clones B1 to B12). Dots indicate residues identical to those of the consensus sequences. Arrowheads indicate amino acid changes in the consensus sequences of patients A and B. Reported HLA class I A-restricted epitopes are underlined, and B-restricted epitopes are shown in italics. HLA class II-restricted CD4 epitopes are indicated between vertical lines and labeled according to published reports (9, 10, 62).

FIG. 4.

Sequence alignment of the complete 631 amino acids of NS3 in 12 clones of patient A close to the time of transmission to patient B (sample A-10/1998, clones A1 to A12) and in 12 clones from patient B (B-9/1998, clones B1 to B12). Dots indicate residues identical to those of the consensus sequences. Arrowheads indicate amino acid changes in the consensus sequences of patients A and B. Reported HLA class I A-restricted epitopes are underlined, and B-restricted epitopes are shown in italics. HLA class II-restricted CD4 epitopes are indicated between vertical lines and labeled according to published reports (9, 10, 62).

FIG. 4.

Sequence alignment of the complete 631 amino acids of NS3 in 12 clones of patient A close to the time of transmission to patient B (sample A-10/1998, clones A1 to A12) and in 12 clones from patient B (B-9/1998, clones B1 to B12). Dots indicate residues identical to those of the consensus sequences. Arrowheads indicate amino acid changes in the consensus sequences of patients A and B. Reported HLA class I A-restricted epitopes are underlined, and B-restricted epitopes are shown in italics. HLA class II-restricted CD4 epitopes are indicated between vertical lines and labeled according to published reports (9, 10, 62).

FIG. 4.

Sequence alignment of the complete 631 amino acids of NS3 in 12 clones of patient A close to the time of transmission to patient B (sample A-10/1998, clones A1 to A12) and in 12 clones from patient B (B-9/1998, clones B1 to B12). Dots indicate residues identical to those of the consensus sequences. Arrowheads indicate amino acid changes in the consensus sequences of patients A and B. Reported HLA class I A-restricted epitopes are underlined, and B-restricted epitopes are shown in italics. HLA class II-restricted CD4 epitopes are indicated between vertical lines and labeled according to published reports (9, 10, 62).

FIG. 5.

Consensus phylogenetic tree showing frequencies of >70% in bootstrap analysis. Branch lengths are drawn to scale.