Constraints on viral evolution during chronic hepatitis C virus infection arising from a common-source exposure

Abstract

Extraordinary viral sequence diversity and rapid viral genetic evolution are hallmarks of hepatitis C virus (HCV) infection. Viral sequence evolution has previously been shown to mediate escape from cytotoxic T-lymphocyte (CTL) and neutralizing antibody responses in acute HCV infection. HCV evolution continues during chronic infection, but the pressures driving these changes are poorly defined. We analyzed plasma virus sequence evolution in 5.2-kb hemigenomes from multiple longitudinal time points isolated from individuals in the Irish anti-D cohort, who were infected with HCV from a common source in 1977 to 1978. We found phylogenetically distinct quasispecies populations at different plasma time points isolated late in chronic infection, suggesting ongoing viral evolution and quasispecies replacement over time. We saw evidence of early pressure driving net evolution away from a computationally reconstructed common ancestor, known as Bole1b, in predicted CTL epitopes and E1E2, with balanced evolution toward and away from the Bole1b amino acid sequence in the remainder of the genome. Late in chronic infection, the rate of evolution toward the Bole1b sequence increased, resulting in net neutral evolution relative to Bole1b across the entire 5.2-kb hemigenome. Surprisingly, even late in chronic infection, net amino acid evolution away from the infecting inoculum sequence still could be observed. These data suggest that, late in chronic infection, ongoing HCV evolution is not random genetic drift but rather the product of strong pressure toward a common ancestor and concurrent net ongoing evolution away from the inoculum virus sequence, likely balancing replicative fitness and ongoing immune escape.

Fig 1

Timeline of anti-D cohort plasma sample isolation. Hemigenomes (5.2 kb) were amplified from inoculum virus (time A) and plasma virus from 10 infected subjects from two time points (time B and time C) during chronic infection.

Fig 2

Viruses isolated at longitudinal time points are phylogenetically distinct. Phylogenetic trees of anti-D HCV sequences spanning 1,651 amino acids from the Core through NS3 proteins. (A) Neighbor-joining amino acid tree with anti-D inoculum (purple circles), chronic anti-D sequences, unrelated genotype 1b sequences, and Bole1b (green circle). (B) Neighbor-joining amino acid trees of anti-D sequences. Center tree contains Bole 1b (green circle) and time A (inoculum) sequences (purple circles). Time B and time C sequences are shown for each study subject, with each color indicating sequences from a different subject. Dots at proximal nodes indicate bootstrap values of >94. For outer trees, green circles indicate Bole 1b, purple circles indicate time A sequences, blue circles indicate sequences amplified from time B plasma, and red circles indicate sequences amplified from time C plasma. Asterisks indicate subjects with statistically significant phylogenetic separation between time B and time C sequences.

Fig 3

Purifying selection dominates evolution late in chronic infection. Sliding window analysis of nonsynonymous and synonymous nucleotide changes from time A to time B and time A to time C. An average nonsynonymous and synonymous distance for all pairwise comparisons was calculated with a 20-nucleotide sliding window and 1-nucleotide steps. Average synonymous change from time A to all time B sequences is indicated by a light blue line and synonymous change from time A to time C by a light red line. Nonsynonymous change from time A to time B is indicated by a dark blue line and nonsynonymous change from time A to time C by a dark red line. Borders of each gene and HVR1 (dashed lines) are indicated. Synonymous change exceeds nonsynonymous change in all regions except HVR1.

Fig 4

Evolution toward Bole1b accelerates later in chronic infection in all regions except HVR1. Rate of amino acid change from time A (inoculum) to time B and time B to time C relative to Bole1b. Total amino acid changes were counted for all pairwise comparisons between 4 time A sequences and 10 time B sequences for each subject and between 10 time B and 10 time C sequences for each subject. Each change was characterized as either away from, toward, or tangential to the Bole1b amino acid sequence. These values were then divided by the number of comparisons, the number of amino acids in the region in question, and the number of years between time A and time B or time B and time C for each subject. Each symbol indicates the rate for a single subject. Horizontal lines indicated medians. (A) Rate of amino acid change in non-E1E2 (Core, P7, NS2, and NS3 proteins; 1,096 sites). (B) Rate of amino acid change in E1E2 without HVR1 (529 sites). (C) Rate of amino acid change in HVR1 (26 sites).

Fig 5

T cell epitope evolution occurs early. The location of amino acid changes relative to HLA-matched and HLA-unmatched class I epitopes. Total amino acid changes, changes away from the Bole 1b amino acid sequence, changes toward Bole1b, and changes tangential to Bole 1b were mapped and identified as falling within HLA-matched (black bars) or HLA-unmatched epitopes (gray bars) for each study subject. The total number of changes of each type was added for all study subjects and divided by the total number of amino acids analyzed. P values were calculated by comparison of proportions (z test). An asterisk indicates P < 0.0001 after correction for multiple comparisons. (A) Changes in non-E1E2 (Core, P7, NS2, and NS3 proteins) from time A to time B. (B) Changes in non-E1E2 (Core, P7, NS2, and NS3 proteins) from time B to time C. (C) Changes in E1E2 (without HVR1) from time A to time B. (D) Changes in E1E2 (without HVR1) from time B to time C.

Fig 6

Evolution away from inoculum continues late in infection. Rate of amino acid change from time B to time C relative to the time A (inoculum) virus amino acid sequence. Amino acid changes were counted for all pairwise comparisons between 10 time B and 10 time C sequences for each subject, and then each change was characterized as either away from, toward, or tangential to time A virus amino acid sequence. These values were divided by the number of sequence comparisons performed, the number of amino acids in the region in question, and the number of years between time B and time C for each subject. Each symbol indicates the rate for a single subject. Horizontal lines indicate medians. (A) Rate of amino acid change in non-E1E2 (Core, P7, NS2, and NS3 proteins; 1,096 sites). (B) Rate of amino acid change in E1E2 (without HVR1) (529 sites).

Fig 7

Late evolution away from the time A virus sequence does not localize to class I-restricted epitopes. Time B to time C amino acid changes relative to time A virus sequence mapped to HLA-matched and HLA-unmatched class I epitopes. Total amino acid changes, changes away from time A virus amino acid sequence, changes toward time A virus sequence, and changes tangential to time A virus sequence were mapped and identified as falling within HLA-matched (black bars) or HLA-unmatched epitopes (gray bars) for each study subject. The total number of changes of each type was added for all study subjects and divided by the total number of amino acids analyzed. P values were calculated by comparison of proportions (z test). An asterisk indicates P < 0.0001 after correction for multiple comparisons. (A) Changes in non-E1E2 (Core, P7, NS2, and NS3 proteins) from time B to time C. (B) Changes in E1E2 (without HVR1) from time B to time C.