Divergent and convergent evolution after a common-source outbreak of hepatitis C virus

Abstract

The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune-driven selection and, in the absence of immune pressure, reversion toward an ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18-22 yr after infection to sequences present in the shared inoculum and found that HCV evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). These data suggest that vaccines for genetically diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.

Figure 1.

Phylogenetic analysis of HCV 18–22 yr after common-source outbreak. (A) Phylogenetic tree of 5.2-kb sequence alignment placing outbreak clade (*) in context with reference sequences for all major subtypes. (B) Detailed analysis of the outbreak clade, using 10 cDNA clones from each study subject to obtain the sequence of a 698-nt region spanning the E1–E2 junction. The label “inoculum” indicates 20 clones from inoculum source plasma (10 each from 2 specimens), and a full-length clone (AF313916) obtained in an independent study of this material using smaller amplicons. For both trees, numbers at nodes are bootstrap values, indicating the percentage of 1,000 permuted trees that supported the presence of that node. Bootstrap support was 100% for each of the major clades in A; in B, only bootstrap values >80% are shown, and values for nodes within a study subject's clade were omitted for clarity. Boxes highlight two subjects whose sequences were segregated into two separate clades.

Figure 1.

Phylogenetic analysis of HCV 18–22 yr after common-source outbreak. (A) Phylogenetic tree of 5.2-kb sequence alignment placing outbreak clade (*) in context with reference sequences for all major subtypes. (B) Detailed analysis of the outbreak clade, using 10 cDNA clones from each study subject to obtain the sequence of a 698-nt region spanning the E1–E2 junction. The label “inoculum” indicates 20 clones from inoculum source plasma (10 each from 2 specimens), and a full-length clone (AF313916) obtained in an independent study of this material using smaller amplicons. For both trees, numbers at nodes are bootstrap values, indicating the percentage of 1,000 permuted trees that supported the presence of that node. Bootstrap support was 100% for each of the major clades in A; in B, only bootstrap values >80% are shown, and values for nodes within a study subject's clade were omitted for clarity. Boxes highlight two subjects whose sequences were segregated into two separate clades.

Figure 2.

HCV divergence and convergence after a common-source outbreak. The upper panel shows a sliding-window analysis of nonsynonymous (red) and synonymous (blue) variation, calculated by comparing the mean pairwise distance between the inoculum (2 cDNA clones obtained from two inoculum source plasma specimens obtained 1 wk apart in 1977) to 44 clones (2 per study subject) obtained from chronically infected women 18–22 yr after exposure, in a sliding window 20 codons wide, moving in 1-codon increments, generated using VarPlot. Horizontal bars indicate average distance for each gene region. The lower panel is composed of sequence logos displaying the variability in HVR1 (the region indicated by dashed lines in the upper panel), with numbers indicating position relative to the H77 polyprotein. The first three rows show the subtype 1b reference sequences, inoculum sequences (20 clones from 2 plasma specimens obtained 1 wk apart), and 220 recipient sequences (10 per study subject) respectively, with the height of each single-letter amino acid code proportional to its frequency. The residue at the top of the stack in each position is the most-frequently observed, and hence the consensus residue, at that position. The fourth row shows differences between the amino acid frequencies in the recipient sequences versus the inoculum sequences as a type 2 logo, in which the height of each amino acid is determined by the log₂ relative risk of observing it, with the scale indicated. Empty spaces indicate a distribution highly similar to the inoculum distribution, because the logarithm of a relative risk of one is zero.

Figure 3.

Escape versus reversion in the presence versus absence of the restricting HLA allele. (A) Amino acid alignment of a region in NS3 (positions 1388 to 1431 relative to the H77 polyprotein (GenBank/EMBL/DDBJ accession no. AF009606) showing sites with polymorphism. Study subjects are listed in arbitrary order. Identity to the inoculum sequence (“inoc”) is indicated by “.”. (B) Sorting the subjects by the presence of HLA A*02, G1409S substitution in the 4th position of a frequently recognized HLA A*02-restricted epitope at 1406–1415 is limited to subjects having the HLA A*02 allele. The subtype 1b consensus sequence for this epitope is shown below the alignment, and has been shown to be recognized as readily as the prototype (subtype 1a) KLVALGINAV sequence (27). Subject AD17 (HLA A*01, A*11) had G1409D substitution, the impact of which on recognition is unknown. (C) Variation resulting in reversion to a HLA B*08-restricted epitope. Sorting the subjects by presence of HLA B*08, R1397K substitution in the 3rd position of a frequently recognized HLA B*08- restricted epitope at 1395–1403 is limited to subjects lacking the HLA B*08 allele. The inoculum sequence differs from the prototypical epitope (HSKKKCDEL) at the 3rd position. Reversion to consensus (and the prototype epitope) occurred only in study subjects lacking the HLA B*08 allele.