Nonrandom distribution of hepatitis C virus quasispecies in plasma and peripheral blood mononuclear cell subsets

Abstract

The existence of an extrahepatic reservoir of hepatitis C virus (HCV) is suggested by differences in quasispecies composition between the liver, peripheral blood mononuclear cells, and serum. We studied HCV RNA compartmentalization in the plasma of nine patients, in CD19(+), CD8(+), and CD4(+) positively selected cells, and also in the negatively selected cell fraction (NF). HCV RNA was detected in all plasma samples, in seven of nine CD19(+), three of eight CD8(+), and one of nine CD4(+) cell samples, and in seven of eight NF cells. Cloning and sequencing of HVR1 in two patients showed a sequence grouping: quasispecies from a given compartment (all studied compartments for one patient and CD8(+) and NF for the other) were statistically more genetically like each other than like quasispecies from any other compartment. The characteristics of amino acid and nucleotide substitutions suggested the same structural constraints on HVR1, even in very divergent strains from the cellular compartments, and homogeneous selection pressure on the different compartments. These findings demonstrate the compartmental distribution of HCV quasispecies within peripheral blood cell subsets and have important implications for the study of extrahepatic HCV replication and interaction with the immune system.

FIG. 1

Phylogenetic tree, patients A (A) and B (B). Abbreviations: T, CD8⁺ lymphocytes; B, CD19⁺ lymphocytes; X, negative cell fraction (CD19⁻, CD8⁻, and CD4⁻); S, plasma. The phylogenetic grouping of sequences from all four compartments in patient A and from CD8⁺ cells in patient B was confirmed by a significant correlation between genetic proximity and compartment appurtenance (P < 0.0001, by Mantel’s test; see Materials and Methods). Variants in NF cells from patient B were not included in the same node of the phylogenetic tree but were overall more genetically identical to each other than to variants from any other compartment (P < 0.0001). These NF cells have at least three phenotypes: monocytes/macrophages, natural killer cells, and granulocytes.

FIG. 1

Phylogenetic tree, patients A (A) and B (B). Abbreviations: T, CD8⁺ lymphocytes; B, CD19⁺ lymphocytes; X, negative cell fraction (CD19⁻, CD8⁻, and CD4⁻); S, plasma. The phylogenetic grouping of sequences from all four compartments in patient A and from CD8⁺ cells in patient B was confirmed by a significant correlation between genetic proximity and compartment appurtenance (P < 0.0001, by Mantel’s test; see Materials and Methods). Variants in NF cells from patient B were not included in the same node of the phylogenetic tree but were overall more genetically identical to each other than to variants from any other compartment (P < 0.0001). These NF cells have at least three phenotypes: monocytes/macrophages, natural killer cells, and granulocytes.

FIG. 2

Peptide alignment, patient A (A) and patient B (B). The lefthand column indicates the origin and number of identical clones: T, CD8⁺ cells; B, CD19⁺ cells; X, negative fraction cells; S, plasma. A consensus sequence was generated for each patient. Antigenic grouping was done manually and blindly with regard to the origin of the clones.

FIG. 3

Antigenic grouping of HVR1 variants from patient A (A) and patient B (B). Antigenic profiles were obtained by using the Parker’s algorithm. HVR1 amino acid sequences composing the signature of antigenic groups are aligned with the consensus sequence.