Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome

Abstract

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.

FIG. 1.

Alignment of the putative E1/E2 amino acid sequences (codons 340 to 420) in the plasma, PBMC, and cryoprecipitate of patient 6. The arrows indicate HVR1. The 5-amino-acid insertion is boxed. Lane 1, reference sequence (HCV2c: D31972); lanes 2 to 11, clones obtained from patient plasma; lanes 12 to 21, clones obtained from patient PBMC; lanes 22 to 31, clones obtained from patient cryoprecipitate.

FIG. 2.

Unrooted neighbor-joining tree, including the 228-nucleotide E1/E2 sequences isolated from the plasma, PBMC, and cryoprecipitates of 10 MC patients and 8 CH controls. The dark ellipses correspond to the clones of individual MC patients (see number), and the clear ellipses correspond to the CH controls. The nine reference sequences representative of the major HCV genotypes circulating in Italy included in the analysis are indicated. The between-sample bootstrap values were always ≥75.

FIG. 3.

Percentage of patients showing positive selection at each site. Columns: ▪, subjects with genotype 2 HCV; □, subjects with genotype 1 HCV. HVR-1 region, codons 384 to 410.

FIG. 4.

Prototypical unrooted neighbor-joining trees of the clones isolated from MC patients and controls. The clones obtained from different organic compartments are shown by different symbols (squares, plasma; stars, PBMC; circles, cryoprecipitate). The numbers on the branches denote bootstrap value percentages with 1,000 replicates (bootstrap values of ≥65%). PBMC and cryoprecipitated clones showing significant compartmentalization are enclosed in ellipses. (A) Patient 8; (B) patient 6; (C) patient 2; (D) control subject 15; (E) patient 9.

FIG. 4.

Prototypical unrooted neighbor-joining trees of the clones isolated from MC patients and controls. The clones obtained from different organic compartments are shown by different symbols (squares, plasma; stars, PBMC; circles, cryoprecipitate). The numbers on the branches denote bootstrap value percentages with 1,000 replicates (bootstrap values of ≥65%). PBMC and cryoprecipitated clones showing significant compartmentalization are enclosed in ellipses. (A) Patient 8; (B) patient 6; (C) patient 2; (D) control subject 15; (E) patient 9.

FIG. 4.

Prototypical unrooted neighbor-joining trees of the clones isolated from MC patients and controls. The clones obtained from different organic compartments are shown by different symbols (squares, plasma; stars, PBMC; circles, cryoprecipitate). The numbers on the branches denote bootstrap value percentages with 1,000 replicates (bootstrap values of ≥65%). PBMC and cryoprecipitated clones showing significant compartmentalization are enclosed in ellipses. (A) Patient 8; (B) patient 6; (C) patient 2; (D) control subject 15; (E) patient 9.

FIG. 4.

Prototypical unrooted neighbor-joining trees of the clones isolated from MC patients and controls. The clones obtained from different organic compartments are shown by different symbols (squares, plasma; stars, PBMC; circles, cryoprecipitate). The numbers on the branches denote bootstrap value percentages with 1,000 replicates (bootstrap values of ≥65%). PBMC and cryoprecipitated clones showing significant compartmentalization are enclosed in ellipses. (A) Patient 8; (B) patient 6; (C) patient 2; (D) control subject 15; (E) patient 9.

FIG. 4.

Prototypical unrooted neighbor-joining trees of the clones isolated from MC patients and controls. The clones obtained from different organic compartments are shown by different symbols (squares, plasma; stars, PBMC; circles, cryoprecipitate). The numbers on the branches denote bootstrap value percentages with 1,000 replicates (bootstrap values of ≥65%). PBMC and cryoprecipitated clones showing significant compartmentalization are enclosed in ellipses. (A) Patient 8; (B) patient 6; (C) patient 2; (D) control subject 15; (E) patient 9.

FIG. 5.

Likelihood mapping of three representative MC patients showing the compartmentalization of the clones obtained from concentrated cryoglobulins. The clones of patients 8 and 9 were divided into two groups: plasma (a) and cryoprecipitated quasispecies (b). The sequences of patient 2 were divided into three groups: plasma (a) and cryoprecipitated clones more (subgroup b) or less (subgroup c) closely related to the plasma quasispecies.