Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells

Abstract

Differences in hepatitis C virus (HCV) variants of the highly conserved 5' untranslated region (UTR) have been observed between plasma and peripheral blood mononuclear cells (PBMC). The prevalence and the mechanisms of this compartmentalization are unknown. Plasma and PBMC HCV variants were compared by single-strand conformation polymorphism (SSCP) and by cloning or by genotyping with a line probe assay (LiPA) in 116 chronically infected patients, including 44 liver transplant recipients. SSCP patterns differed between compartments in 43/109 analyzable patients (39%). Differences were significantly more frequent in patients with transplants (21/38 [55%] versus 22/71 [31%]; P < 0.01) and in those who acquired HCV through multiple transfusions before 1991 (15/20; 75%) or through drug injection (16/31; 52%) than in those infected through an unknown route (7/29; 24%) or through a single transfusion (5/29; 17%; P < 0.001). Cloning of the 5' UTR, LiPA analysis, and nonstructural region 5B sequencing revealed different genotypes in the two compartments from 10 patients (9%). In nine patients, the genotype detected in PBMC was not detected in plasma and was weak or undetectable in the liver in three cases. This genotypic compartmentalization persisted for years in three patients and after liver transplantation in two. The present study shows that a significant proportion of HCV-infected subjects harbor in their PBMC highly divergent variants which were likely acquired through superinfections.

FIG. 1.

(A) 5′-UTR SSCP analysis of paired plasma (P) and PBMC HCV RNAs. Patients 46, 51, 52, and 70 were considered to have similar SSCPs in both compartments. Patients 3, 5, 15, 16, and 17 had different SSCP patterns (Table 4). (B) 5′-UTR SSCP analysis of plasma (P) HCV RNA and of 10 clones (1 to 10) obtained from the same amplification. SSCP of plasma HCV RNA was composed of the different bands observed for each clone.

FIG. 2.

Phylogenetic analysis of plasma and PBMC HCV variants infecting patient 3. HCV RNA was amplified between nucleotides 100 and 1600 in plasma and PBMC and then cloned. The phylogenetic trees drawn from 5′-UTR sequences (nucleotides 100 to 350) (A) and the E1-E2 region (nucleotides 1370 to 1600) (B) evidenced the same clustering of some PBMC HCV variants that could be assigned to subtype 1a while other variants were assigned to subtype 1b.

FIG. 3.

SSCP, phylogenetic analysis, and line probe assay results for four patients with compartmentalized HCV genotypes. 1, immunocompetent patient, HCV type 3 in plasma, HCV types 3 and 2 in PBMC; 2, immunocompetent patient, HCV types 1 and 3 in plasma, HCV type 1 in PBMC; 18, transplant patient, HCV type 1 in plasma, HCV types 1 and 2 in PBMC; 19, transplant patient, HCV type 2 in plasma, HCV type 1 in PBMC.

FIG. 4.

SSCP 5′ UTR on 1:1 mixtures of genotypes 1 and 3: 100,000, 10,000, and 1,000 RNA copies of each genotype (lanes 1, 2, and 3). SSCP of genotype 1 alone (lane 4) and genotype 3 alone (lane 5). L, ladder.

FIG. 5.

Follow-up of patients with HCV genotypic compartmentalization. 2, immunocompetent patient sampled before and after interferon therapy, disappearance of plasma type 3 HCV and persistence of HCV type 1 in plasma and PBMC; 4, immunocompetent untreated patient sampled at 3-year intervals, persistence of the genotypic compartmentalization (genotype 4 in plasma and PBMC and genotype 1 in PBMC); 19 and 20, patients sampled before and after liver transplantation, persistence of the genotypic compartmentalization.

FIG. 6.

Line probe assays of plasma, PBMC, and liver 5′ UTRs in patients 4, 19, and 20. The plasma genotype was found in the liver in all cases. The PBMC genotype was detected in the liver in patients 4 and 20. Traces of a third genotype (type 1) not found in plasma or PBMC was detected in the liver of patient 20. In patient 4, 5′-UTR SSCPs are shown for plasma, PBMC, and CD4⁺, CD19⁺, CD14⁺, and CD8⁺ cells. Specific bands observed in PBMC are also observed in CD19⁺ and CD8⁺ fractions and corresponded to the presence of genotype 1.