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Article Figures & Data

Figures

  • Fig. 1.

    Analysis of the genotype distribution in the serum and PBMC of a HCV patient harboring a dual infection. Nested RT-PCR was performed using Cap derived primers for the amplification of genotype specific products either from the positive or the negative strand viral RNA. Control human sera included positive strand RNA amplified from patients infected with genotypes 1a, 1b, 2(a/c), 3 and 4 (lanes 1, 2, 3, 4, and 5, respectively). Serum (S, lanes 6 and 8) and total PBMC (PB, lanes 7 and 9) from a patient infected with a genotype 1a and a genotype 2 were used for amplification of both the positive (+) and the negative (−) strand RNA. PCR products were fractionated on a 3% agarose gel and stained by ethidium bromide. Markers are shown on the left hand side (base pair, bp) while expected sizes of amplified products specific for the different genotypes are shown on the right hand side (bp).

  • Fig. 2.

    Efficiency of detection of positive (A) and negative (B) strand RNA from genotype 1 to 4 derived templates. Synthetic RNA, encompassing the near full length 5' NCR and CAP sequences from genotypes 1 to 4 HCV sequences, were derived as described in Materials and Methods and used in serial dilution assays. Assays included amplification of 0 to 10 6 RNA copies per reaction. PCR products obtained after single rounds of amplification (positive strand RNA, 1A) or nested rounds of amplification (negative strand RNA, 1B) were fractionated on 2.5% agarose gel and stained with ethidium bromide.

  • Fig. 3.

    Percent of patients harboring HCV RNA sequences in total PBMC: correlation with viral load and viral genotype. Serum (250 μL) and PBMC (2 × 106 cells) from 38 patients were processed as described in Materials and Methods. Titration of HCV RNA in serum was performed by means of the bDNA assay (HCV RNA 2.0 assay). All indicated genotypes were deduced fom concordant results obtained from three genotyping assays. Titers < 2 × 105 genome equivalents/ml (Eq/mL) were considered equal to 2 × 105 Eq/ml for representation in the figure. (2A): detection of the positive strand RNA; (2B): detection of the negative strand RNA. Subtype distribution in co-infected patients was: subtype 1a, n = 5, subtype 1b, n = 1. Open circles indicate individual viral titers for each patient while black symbols represent the mean titers for each group of patients represented.

Tables

  • Table 1.

    Detection of HCV Positive and Negative Strand RNA in Different Subsets of Peripheral Hematopoietic Cells

    Cells*PhenotypeRT-PCR Strand Specificity Cell Range (×106 cells)
    Positive Negative
    Total PBMC x 11/11 (100%)3/11 (27%) 1.30 to 15.00
    GranulocytesCD15+ 8/9 (89%) 3/10 (30%)0.03 to 12.50
    Monocytes/Macrophages CD14+ 5/9 (56%) 1/10 (10%) 0.03 to 4.00
    B lymphocytesCD19+ 5/8 (63%) 2/9 (22%)0.06 to 1.80
    T lymphocytes CD3+0/7 (0%) 0/7 (0%) 0.34 to 4.40
    Thrombocytesnegative fraction1-153 0/6 (0%) 0/6 (0%)x
    • *Cellular subsets are deduced according to their phenotype.

    • Monoclonal antibodies directed against those surface molecules were used to purify corresponding cell type as described in Materials and Methods.

    • Range of cells used in the PCR assays.

    • F1-153 Negative fraction collected at the end of the magnetic selection and corresponding to the CD15, CD14, CD19, and CD3 cells. This fraction was depleted in nucleated cells and contained principally thrombocytes.

  • Table 2.

    FACscan Analysis of Immunofluorescence Assays Performed on PBMC and Cell Subsets Purified From HCV-Infected Patients

    Antibodies*CD Tested Cell-151Percent Detection
    Before Selection After Selection Using Antibodies Directed Against*:
    Total EventsCD45+-152CD3 CD15 CD19 CD14Neg Fraction-153
    M833 CD45RB PBMC 62.3 100100 100 95-155 100 38¶
    Leu4CD3 T lymphocytes 35.3 59.3 99 3 <1<1 37
    Leu12 CD19 B lymphocytes 6.9 11.6<1 <1 94 <1 <1
    LeuM1 CD15Granulocytes 20.5 24.9 <1 96 <1 <1<1
    LeuM3 CD14 Monocytes 8.2 13.8 <1<1 <1 79 <1
    CD42a Platelets 9.5x <1 <1 <1 <1 52
    • *Immunomagnetic selection and antibodies as described in Materials and Methods, 10,000 events were counted and analyzed for each antibody.

    • F0-151 Subset of cells expressing the corresponding CD.

    • F0-152 Percentage of fluorescent cells when CD45+ cells = 100%.

    • F0-153 Negative fraction collected at the end of the last immunomagnetic selection.

    • F0-155 The CD45 cells were dead cells (<5%) and erythrocytes (23% of the events).

    • ¶The CD45 cells were dead cells.