Productive Replication of Hepatitis C Virus in Perihepatic Lymph Nodes In Vivo: Implications of HCV Lymphotropism

GASTROENTEROLOGY 2006;130:1107–1116

Productive Replication of Hepatitis C Virus in Perihepatic Lymph Nodes In Vivo: Implications of HCV Lymphotropism SAMPA PAL,* DANIEL G. SULLIVAN,* SEAN KIM,‡ K. KAY–YIN LAI,* JOHN KAE,* SCOTT J. COTLER,§ ROBERT L. CARITHERS Jr,¶ BRENT L. WOOD,* JAMES D. PERKINS,储 and DAVID R. GRETCH*,¶ *Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington; ‡Department of Developmental and Cell Biology, UC Irvine, Irvine, California; §Section of Hepatology, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois; ¶Department of Medicine, University of Washington Medical Center, Seattle, Washington; and 储Department of Surgery, University of Washington Medical Center, Seattle, Washington

Background & Aims: The pathogenesis of chronic hepatitis C is poorly understood. This study examines the ability of hepatitis C virus (HCV) to infect, replicate in, and produce progeny virus from perihepatic lymph nodes in vivo. Methods: Lymph node (LN) biopsy specimens were taken from 20 patients with HCV genotype 1 infection and end-stage liver disease and 20 noninfected negative controls. Sections were probed with HCV RNA strand-specific riboprobes and antibodies specific for HCV core and nonstructural region 3 antigens plus B-cell (CD20) and T-cell (CD2) antigens. In a selected case, HCV quasispecies in serum, peripheral blood mononuclear cells, liver, and perihepatic lymph nodes were analyzed by clonal frequency analysis and sequencing. Results: HCV infection was confirmed in 17 of 20 (85%) of lymph node specimens by in situ hybridization, and HCV replication was confirmed in 50% of cases by detection of HCV replicative intermediate RNA. HCV core and nonstructural 3 antigens were detected in lymph nodes by immunocytochemistry. Infected cell phenotypes were primarily CD20 B cells, although other cell types were positive for HCV replication markers. Quasispecies analysis in one case indicated that 68% of variants circulating in serum were also present in lymphoid tissues, and only 40% of serum variants were identified in liver, documenting a major contribution of lymphoid replication to HCV viremia. Conclusions: HCV lymphotropism provides new insights into the complex pathobiology of chronic hepatitis C in humans. We demonstrate for the first time a major contribution of extrahepatic HCV replication to circulating virus in serum (viremia).

epatitis C virus (HCV) is a major cause of chronic liver disease in humans.1 Primary HCV infection results in long-term persistence of viremia in approximately 80% of cases, which is associated with

H

progression from chronic liver disease to cirrhosis in approximately 20% of infected individuals. In addition, HCV infection is strongly associated with several extrahepatic disease syndromes including essential mixed cryoglobulinemia and membranoproliferative glomerulonephritis.2 However, the mechanisms of HCV persistence in humans and the pathogenesis of HCV-associated disease syndromes are poorly understood because of a lack of animal and tissue culture models. HCV replicates by enzymatically converting its positivestrand RNA genome into a complementary or minus-strand replicative intermediate (RI) RNA and then copying the minus-strand RNA to produce new progeny positive-strand RNA, as has been well described for closely related Flaviviridae.3 For positive-strand viruses such as HCV, RI RNA is a highly specific index of active viral replication. Although liver is considered the primary site of HCV replication in vivo, results from in vitro studies indicate that HCV is capable of low-efficiency replication in hematopoietic cells under special conditions.4 –7 Numerous investigators have sought evidence of hematopoietic HCV replication in vivo by studying peripheral blood mononuclear cells (PBMCs) for the viral RI RNA using highly sensitive and strand-specific reverse-transcription polymerase chain reaction (RT-PCR) methods. However, results using this approach have yielded conflicting results, possibly because of the problem of nonspecific priming during RT-PCR of the HCV 5= noncoding region of HCV, and thus the question of HCV in vivo hematotropism remains controversial.8 –13 Abbreviations used in this paper: E1, envelope 1; HVR, hypervariable region; ICC, immunocytochemistry; ISH, in situ hybridization; NS3, HCV nonstructural region 3; RI, replicative intermediate. © 2006 by the American Gastroenterological Association Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2005.12.039

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In the current study, we tested the hypothesis of extrahepatic HCV replication in vivo using a highly sensitive and strand-specific in situ hybridization (ISH) assay, which was designed to detect and differentiate specifically between HCV genomic and RI RNAs within infected cells. Strand-specific ISH, one of the wellaccepted “gold standard” methods for assessing viral replication in tissue, involves direct hybridization of probes with viral RNA molecules and does not require PCR amplification of viral sequences for sensitive detection.14,15 Immunocytochemistry (ICC) was used to assay for viral structural and nonstructural protein expression and cell surface markers associated with HCV replication. HCV quasispecies variants were characterized in multiple tissue compartments and serum to test for evidence of tissue-restricted quasispecies production. We studied proximal lymph node tissues from 20 consecutive HCV genotype 1-infected patients undergoing orthotopic liver transplantation for end-stage hepatitis C at our institution.

Materials and Methods Patients Twenty consecutive patients with HCV genotype 1 infection and end-stage liver disease were recruited and consented via human subjects–approved protocol. Lymph node biopsy specimens were taken at the time of liver transplantation and immediately snap frozen in ornithine carbamyl transferase (OCT) buffer. Lymph node biopsy specimens were also submitted for histologic evaluation. Twenty negative control lymph node biopsy specimens snap frozen in OCT were obtained from the University of Washington Medical Center hematopathology laboratory via a human subjects–approved protocol; the specimens were randomly selected from archived clinical material by hematopathology laboratory technologists, who subsequently stripped all patient identifiers prior to sending them to study investigators. Thin sections of all biopsy specimens were screened with probes for the HCV actin and HPRT (hypoxanthine-guanine phosphoribosyl transferase) RNA to ensure the intact nature of cellular RNA. Parallel sections were then probed with the HCV genotype–specific riboprobes described below and were also stained for HCV core and HCV nonstructural region 3 (NS3) antigens plus B-cell (CD19 and PAX) and T-cell (CD2) antigens by ICC (see below).

Generation of Probes Generation of clones and riboprobes for in situ (ISH) hybridization experiments has been described in detail previously.15 For both HCV genotypes 1a and 1b, HCV 5= non-translated region, core, and envelope 1 (E1) genes were amplified by RT-PCR and cloned into plasmid pDP19 (Invitrogen, Carlsbad, CA). To generate digoxigenin (Dig)-la-

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beled riboprobes, RNA were synthesized by runoff transcription with T7 or T3 polymerase in the presence of Dig-UTP according to the manufacturer’s protocol (Roche Diagnostics, Indianapolis, IN). The production of RNA and the subsequent removal of the DNA template by DNase I were monitored by agarose gel electrophoresis, RNA denaturing gel electrophoresis, and a sensitive dot blot hybridization assay. Dig-labeled riboprobes were further broken down to an average size of 100 nucleotides by alkaline hydrolysis. The final riboprobe was precipitated and dissolved in 0.1% sodium dodecyl sulfate. The yield of each newly synthesized Dig-labeled riboprobe was evaluated against a known, standard Dig-labeled RNA according to the manufacturer’s protocol (Roche Diagnostics). The concentrations of experimental riboprobes were determined by comparing spot intensities of the standard control and the experimental dilutions. Probe concentrations were further optimized by Northern dot blot hybridization, and probe concentrations were adjusted to equivalent specific activity for all ISH experiments.

Generation of Control Cell Lines for ISH To generate control cell lines expressing either HCV positive-strand or negative-strand RNA, DNA containing the HCV genotype 1a core plus E1 gene and HCV genotype 1b core plus E1 gene were amplified by PCR, cloned into plasmid pDP19 (Invitrogen), and then subcloned into the eukaryotic expression vector pTRE2hyg in both sense and antisense orientations relative to the promoter enhancer of human cytomegalovirus. HeLa tet off cells were transfected by electroporation, and positive cell lines were selected by culturing in the presence of hygromycin B (Calbiochem, San Diego, CA).

ISH Assay The strand-specific ISH assay has been described in detail previously.15 In brief, frozen sections (6 ␮m) were heat thawed, fixed in 10% neutral buffered formalin, and washed with 1X phosphate-buffered saline (PBS). The tissue sections were treated with 0.2 N HCl and proteinase K (1 ␮g/mL) and soaked in equilibration solution followed by prehybridization solution (Novagen, Madison, WI) at 50°C for 1 hour. Approximately 15 ␮L Dig-labeled riboprobes were applied to each slide at a final concentration of 2 to 4 ng/␮L in hybridization buffer. For analysis of HCV RNA, mixtures of core and genotype-specific E1 riboprobes were used as HCV antisense (negative-strand) or sense (positive-strand) riboprobes. To ensure stability of cellular RNA and the intact nature of cells, ISH was performed using a mixture of HPRT and ␤-actin antisense riboprobes as a positive control. During the hybridization steps, tissue sections were covered with siliconized coverslips, sealed with rubber cement, and incubated at 50°C in a humidified chamber for 18 hours. After hybridization, sections were treated with RNase (Novagen) (20 ␮g/mL in 2X SSC) at 37°C for 30 minutes to reduce nonspecific background. Subsequently, sections were washed in 50% formamide and

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SSC at 50°C to 65°C for 30 minutes. The wash temperature was optimized by titration for each probe.

Immunologic Detection of Riboprobes in Tissue Tissue sections were soaked in blocking buffer (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature, followed by incubation with anti-Dig-alkaline phosphatase conjugate (1:250 dilution) at 4°C overnight in a humidified chamber. Sections were washed twice with 100 mmol/L Tris buffer at room temperature. Vector red substrate (Vector Laboratories) supplemented with 1.25 mmol/L levamisole (Sigma Chemical Co, St Louis, MO) was added for 30 minutes before the reaction was terminated by Tris-HCl buffer, 10 mmol/L (pH 8.0), and 1 mmol/L EDTA. Slides were counterstained with 2% methyl green and dehydrated by successive washings with 95% ethanol, 100% ethanol, and xylene before permanent mounting. Slides were examined and photographed using bright-field microscopy. Whenever adequate tissue was available, multiple independent ISH experiments with both sense and antisense riboprobes were performed, and 3 different technologists blinded to infection status analyzed the data. Negative control lymph node biopsy sections obtained clinically from patients without hepatitis C were also stained using HCV riboprobes and antibodies to ensure the specificity of the ISH and ICC assays using lymph node tissue.

ICC Snap frozen lymph node sections were fixed in 10% neutral-buffered formalin and subjected to immunohisto-

Figure 1. Staining of positive control cell lines by in situ hybridization (ISH). Positive control HeLa cell lines expressing either positive strand (A and C) or negative strand (E and G) subgenomic HCV RNA and negative control HeLa cells (B, D, F, and H) were stained with HCV riboprobes of opposite polarity, followed by immunologic detection of bound probes and enzymatic colorometry as described in the Materials and Methods section. Positive signal is red; nuclei are counterstained green. A and E demonstrate detection of genotype 1a-positive and -negative strand RNA using genotype 1a antisense and sense riboprobes, respectively, and C and G demonstrate detection of genotype 1b-positive and -negative strand RNA with genotype 1b antisense and sense riboprobes, respectively. Negative control HeLa cell lines were stained with the same HCV sense and antisense HCV riboprobe preparation used in the experiment presented in the left adjacent panel. Original magnification, ⫻40.

Figure 2. Immunocytochemistry (ICC) staining of HCV replicon cell lines with anti-HCV core and NS3 antibodies. (A) Huh7-replicon cells stained with anti-NS3 antibodies followed by detection with DAB (brown signal). (B) Huh7-replicon cells stained with anticore antibodies followed with vector red (VR) detection (red signals). (C) Huh7replicon cells stained with a mixture of anticore and anti-NS3 antibodies; core and NS3 antigens were simultaneously detected using different substrate systems, as described for A and B. (D) Huh7 cells without replicon, stained with the same antibodies as in C. Original magnification, ⫻100.

chemistry. Mouse monoclonal antibodies against HCV core (Affinity BioReagents, Golden, CO), NS3 (Vision Biosystem, MA), and CD20 and CD2 (Cymbus Biotech, CA) were used at 1:50 dilution for 40 minutes, followed by biotinylated goat anti-mouse immunoglobulins (dilution 1:200) for 30 minutes at room temperature. Sections were incubated with the Vectastain ABC alkaline phosphatase kit (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature and revealed by using a vector red substrate kit (Vector Laboratories). Methyl green (Vector Laboratories) was used to counterstain the sections. HCV replicon cells16 (a gift from C. Rice, Rockefeller Institute, NY) served as positive controls for ICC experiments, and both Huh7 cells lacking HCV replicon and liver biopsy specimens obtained from HCV-negative subjects served as negative controls.

Interpretation of ISH and ICC Results of ISH and ICC are presented as the consensus result of a minimum of 3 independent experiments on parallel lymph node biopsy sections in all cases. Each ISH and ICC experiment involved hybridization of a set of probes (ie, HCV sense and antisense riboprobes) to parallel sections of the same lymph node biopsy specimen, to a negative control lymph node biopsy specimen, and to the positive and negative control cell lines described previously. Experiments were only considered valid if the positive and negative controls all gave the expected results as illustrated in Figures 1 and 4. A specimen had to be

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Table 1. Results of In Situ Hybridization Analysis of Pretransplantation Lymph Node Specimens and Posttransplantation Histologic Course of Chronic Hepatitis C ISH resultsa Patient No.

HCV genotype

1 1a 2 1a 3 1a 4 1a 5 1a 6 1a 7 1a 8 1b 9 1b 10 1b 11 1a 12 1a 13 1a 14 1a 15 1a 16 1b 17 1a 18 1a 19 1a 20 1a No. positive/total (% positive)

G

RI

⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ 17/20 (85)

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ 10/20 (50)

Posttransplantation months of F/U

Histologic diagnosisb

6 6 24 6 6 12 17 20 26 30 ND ND ND 6 6 12 12 13 18 24

0 0 0 1 0 4 0 4 1 2 ND ND ND 0 2 1 1 2 4 2

F/U, follow-up; ND, No data. aIn situ hybridization (ISH) assay results for HCV genomes (G) and replicative intermediate RNA (RI). (⫺) indicates negative for HCV RNA. bLiver fibrosis score: 0, no fibrosis; 1, minimal fibrosis; 2, moderate fibrosis; 3, bridging fibrosis; 4, cirrhosis.

called unequivocally positive by 3 independent and blinded readers in at least 2 out of 3 experiments to be considered positive for the purposes of the study.

HCV Quasispecies Analysis The HCV envelope 2-gene hypervariable region (HVR1) was amplified by RT-PCR, and amplification products were cloned into the pCR 2.1 vector. A series of clones derived from serum were screened to determine the quasispecies major variant, as described previously.17 The HVR1 sequence of the quasispecies major variant was end labeled with 32P and used as a probe that was hybridized to unlabeled heterogeneous HVR1 PCR products derived from serum, liver, lymph node, or PBMC specimens or to homogenous (ie, cloned) PCR products derived from the respective HVR1 amplicons. Hybrids were then analyzed by gel electrophoresis followed by autoradiography. After analysis of 112 clones by the clonal frequency analysis (CFA) technique, 33 unique variants were identified and directly sequenced using the Applied Biosystems model 373A automated sequencer. Nucleotide sequences were optimally aligned using the CLUSTAL X program (Accelrys Inc, San Diego, CA). Phylogenetic analysis was performed using programs for the PHYLIP package version 3.5c (available at packahttp://evolution. genetics.washington.edu/phylip.html).

Results Characterization of Positive and Negative Control Cell Lines for ISH and ICC Experiments Permanent cell lines expressing subgenomic regions of HCV sense and antisense RNAs were established from genotypes 1a and 1b clinical isolates for use as positive and negative controls in our strand-specific ISH assay. Figure 1A, 1C, 1E, and 1G illustrates positive staining of plus- and minus-strand HCV RNA expressed in HeLa cells using probes of opposite strand polarity and same genotype. Red punctate signals were evenly distributed throughout the cell cytoplasm, with occasional signal over nuclear regions (green) that likely reflects cytoplasmic signal because of folding or inclusion of cytoplasm above nuclei during sectioning. Figure 1B, 1D, 1F, and 1H demonstrates negative staining using the same probes applied to HeLa control cell lines lacking HCV RNA expression. The riboprobes were both strand specific and HCV genotype specific in both ISH and Northern dot blot experiments as previously reported.15 The HCV replicon system16,18 was used as a control for ICC experiments. Figure 2 demonstrates ICC staining of replicon HeLa cells for HCV core (struc-

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Figure 3. Detection of HCV genomes in follicular regions of human lymph node biopsy specimens by strand-specific ISH. Lymph node biopsy specimens were obtained at the time of liver transplantation, fixed and frozen in OCT buffer, and frozen sections were stained by ISH using antisense HCV riboprobes as in Figure 1, as described in the Materials and Methods section. A shows a low-power image of positive-staining HCV genomes in a lymph node follicle obtained from an HCV-infected patient (original magnification, ⫻10), and B shows detection of abundant HCV genomes (red punctate signals) throughout the cytoplasms of infected intrafollicular lymphocytes at high power (original magnification, ⫻100). Cell nuclei are counterstained blue. C shows negative ISH results using the antisense riboprobe and an uninfected lymph node specimen (original magnification, ⫻10). D illustrates negative results when staining a lymph node biopsy specimen obtained from an HCV-positive individual with an irrelevant riboprobe synthesized from the human MxA gene coding strand (original magnification, ⫻100). Note that neither the nucleus-associated MxA gene nor cytoplasmic MxA transcripts were recognized by the plus strand MxA riboprobe, confirming specificity of the ISH method for RNA of the opposite strand polarity. E and F illustrate ISH staining of human liver biopsy specimens from an HCV-infected patient (E) and an HCV-negative individual (F) using the antisense HCV riboprobes (original magnification, ⫻40).

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riboprobes in 17 out of 20 (85%) lymph node specimens from HCV-infected subjects (Table 1). Furthermore, 10 of 20 (50%) specimens were positive for HCV RI RNA when stained using sense riboprobes. These data provide the first documentation HCV infection and replication in perihepatic lymph nodes during natural infection. Figure 3 presents a representative ISH analysis of HCV genomic RNA in infected and uninfected lymph node specimens, using the antisense riboprobes described for Figure 1. Figure 3A shows detection of HCV genomes within a B-cell-rich lymphoid follicle at low power, whereas Figure 3B shows a higher magnification of the same image. The red punctate signals represent HCV genomic RNA. Figure 3C illustrates negative ISH results in an uninfected lymph node specimen using HCV antisense riboprobes, whereas Figure 3D illustrates negative ISH results in an infected lymph node specimen using an irrelevant non-HCV riboprobe, as detailed in the legend to Figure 3. Figure 3E and 3F illustrates positive and negative ISH results, respectively, in control liver biopsy specimens from an HCV-infected subject (Figure 3E) and an uninfected subject (Figure 3F). Figure 4 illustrates staining of HCV RI RNA in 2 lymph node specimens using the HCV sense riboprobes described in Figure 1. Significant levels of HCV replication are evident in the lymph node specimens upon inspection of the images captured in Figure 4A– 4C.

tural) and NS3 (nonstructural) antigens. The association of HCV proteins with cell cytoplasm is apparent in Figure 2A (brown signals are NS3 antigen) and Figure 2B (red signals are core antigen). A dual-label ICC experiment using replicon HeLa cells was performed by adding a mixture of anticore (red signal) and anti-NS3 antibodies (brown signal) and is shown in Figure 2C. ICC staining of Huh7 cells lacking the HCV replicon with the same antibody mixture showed negative results (Figure 2D). ISH Analysis of HCV Genomes and Replicative Intermediate RNA in Human Lymph Node Specimens After establishing reliable controls for ISH and ICC experiments, our next objective was to test for evidence of HCV genomes and RI RNA in a panel of 20 perihepatic lymph node biopsy specimens obtained from 20 consecutive HCV genotype 1–infected patients with end-stage hepatitis C (Table 1). HCV genomic RNA was detected within mononuclear cell cytoplasm by antisense

Figure 4. Detection of HCV replicative intermediate RNA in human lymph node specimens. In A–C, HCV sense riboprobes were used to detect the viral replicative intermediate RNA by strand-specific ISH. A and C are ⫻100 magnification; the image in B was photo enlarged. Replicative intermediate RNA were detected at relatively high levels in a lower percentage of cells than genomic RNA in most infected lymph nodes. D shows staining of a lymph node biopsy specimen obtained from an HCV-negative individual with the HCV sense riboprobes (original magnification, ⫻100). E illustrates abundant positive ISH staining of HCV replicative intermediate RNA in periportal inflammatory cells within a liver biopsy specimen from an HCV-infected individual, and F shows negative staining of a similar lesion in a liver biopsy specimen from an uninfected patient (original magnification, ⫻40).

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Figure 5. Identification of HCV core and NS3 antigens within lymph node follicles by immunocytochemistry (ICC). The lymph node specimens in A and B were immunostained with anticore antibodies, and the specimens in C and D were stained with anti-NS3 antibodies followed by detection with VectorRed (VR). Abundant positive signals were evident (red) at ⫻100 magnification in the lymph node specimen taken from an HCV-infected subject (A and C) but not the lymph node specimen from an uninfected control subject (B and D). E illustrates negative staining of an HCV-infected lymph node specimen using an isotype-matched antibody specific for the HIV nef protein. F illustrates positive identification of B cells using anti-CD20, a B-cell marker, and G illustrates dual-label ICC staining of an infected lymph node specimen with a mixture of anticore (brown signal) and anti-CD20 (red signal) antibodies. The results demonstrate HCV core localization in the CD20-positive cells. All images are ⫻100 magnifications, except for F (⫻40).

Figure 4D illustrates negative staining of an uninfected lymph node specimen with the same HCV sense riboprobes. Finally, Figure 4E shows intense staining of HCV RI RNA in periportal inflammatory cells within a liver biopsy specimen taken from an HCV-infected individual, whereas negative ISH results were universally observed in similar lesions when using liver specimens obtained from uninfected patients (Figure 4F). In our ISH study of the perihepatic lymph nodes of HCV-infected patients, viral RNA were most frequently observed within the intrafollicular B-cell-rich regions of lymph node biopsy specimens, although we also observed HCV genomes and RI RNA in nonfollicular T-cell-rich regions in some cases. In such cases, positive cells usually resembled T lymphocytes morphologically; however, positive staining was also observed in cells with elongated nuclear morphology, suggesting that cell types other than lymphocytes may harbor replicating HCV in lymph node tissue. ICC Staining of HCV Core and NS3 Antigens Based on the abundance of HCV RNA in some infected lymph node specimens, we expected to be able to detect HCV antigens in situ using ICC methods. Figure 5A and 5C demonstrates positive staining for HCV core (Figure 5A) and NS3 (Figure 5C) antigens in the follicular region of an infected lymph node specimen that had strong signals for both HCV genomes and RI RNA. The NS3-staining pattern was typically more diffuse and yielded a much finer signal density than the staining pattern for HCV core antigen. As expected, the majority of lymphocytes in the follicles were B cells

(Figure 5F), with accumulation of T cells in the lymph node mantle and perifollicular areas (not shown). Negative staining of uninfected lymph node tissue with anticore and anti-NS3 antibodies is shown in Figure 5B and 5D, whereas negative control staining of an HCVpositive lymph node specimen using an isotype-matched antibody specific for the HIV nef protein is shown in Figure 5E. Dual-label ICC experiments gave equivocal results, which limited our ability to resolve fully the phenotypes of infected cells in this study. Figure 5G shows dual-ICC staining for HCV core and CD20, a B-cell marker, in an infected lymph node follicular region. Core signal is indicated by brown pigment, and CD20 signal is indicated by red. Distinction of the 2 colors in this experiment was extremely difficult; significant quenching of the CD20 signal is evident when comparing Figure 5F and 5G. Cells in the perifollicular region that lacked B-cell markers also stained positive for HCV core (not shown). Taken together, the ICC and morphologic data implicate B cells as the primary site of HCV replication in perihepatic lymph nodes. However, there is also morphologic evidence that T cells and perhaps other cell types also support HCV replication in lymph nodes, a question that remains to be resolved. Characterization of HCV Quasispecies Variant Populations in Liver, Perihepatic Lymph Nodes, and PBMCs HCV replicates in humans as a genetically diverse population of viral genomes referred to as a quasispecies.19 We therefore explored the possibility that

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Figure 6. Analysis of HCV quasispecies in serum, liver, lymph node, and PBMC compartments. (A) Autoradiograms showing the complex nature of HCV hypervariable regions amplified and cloned from serum, liver, PBMC, and lymph node tissue compartments in 1 case. Quasispecies variants were analyzed by the clonal frequency analysis technique described in the Materials and Methods section. Each unique band profile represents a unique quasispecies variant. (B) Bar graph summarizing the frequencies of the 5 predominant quasispecies variants in the 4 tissue compartments. Note that, whereas variants A and E were recovered from all 4 compartments, variant B was only recovered from lymph node (31% of clones) and serum (22% of clones) compartments, and variant C was only recovered from PBMCs (2% of clones) and serum (14% of clones). (C) Phylogenetic tree of 33 unique quasispecies variants isolated from the 4 compartments. Colored ovals indicate variants isolated from hematopoietic compartments; boxes with letters identify the 5 predominant quasispecies variants identified in the patient. See text for details.

lymph nodes and/or PBMCs may harbor unique quasispecies compared with liver in a single case with a complex serum quasispecies profile. HVR sequences were amplified from liver, lymph node, PBMC, and serum specimens obtained at the time of liver transplantation by RT-PCR, and individual HVR clones were generated and analyzed by clonal frequency analysis (CFA) and nucleotide sequencing as described in the Materials and Methods section. By screening 112 HVR clones from the 4 tissue compartments (Figure 6A), we identified 5 predominant quasispecies variants infecting this patient. These 5 quasispecies variants, designated A, B, C, D, and E, were each present in 1 or more tissue compartment and collectively represented 67% of quasispecies variants circulating in the patient’s serum at the time of tissue harvest. Figure 6B summarizes the distribution frequency of the 5 predominant variants in the serum, liver, lymph node, and PBMC compartments at the time of tissue harvest. Variant A was found in all 4 reservoirs we tested, representing 38% of clones recovered from liver, 27% of clones from lymph node, 22% of clones from PBMCs, and 22% of clones from serum. Likewise, variants D and E were both detected in serum (each represented 4% of clones), liver (11% and 3% of clones, respectively), and the lymph node reservoir (8% and 3% of clones, respec-

tively). Variant E (but not D) was also detected in the PBMC-associated quasispecies (7% of clones). In summary, variants A, D, and E were each found in serum and liver and at least 1 hematopoietic compartment. In contrast to the above findings, variants B and C were both identified in hematopoietic and serum compartments, but neither were recovered from liver. Variant B was the highest frequency variant in the lymph node– associated quasispecies (31% of clones) and was present in the serum in equal proportion to variant A (22% of clones), but no clones of variant B were identified in either liver or PBMC compartments after analysis of 30 clones per compartment. Variant C was also found at relatively high frequency in the serum (14% of clones), and at low frequency in the PBMC compartment (2% of clones), but was not detected in the liver or lymph node–associated quasispecies. Overall, 68% of clones isolated from serum were identified in lymphoid reservoirs, and only 40% of serum clones were detected in liver. Finally, 21% of clones in serum were not identified in any of the 3 tissue reservoirs studied. Our next experiment analyzed the genetic relatedness of 33 different quasispecies variants identified during this individual case study. Figure 6C shows a phylogram of 33 unique hypervariable region sequences isolated from the 3 tissue compartments and serum in this case.

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The green, blue, and purple ovals identify variants isolated only in hematopoietic compartments. Three genetically distinct quasispecies clusters were evident. The upper cluster in Figure 6C contained variants isolated from each of the 4 tissue compartments, including predominant variant D, which was isolated from serum, liver, and lymph node compartments (Figure 6B), and several variants that were only identified in hematopoietic compartments. The middle cluster also contained quasispecies variants isolated from each of the 4 compartments, including predominant variants A, C, and E. Again, several variants in this cluster were only isolated from hematopoietic compartments. Within this cluster, the phylogenetic evidence indicates close genetic relatedness between predominant variants A and E, which is remarkable because these were the only variants found in all 4 compartments. Furthermore, variant C, found only in the PBMC and serum compartments, was closely related to variant E. The third quasispecies cluster (bottom branches in Figure 6C) consisted only of 2 variants found in hematopoietic compartments; neither of these variants was found in liver. This interesting cluster included predominant variant B (22% of serum clones and 31% of lymph node clones), and a fairly divergent minor variant detected in both lymph node tissue and PBMCs, but not in either serum or liver. In summary, the 2 largest quasispecies clusters contained variants isolated from each of the 3 tissue compartments and serum, perhaps indicating broader tropism of variants constituting these clusters. In contrast, the cumulative evidence presented herein argues against hepatotropic potential for the variant B cluster and provides strong indirect evidence that a major component of the serum-associated quasispecies population in this patient was a product of extrahepatic HCV replication.

Discussion The question of whether or not HCV is capable of replication in hematopoietic tissues remains controversial, despite a large number of published studies on the topic. Hematopoietic reservoirs of HCV infection could potentially play an important role in viral persistence through mechanisms such as immune escape and viral modulation of host immune responses. From a clinical perspective, hematopoietic infection may help explain the diverse clinical biology of chronic hepatitis C, including the existence of multiple extrahepatic disease syndromes, the association with non-Hodgkin’s lymphoma, resistance to antiviral therapy, relapse after successful therapy, and recurrent infection after liver trans-

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plantation. The major contribution of the present study is the novel finding that HCV commonly infects and replicates in perihepatic lymph nodes in vivo, documenting a new viral reservoir during natural infection in man. In the 1 case we analyzed, 68% of quasispecies genomes circulating in the serum were identified in lymph node and/or PBMC compartments. Surprisingly, only 40% of serum clones were detected in liver, which implies in this case a remarkable contribution of hematopoietic replication to HCV viremia. This finding was not expected because the phenomenon of orphan quasispecies has not been previously recognized, even though several studies of HCV quasispecies in liver and PBMC compartments have already been reported in the literature. Although it is important not to generalize based on the observations described in the present report until more cases are studied with a similar degree of rigor, the potential implications of these data for kinetic modeling of HCV replication dynamics should be noted. All models to date have been based on the assumption that productive HCV replication occurs in extrahepatic reservoirs at an insignificant level compared with liver.20,21 However, because minor modifications of assumptions can lead to drastic changes in conclusions drawn from mathematical models, the full implications of the present study will not be appreciated until similar studies have been completed on larger numbers of patients. The current results suggest a new model for extension of HCV infection in hepatitis C. Theoretically, B cells and/or other lymphocytes or monocytes may become infected while circulating in blood or perhaps during passage through liver and then establish local infections within lymph nodes. Another possibility is that HCV infection may spread locally through the lymphatics to perihepatic lymph nodes, at which time peripheral immune cells might become productively infected prior to recirculation. In the 1 case in which time both perihepatic and distal lymph node biopsy specimens were available in our study, both sites were infected, although levels of HCV RI RNA were not detectable in the distal node, even though high replication levels were evident in lymph nodes, proximal to the liver. It is possible that critical factors draining from liver into perihepatic lymph nodes facilitate HCV replication only in the more proximal tissues. Given the evidence that HCV replication is usually not detected in PBMCs,9 –11,13 we propose a model that circulating cells contain primarily quiescent HCV genomes, some of which represent lymph node– derived quasispecies variants. Two implications of our phylogenetic analyses are (1) that some HCV quasispecies populations are capable of replicating in either liver or hematopoietic tissues (ie,

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unrestricted tropism) and (2) that serum major variant B replicated preferentially in lymph nodes, at which it was the most predominant clone isolated, and either at reduced levels or not at all in liver because no HVR sequences were recovered from the latter reservoir. A previous report by Laskus et al10 identified common HCV sequences in liver and PBMC compartments and interpreted the data as an argument against extrahepatic HCV replication. However, unrestricted HCV replication in both liver and hematopoietic tissues was not ruled out by their results. Furthermore, a more common finding in the literature is evidence of unique quasispecies populations in PBMCs compared with liver,12,22,23 supporting the hypothesis of HCV compartmentalization in vivo. Finally, the elegant infectivity studies of Shimizu et al4 provided strong evidence of HCV hematotropism in both cell culture and the chimpanzee model. Based on previous studies and our present findings, the evidence supporting tissue compartmentalization of HCV quasispecies replication in vivo is overwhelming. However, further experimentation using the infectivity approach described by Shimizu et al4 and well-characterized clinical samples such as the sera described herein is required to take our understanding of HCV tropism to the next level. Our findings have significant clinical implications. Three previous studies have found that perihepatic lymphadenopathy is a common finding in patients with chronic hepatitis C.24 –26 Although it was originally assumed that perihepatic lymphadenopathy was a direct result of liver inflammation, our findings raise the possibility that viral infection may play a direct role in perihepatic lymph node hyperplasia, possibly contributing to HCV-associated non-Hodgkin’s lymphoma and other B-cell lymphoproliferative disorders,27–30 although this association is also controversial.31–33 A recent study by Sung et al7 provided a strong link between HCV infection and lymphoma by demonstrating productive HCV replication in patient-derived lymphoma cells in vitro. Finally, the highly significant association between hepatitis C and immune complex disorders may be a direct consequence of HCV infection of B cells in perihepatic lymph nodes, a question that deserves further study. Whether or not HCV infection of perihepatic lymph nodes results in altered immune responses in subjects with chronic hepatitis C is another important question to address. It is also possible that lymph node and/or PBMC infection is a barrier to successful treatment with interferon regimens. We and others have previously reported that HCV quasispecies show dramatic shifts during interferon monotherapy and interferon plus ribavirin com-

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bination therapy in nonresponders.34 –36 Thus, we postulate that hematopoietic tissues may harbor treatmentresistant quasispecies populations or that these tissues are inherently resistant to interferon, which would explain the dramatic shifts in HCV quasispecies observed during treatment of nonresponders. If confirmed, pharmacologic preparations targeting lymph node infection might be a reasonable approach to consider when designing strategies to treat hepatitis C in nonresponders to conventional therapy. In summary, the present study documents HCV replication in perihepatic lymph nodes as a common event in patients with progressive hepatitis C. Generalization of this finding to subjects with mild hepatitis C is obviously important. As discussed above, the present study adds important confirmatory evidence to the concept of hematopoietic HCV replication from both virologic and clinical perspectives, extending our understanding of the enormously complex pathobiology of chronic hepatitis C in humans.

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Received July 21, 2005. Accepted December 14, 2005. Address requests for reprints to: David R. Gretch, MD, PhD, Viral Hepatitis Laboratory, Room 706, UW Research and Training Building, Harborview Medical Center, Box 359690, 325 Ninth Avenue, Seattle, Washington 98104. e-mail: [email protected]; fax: (206) 3415203. Supported by National Institutes of Health grants 61-0311, 612154, and 62-2845.