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• W2038832956 abstract "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). 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). Hepatitis C virus (HCV) is a major cause of chronic liver disease in humans.1Seeff L.B. Miller R.N. Rabkin C.S. Buskell-Bales Z. Straley-Eason K.D. Smoak B.L. Johnson L.D. Lee S.R. Kaplan E.L. 45-year follow-up of hepatitis C virus infection in healthy young adults.Ann Intern Med. 2000; 132: 105-111Crossref PubMed Scopus (325) Google Scholar Primary HCV infection results in long-term persistence of viremia in approximately 80% of cases, which is associated with 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.2Manns M.P. Rambusch E.G. Autoimmunity and extrahepatic manifestations in hepatitis C virus infection.J Hepatol. 1999; 31: 39-42Abstract Full Text PDF PubMed Google Scholar 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 positive-strand 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.3Lindenbach B.D. Rice C.M. Molecular biology of flaviviruses.Adv Virus Res. 2003; 59: 23-61Crossref PubMed Scopus (591) Google Scholar 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.4Shimizu Y.K. Igarashi H. Kiyohara T. Shapiro M. Wong D.C. Purcell R.H. Yoshikura H. Infection of a chimpanzee with hepatitis C virus grown in cell culture.J Gen Virol. 1998; 79: 1383-1386Crossref PubMed Scopus (51) Google Scholar, 5Kato N. Nakazawa T. Mizutani T. Shimotohno K. Susceptibility of human T-lymphotropic virus type I infected cell line MT-2 to hepatitis C virus infection.Biochem Biophys Res Commun. 1995; 206: 863-869Crossref PubMed Scopus (113) Google Scholar, 6Morsica G. Tambussi G. Sitia G. Novati R. Lazzarin A. Lopalco L. Mukenge S. Replication of hepatitis C virus in B lymphocytes (CD19+).Blood. 1999; 94: 1138-1139PubMed Google Scholar, 7Sung V.M. Shimodaira S. Doughty A.L. Picchio G.R. Can H. Yen T.S. Lindsay K.L. Levine A.M. Lai M.M. Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro the apoptotic effects of virus infection.J Virol. 2003; 77: 2134-2146Crossref PubMed Scopus (231) Google Scholar 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.8Laskus T. Radkowski M. Jablonska J. Kibler K. Wilkinson J. Adair D. Rakela J. Human immunodeficiency virus facilitates infection/replication of hepatitis C virus in native human macrophages.Blood. 2004; 103: 3854-3859Crossref PubMed Scopus (78) Google Scholar, 9Lanford R.E. Chavez D. Chisari F.V. Sureau C. Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rTth reverse transcriptase PCR.J Virol. 1995; 69: 8079-8083PubMed Google Scholar, 10Laskus T. Radkowski M. Wang L.F. Cianciara J. Vargas H. Rakela J. Hepatitis C virus negative strand RNA is not detected in peripheral blood mononuclear cells and viral sequences are identical to those in serum a case against extrahepatic replication.J Gen Virol. 1997; 78: 2747-2750Crossref PubMed Scopus (74) Google Scholar, 11Mellor J. Haydon G. Blair C. Livingstone W. Simmonds P. Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells.J Gen Virol. 1998; 79: 705-714Crossref PubMed Scopus (104) Google Scholar, 12Lerat H. Rumin S. Habersetzer F. Berby F. Trabaud M.A. Trepo C. Inchauspe G. In vivo tropism of hepatitis C virus genomic sequences in hematopoietic cells influence of viral load, viral genotype, and cell phenotype.Blood. 1998; 91: 3841-3849Crossref PubMed Google Scholar, 13Boisvert J. He X.S. Cheung R. Keeffe E.B. Wright T. Greenberg H.B. Quantitative analysis of hepatitis C virus in peripheral blood and liver replication detected only in liver.J Infect Dis. 2001; 184: 827-835Crossref PubMed Scopus (73) Google Scholar 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 well-accepted “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.14Brahic M. Haase A.T. Detection of viral sequences of low reiteration frequency by in situ hybridization.Proc Natl Acad Sci U S A. 1978; 75: 6125-6129Crossref PubMed Scopus (345) Google Scholar, 15Chang M. Marquardt A.P. Wood B.L. Williams O. Cotler S.J. Taylor S.L. Carithers Jr, R.L. Gretch D.R. In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.J Virol. 2000; 74: 944-955Crossref PubMed Scopus (55) Google Scholar 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. 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 clones and riboprobes for in situ (ISH) hybridization experiments has been described in detail previously.15Chang M. Marquardt A.P. Wood B.L. Williams O. Cotler S.J. Taylor S.L. Carithers Jr, R.L. Gretch D.R. In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.J Virol. 2000; 74: 944-955Crossref PubMed Scopus (55) Google Scholar 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)-labeled 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. 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). The strand-specific ISH assay has been described in detail previously.15Chang M. Marquardt A.P. Wood B.L. Williams O. Cotler S.J. Taylor S.L. Carithers Jr, R.L. Gretch D.R. In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.J Virol. 2000; 74: 944-955Crossref PubMed Scopus (55) Google Scholar In brief, frozen sections (6 μm) 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 μg/mL) and soaked in equilibration solution followed by prehybridization solution (Novagen, Madison, WI) at 50°C for 1 hour. Approximately 15 μL Dig-labeled riboprobes were applied to each slide at a final concentration of 2 to 4 ng/μL 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 β-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 μg/mL in 2X SSC) at 37°C for 30 minutes to reduce nonspecific background. Subsequently, sections were washed in 50% formamide and SSC at 50°C to 65°C for 30 minutes. The wash temperature was optimized by titration for each probe. 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. Snap frozen lymph node sections were fixed in 10% neutral-buffered formalin and subjected to immunohistochemistry. 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 cells16Blight K.J. McKeating J.A. Marcotrigiano J. Rice C.M. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture.J Virol. 2003; 77: 3181-3190Crossref PubMed Scopus (289) Google Scholar (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. 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 Figure 1, Figure 4. A specimen had to be 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.Figure 4Detection 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.17Wilson J.J. Polyak S.J. Day T.D. Gretch D.R. Characterization of simple and complex hepatitis C virus quasispecies by heteroduplex gel shift analysis correlation with nucleotide sequencing.J Gen Virol. 1995; 76: 1763-1771Crossref PubMed Scopus (65) Google Scholar 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). 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.15Chang M. Marquardt A.P. Wood B.L. Williams O. Cotler S.J. Taylor S.L. Carithers Jr, R.L. Gretch D.R. In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.J Virol. 2000; 74: 944-955Crossref PubMed Scopus (55) Google Scholar The HCV replicon system16Blight K.J. McKeating J.A. Marcotrigiano J. Rice C.M. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture.J Virol. 2003; 77: 3181-3190Crossref PubMed Scopus (289) Google Scholar, 18Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.Science. 1999; 285: 110-113Crossref PubMed Scopus (2474) Google Scholar was used as a control for ICC experiments. Figure 2 demonstrates ICC staining of replicon HeLa cells for HCV core (structural) 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). 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 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.Table 1Results of In Situ Hybridization Analysis of Pretransplantation Lymph Node Specimens and Posttransplantation Histologic Course of Chronic Hepatitis CPatient No.HCV genotypeISH resultsaIn situ hybridization (ISH) assay results for HCV genomes (G) and replicative intermediate RNA (RI). (−) indicates negative for HCV RNA.Posttransplantation months of F/UHistologic diagnosisbLiver fibrosis score: 0, no fibrosis; 1, minimal fibrosis; 2, moderate fibrosis; 3, bridging fibrosis; 4, cirrhosis.GRI11a−−6021a−−6031a−−24041a+−6151a+−6061a+−12471a++17081b+−20491b+−261101b+−302111a++NDND121a++NDND131a++NDND141a++60151a++62161b++121171a++121181a++132191a++184201a++242No. positive/total (% positive)17/20 (85)10/20 (50)F/U, follow-up; ND, No data.a In situ hybridization (ISH) assay results for HCV genomes (G) and replicative intermediateRNA (RI). (−) indicates negative for HCV RNA.b Liver fibrosis score: 0, no fibrosis; 1, minimal fibrosis; 2, moderate fibrosis;3, bridging fibrosis; 4, cirrhosis. Open table in a new tab F/U, follow-up; ND, No data. 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. 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. 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 HCV-positive 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. HCV replicates in humans as a genetically diverse population of viral genomes referred to as a quasispecies.19Weiner A.J. Geysen H.M. Christopherson C. Hall J.E. Mason T.J. Saracco G. Bonino F. Crawford K. Marion C.D. Crawford K.A. et al.Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants potential role in chronic HCV infections.Proc Natl Acad Sci U S A. 1992; 89: 3468-3472Crossref PubMed Scopus (651) Google Scholar We therefore explored the possibility that 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 represe" @default.
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• W2038832956 date "2006-04-01" @default.
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• W2038832956 title "Productive Replication of Hepatitis C Virus in Perihepatic Lymph Nodes In Vivo: Implications of HCV Lymphotropism" @default.
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• W2038832956 doi "https://doi.org/10.1053/j.gastro.2005.12.039" @default.
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