VIRAL HEPATITIS

Hepatitis C Virus Persisting After Clinically ApparentSustained Virological Response to Antiviral Therapy

Retains Infectivity In VitroSonya A. MacParland,1 Tram N. Q. Pham,1 Clifford S. Guy,1 and Tomasz I. Michalak1,2

Hepatitis C virus (HCV) can persist in the liver, lymphoid cells, and serum of individualswith apparently complete spontaneous or therapy-induced resolution of hepatitis C and canreplicate in vivo and in vitro in human T cells. The current study was aimed at assessing theinfectivity of HCV persisting at very low levels using the previously established HCV infec-tion system in human T cells. Naive lymphoid cells were exposed to plasma and/or super-natants from cultured peripheral blood mononuclear cells from nine individuals withapparent sustained virological response after completion of antiviral therapy. Exposed cellswere analyzed for HCV RNA–positive and HCV RNA–negative strands and, in selectedcases, for HCV nonstructural protein 5a (NS5a), the appearance of HCV variants, and therelease of virions by immunoelectron microscopy (IEM). The results showed that 11 of the12 established cultures became HCV RNA–positive strand–reactive, whereas 4 also ex-pressed the virus replicative strand. NS5a protein was detected in the de novo infected cells,and clonal sequencing revealed HCV variants not found in inocula. IEM demonstratedenveloped HCV particles in plasma used as inocula and in culture supernatant from T cellsexposed to that plasma. Overall, HCV carried in three of the nine individuals studied elicitedproductive infection in vitro. Conclusion: HCV persisting at very low levels long aftertherapy-induced resolution of chronic hepatitis C can remain infectious. The retained bio-logical competence of the virus might have implications with respect to the mechanisms of itspersistence and the epidemiology of HCV infection. (HEPATOLOGY 2009;49:1431-1441.)

Hepatitis C virus (HCV) is a single-strandedRNA virus that chronically infects approxi-mately 170 million people worldwide. Up to85% of the infected individuals may develop chronic hep-atitis C (CHC). HCV is infectious even in trace amounts,with approximately 20 virus copies capable of transmit-

ting infection in chimpanzees.1 Recently, the introduc-tion of nucleic acid amplification assays detecting HCVgenomes with enhanced sensitivity, which has reached inour laboratory �10 virus genomes or virus genome equiv-alents (vge)/mL or �2 IU/mL, has revealed that HCV canpersist at low levels in individuals with apparently com-

Abbreviations: 5�-UTR, 5�-untranslated region; CHC, chronic hepatitis C; F, female; FACS, fluorescence activated cell sorting; HCV, hepatitis C virus; HCV sRNA,synthetic hepatitis C virus RNA; IEM, immunoelectron microscopy; IFN�, interferon alpha; IL-2, interleukin-2; IVDU, intravenous drug use; M, male; mAb, monoclonalantibody; NAH, nucleic acid hybridization; ND, not detectable; NS5a, nonstructural protein 5a; P-IFN, pegylated interferon alpha; PBMC, peripheral blood mononuclearcell; PCR, polymerase chain reaction; PHA, phytohemagglutinin; R, ribavirin; rHCV UTR-E2, recombinant hepatitis C virus 5�-untranslated region E2 fragment;RT-PCR, reverse-transcription polymerase chain reaction; SVR, sustained virological response; vge, virus genome equivalents.

From the 1Molecular Virology and Hepatology Research Group, Division of Biomedical Sciences, and 2Discipline of Laboratory Medicine, Faculty of Medicine, HealthSciences Centre, Memorial University, St. John’s, Newfoundland, Canada.

Received May 3, 2008; accepted December 7, 2008.This work was supported by operating grant MOP-77544 from the Canadian Institutes of Health Research (to Tomasz I. Michalak). Sonya A. MacParland is supported

by doctoral fellowship awards from the Canadian Liver Foundation and the National Canadian Research Training Program in Hepatitis C, Tram N. Q. Pham is arecipient of a postdoctoral fellowship award from the Canadian Association for the Study of the Liver/Hoffmann-La Roche/Astellas Pharma Canada, and Clifford S. Guywas supported in part by a doctoral fellowship from the Canadian Liver Foundation. Tomasz I. Michalak holds the Canada Research Chair (Tier 1) in ViralHepatitis/Immunology, which is sponsored by the Canada Research Chair Program and funds from the Canadian Institutes for Health Research and the CanadaFoundation for Innovation.

Address reprint requests to: Tomasz I. Michalak, M.D., Ph.D., Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Centre,Memorial University, St. John’s, Newfoundland, Canada A1B 3V6. E-mail: timich@mun.ca; fax: 709-777-8279.

Copyright © 2009 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.22802Potential conflict of interest: Nothing to report.

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plete resolution of hepatitis C occurring either spontane-ously or because of antiviral therapy.2-7 In general, occultHCV infection is considered when small quantities ofHCV RNA are identifiable in the serum (usually below100 vge/mL), peripheral blood mononuclear cells (PB-MCs), and/or liver of individuals who are repeatedly se-rum HCV RNA–nonreactive by clinical laboratory testswith sensitivities ranging between 52 and 1000 vge or 10and 615 IU/mL and have no clinical or biochemical evi-dence of liver disease.2,3,5-7 In this silent form of HCVinfection, the detection of HCV RNA replicative (nega-tive) strand is not uncommon, particularly when ex vivoactivated PBMCs are tested by sensitive HCV RNA–neg-ative strand–specific nested reverse-transcription poly-merase chain reaction (RT-PCR) combined with nucleicacid hybridization (NAH) analysis of the resulting ampli-cons.2,8

Although originally thought to be strictly hepato-tropic, HCV has been shown in numerous studies to alsoinvade and replicate in immune cells.9-11 In our recentwork, conclusive evidence of HCV replication in CD4�and CD8� T lymphocytes, B cells, and monocytes waspresented.7 It has also been shown that the same immunecell subsets can be infected in both CHC and persistentlow-level HCV infection continuing after resolution ofCHC.7 Furthermore, primary T lymphocytes fromhealthy individuals have been found to be susceptible toHCV infection in vitro.9 Along this line, mathematicalmodeling has independently predicted that HCV origi-nating from extrahepatic reservoirs, possibly the immunesystem, constitutes approximately 3% of the circulatingvirus pool in CHC.12 However, an analysis of HCV qua-sispecies occurring in the liver, plasma, PBMCs, and lym-phoid tissue of a patient waiting for a liver transplantdemonstrated that extrahepatic variants may constitutemore than 50% of those occurring in serum.10

The clinical relevance of low-level HCV carriage, in-cluding its potential ability to transmit infection, is yet tobe determined. Nonetheless, HCV reactivation has beenreported in patients with HCV RNA clearance confirmedby standard clinical laboratory assays prior to liver trans-plantation.13,14 However, the opposite has also been de-scribed, although no details have been given regardingassay sensitivity and the quantity of the template ana-lyzed.15 Also, HCV RNA has been identified in anti-HCV–reactive patients receiving HCV-negative bonemarrow16 or an HCV-negative kidney.17 Taken together,these findings suggest the possibility that occult HCVinfection could have both pathogenic and epidemiologi-cal importance.

At present, sustained virological response (SVR) is de-fined as serum HCV RNA negativity by clinical labora-

tory assays for at least 6 months after completion ofantiviral therapy. However, given that the identificationof low-level (occult) HCV infection is made possible onlyby the use of research tests with a sensitivity much greaterthan that of those applied for clinical use, it is not surpris-ing that low levels of HCV RNA are frequently escapingdetection, giving conflicting results on the occurrence andinfectivity of HCV persisting at trace levels. Implementa-tion of assays detecting HCV RNA with a greater sensi-tivity (preferably �10 vge/mL) for clinical andpopulation-based testing should meaningfully contributeto the identification of the scope of potential problemsassociated with low-level HCV infection.18

An HCV cell culture system allowing for authenticpropagation of wild-type HCV in primary human T cellshas previously been established in this laboratory.9 In thecurrent study, this system was employed to assess the po-tential infectivity of HCV persisting at trace quantities foryears in patients who achieved SVR after completion ofinterferon alpha (IFN�) therapy with or without ribavi-rin. Our investigation has focused on randomly selectedcases that, although repeatedly serum-negative by theclinical test, were found by RT-PCR/NAH to be positivefor HCV RNA in sera and in PBMCs after their ex vivostimulation.2,8 We have discovered that the residual viruscarried by some of the individuals has the capacity to denovo infect and propagate in T cells, and this stronglysuggests that the virus, persisting as an occult infection,can retain its biological competence and thus be poten-tially infectious.

Patients and Methods

HCV Inocula and Cell Targets. Nine patients whoachieved SVR after completion of IFN� or IFN�/ribavi-rin therapy, as defined by repeated serum HCV RNAnegativity by the Roche Amplicor HCV version 2.0 assay(sensitivity, 500 IU/mL or 1000 vge/mL; Roche Molec-ular Diagnostics, Pleasanton, CA) and normal liver func-tion tests assessed at 6- to 12-month intervals, wereinvestigated in this study (Table 1). All patients were anti-HCV antibody–positive by enzyme immunoassay (Ab-bott Diagnostics, Mississauga, Canada). The follow-upperiod after SVR ranged from 24 to 72 months. All nineindividuals were found to carry HCV RNA at the time ofthis study when total RNA isolated from 500 �L of serumwas assayed by highly sensitive RT-PCR/NAH (sensitiv-ity of �10 vge/mL or �2 IU/mL) that was previouslyestablished.2 The estimated HCV RNA loads in the pa-tients’ sera ranged from �40 to 400 vge/mL, with theexception of 6/F (59/F), who carried as much as 1.6 � 103

vge/mL (Table 1). Also, although PBMCs collected from

1432 MACPARLAND ET AL. HEPATOLOGY, May 2009

the patients were seemingly HCV RNA–nonreactive bythe same highly sensitive assay, a 72-hour culture of thePBMCs with phytohemagglutinin (PHA; 5 �g/mL;Sigma, Oakville, Canada) and recombinant interleukin-2(IL-2; 20 IU/mL; Roche)2 enabled detection of the virusgenome in seven individuals at estimated levels between�10 and 300 vge/�g of total RNA (Table 1). Further-more, HCV RNA–negative strand in PBMCs was de-tected in five cases (Table 1).

Plasma from eight patients was used for in vitro infec-tion experiments (see Table 1). In four cases (Table 1),supernatant from PBMCs cultured in the presence ofPHA and IL-2 for 72 hours served as an inoculum for invitro infection. Infectivity of both plasma and PBMC su-pernatants was examined in three cases (Table 1).

Lymphoid cells serving as in vitro HCV targets wereisolated from healthy donors who had no clinical historyor molecular indication of HCV exposure, as confirmedby RT-PCR/NAH assay2 and the absence of anti-HCVantibody by enzyme immunoassay (Abbott).

HCV Infection. Monocyte-depleted lymphoid cellsfrom a healthy donor were treated with 5 �g/mL PHA for48 hours.9 Following stimulation, 7 � 106 lymphoid cellswere exposed to 500 �L of test plasma in 6.5 mL ofculture medium9 or 7 mL of supernatant from in vivo

infected PBMCs, which were cultured as already indi-cated and described in detail previously.9 In parallel, thesame number of target cells was exposed to 250 �L ofplasma from a patient with CHC carrying HCV genotype1b at 7.3 � 105 vge/mL as a positive control and to 500�L of plasma from a healthy donor as a negative control(mock infection). Inocula were removed after 24 hours,and the cells were washed and cultured under alternatingstimulation with PHA and IL-2 (phases A-D) for 14 days,as reported.9 It was previously established that after 14days in culture, approximately 98% of the cells were Tcells.9 Culture supernatants were collected at 1, 4 (phaseA), 7 (phase B), 11 (phase C), and 14 (phase D) dayspost-infection and stored at �80°C, whereas cells recov-ered at 14 days post-infection (phase D) were cryopre-served for analysis.

Modification of HCV Infectivity by Anti-HCV E2,Anti-CD81, and IFN-� Treatments. Neutralization ofHCV was carried out by the incubation in duplicate of250 �L of 48/F plasma with an anti-HCV E2 monoclo-nal antibody (mAb; AP33; provided by Dr. A. Patel, In-stitute of Virology, University of Glasgow, Glasgow,United Kingdom) for 1 hour at 37°C and then for 1 hourat 4°C prior to the addition of T cell targets. Infection wasalso inhibited by pre-incubation of T cell targets with

Table 1. Clinical and Virological Characteristics of Individuals with Occult HCV Infection

CaseAge/Sex

Route ofInfection

AntiviralTreatment

(Weeks)HCV

Genotype

Follow-UpPeriod

After SVR(Months)

HCV RNA

Serum Load(vge/mL)*

PBMCs‡

HCV RNA–PositiveStrand (vge/�g)

HCV RNA–NegativeStrand

1/M‡ 48/M Unknown IFN (24) 4 72 �40 300 �

2/M§ 46/M IVDUIFN/R(24) 1a 60 400 50 ND

3/M‡ 43/M IVDUIFN/R(48) 2a 60 40 �50 ND

4/Fठ43/F IVDUIFN/R(48) Unknown 60 40 ND n.a.

5/F‡§ 44/F InhalationP-IFN/R(48) 1a 42 100 100 �

6/F‡ 59/F UnknownP-IFN/R(48) 1b 36 1.6 � 103 ND n.a.

7/F‡ 48/F EndemicIFN/R(48) 1a 24 50 100 �

8/F‡ 33/F InhalationP-IFN/R(48) 1 24 100 �50 �

9/F‡§ 45/F IVDUP-IFN/R(48) 3a 18 �50 �10 �

Abbreviations: F, female; HCV, hepatitis C virus; IFN, interferon alpha; IL-2, interleukin-2; IVDU, intravenous drug use; M, male; n.a., not applicable; NAH, nucleicacid hybridization; ND, not detectable; P-IFN, pegylated interferon alpha; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; R, ribavirin; RT-PCR,reverse-transcription polymerase chain reaction; SVR, sustained virological response; vge, virus genome equivalents.

*The serum HCV RNA load was determined by real-time RT-PCR.†PBMCs were stimulated with 5 �g/mL PHA and 20 IU/mL IL-2, the HCV RNA–positive strand was measured by nested RT-PCR/NAH, and the RNA-negative strand

was measured by strand-specific RT-PCR/NAH as described in the Patients and Methods section.‡Plasma was used as the HCV inoculum for the in vitro infection.§The supernatant from PBMCs after a 72-hour culture with PHA and IL-2 was used as the HCV inoculum for the in vitro infection.

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anti-CD81 mAb (Pharmingen, San Diego, CA) beforeexposure to 44/F or 48/F plasma, as described previous-ly.9 Appropriate isotype-matched mAbs were used in con-trol experiments. To further reaffirm that active HCVreplication was established in T cells, the cells were treatedin duplicate with 1000 U/mL recombinant human IFN�2b (Research Diagnostics, Flanders, NJ) at the time ofHCV inoculation, as reported.9 The cells exposed to thesame amount of 44/F or 48/F plasma, but not treatedwith IFN�, served as positive controls.

Ultracentrifugation. To concentrate the virus and torecognize its general biophysical properties, plasma (5mL) or pooled T cell culture supernatants (10 mL) col-lected after phases C and D, which were preclarified at400g for 30 minutes in the presence of a protease inhibitorcocktail (1:200; Sigma), were layered onto 1-mL 30%sucrose cushions and centrifuged at 28,000g for 2.5 hoursat 4°C in a TH641 rotor with a Sorvall Discovery 100SEultracentrifuge (Mandel Scientific Co., Inc., Guelph,Canada). On the basis of the findings from precedingexperiments (data not shown), 8.6 mL was removed fromthe top of each tube, and the remaining 2.4 mL was col-lected in 300-�L fractions (n � 8) for the evaluation ofthe HCV RNA content and sucrose density. In someinstances, two 10-mL samples of pooled T cell culturesupernatant collected after phases A to D of the sameinfection experiment were concentrated as indicated pre-viously, and the resulting equivalent fractions were pooledand used for analysis.

RNA Extraction and RT-PCR/NAH Assays. TotalRNA was extracted with Trizol (Invitrogen Life Technol-ogies, Burlington, Canada) from �1 � 107 cells (yielding�10 �g of RNA) or from 150 �L of the 300-�L sucrosefractions. RNA was reversely transcribed with Moloneymurine leukemia virus reverse transcriptase (Invitrogen).HCV RNA–positive and HCV RNA–negative strandswere detected with complementary DNA derived from 1to 2 or 2 to 4 �g of total RNA, respectively, and primers,amplification conditions, and controls were exactly as re-ported in our previous studies.2,7,8 A water sample and amock extraction were always included as contaminationcontrols. Complementary DNA prepared from the mockinfection served as an additional RT-PCR–negative con-trol. Recombinant hepatitis C virus 5�-untranslated re-gion E2 fragment (rHCV UTR-E2) served as a positivecontrol.2 The specificity of the detection and validity ofcontrols were routinely confirmed by NAH (i.e., South-ern blot hybridization) with 32P-labeled rHCV UTR-E2as a probe.2 The sensitivity of the RT-PCR assay for HCVRNA–positive strand detection was �10 vge/mL (�2IU/mL) or 5 vge/�g of total RNA, whereas that for HCVRNA–negative strand detection was 25 to 50 vge/�g of

total RNA.2 As a rule, HCV RNA–negative strand wastested only in RNA samples from T cells that had beenfound reactive for HCV RNA–positive strand.

Clonal Sequencing. The nucleotide sequences of 5�-untranslated region (5�-UTR) HCV amplicons detectedin cultured T cells exposed to 44/F or 48/F plasma werecompared to those amplified from the respective plasmaand from PBMCs isolated from the patients who pro-vided those plasma samples. The amplicons were clonedwith the TOPO-TA cloning system (Invitrogen). Tenclones for each polymerase chain reaction (PCR) productwere sequenced in both directions with M13 primers andthe ABI-Prism 7000 Sequence Detection System (Ap-plied Biosystems, Streetsville, Canada). The resulting se-quences were aligned with the help of Sequenchersoftware version 4.7 (Gene Codes Corp., Ann Arbor,MI).7

Confocal Microscopy and Flow Cytometry. To de-tect HCV nonstructural protein 5a (NS5a) in in vitroinfected T cells and to estimate the number of positivecells, confocal immunofluorescent microscopy and fluo-rescence activated cell sorting (FACS) were applied. Forconfocal microscopy, infected cells were fixed with 4%paraformaldehyde, permeabilized with 0.5% TritonX-100, blocked with 10% normal goat serum, and dou-ble-stained with rat anti-tubulin (Chemicon Interna-tional, Temecula, CA) and with either mouse anti-HCVNS5a mAb (Chemicon) or mouse isotype control.7 Then,cells were incubated with Cy2-labeled donkey anti-mouseand Cy5-labeled donkey anti-rat antibodies (both fromJackson ImmunoResearch Laboratories, Inc., WestGrove, PA). Cultured HCV-naı̈ve T cells, Huh7 cells,and Huh7 cells carrying HCV AB12-A2FL replicon (pro-vided by Dr. C. Richardson and Dr. J. Wilson, OntarioCancer Institute, Toronto, Canada), stained as previouslydescribed, were used as controls. Cells were examined in aFluoView FV300 confocal system (Olympus America,Inc., Melville, NY). Approximately 1000 cells per prepa-ration were examined, and NS5a-positive cells werecounted. For FACS analysis, cells were fixed with 4%paraformaldehyde, permeabilized with 0.5% saponin,and double-stained with anti-NS5a mAb and anti-tubu-lin and then with Cy2-labeled and Cy5-labeled secondaryantibodies. Cells were examined by flow cytometry with aFACSCalibur cytometer (BD Biosciences Pharmingen,San Jose, CA), and the results were analyzed withCellQuest Pro software (BD Biosciences).

Immunoelectron Microscopy (IEM). To determinewhether complete HCV virions can circulate in individ-uals with clinically apparent SVR and be secreted by denovo infected T cell cultures, 500 �L of unfractionated48/F plasma and culture supernatant from T cells exposed

1434 MACPARLAND ET AL. HEPATOLOGY, May 2009

to that plasma was incubated with anti-E2 AP33 mAb asreported.9 In addition, HCV RNA–positive fractions 4and 7 (shown later in Fig. 5A), obtained after centrifuga-tion of 44/F plasma over sucrose, were similarly incubatedwith anti-E2 mAb. Reacting particles were detected withanti-mouse IgG conjugated with 12-nm gold particles(Jackson ImmunoResearch) and counterstained with 1%phosphotungstic acid. Examinations were carried out in aJEM 1200 EX microscope (JEOL, Ltd., Tokyo, Japan).

Results

HCV Genome Expression in T Cells Exposed toPlasma from Individuals with Clinically ApparentSVR. With the previously established system allowingfor de novo infection and propagation of wild-type HCVin vitro,9 the infectivity of residual HCV occurring in theplasma of individuals with clinically apparent SVR wastested. Prestimulated lymphoid cells exposed to plasmafrom eight individuals and then cultured under alternatestimulation became reactive for HCV RNA–positivestrand in seven of the cases (Fig. 1A). HCV RNA–nega-tive strand, indicative of active virus replication, was evi-dent in three of the seven (42.8%) cell cultures that werepositive for the virus-positive strand. The HCV loadswere estimated to be between 1 � 103 and 5 � 104 vge/107 cells.

HCV NS5a Protein in In Vitro Infected T Cells. Todetermine whether expression of HCV RNA in in vitro

infected T cells was accompanied by synthesis of viralprotein, cells exposed to 44/F and 48/F plasma were ex-amined for HCV NS5a protein by confocal microscopy.As illustrated in Fig. 2A, HCV NS5a occurred predomi-nantly as granular intracytoplasmic deposits and at theplasma membrane of the positive cells. The percentages ofNS5a-reactive cells enumerated under a confocal micro-scope were between 0.78% and 1.35%. A flow cytometricanalysis gave comparable results of 1.05% to 1.52% ofHCV NS5a protein–positive cells (Fig. 2B).

Inhibition of HCV Infection by Anti-E2, Anti-CD81, and IFN�. Pre-incubation of HCV present in48/F plasma with anti-HCV E2 mAb, but not with anisotype control, neutralized the virus infectivity, as evi-denced by the absence of HCV RNA–negative stranddetection in the cells exposed to the treated inoculum(Fig. 3A). Similarly, pre-incubation of T cells with anti-CD81 mAb, but not with an isotype control mAb,blocked HCV replication in experiments in which 44/For 48/F plasma was used as inoculum (Fig. 3A). Further-more, treatment with recombinant IFN� prevented es-tablishment of HCV replication in T cells, as shown inFig. 3B.

Unique HCV Variants in De Novo Infected TCells. To assess whether HCV replication in de novo in-fected T cells led to the appearance of variants distinctfrom those present in the plasma used for their inocula-tion, as observed in our previous study,9 5�-UTR ampli-cons from 44/F and 48/F plasma, PBMCs, and culturedT cells exposed to this plasma were cloned, bidirectionallysequenced, and compared. As shown in Fig. 4, clonalsequence analysis of the 147-bp fragment revealed a dele-tion at position 120 in all clones from PBMCs and ineight clones from de novo infected T cells and a C to Tchange at position 249 in all clones from PBMCs and inseven clones from T cells in comparison with the sequenceamplified from 44/F plasma used as an inoculum; thisindicated that unique HCV variants were present in thePBMCs and, importantly, that the same variants emergedin the T cells exposed to the plasma. On the other hand,sequencing of the cloned 5�-UTR amplicons derivedfrom 48/F plasma, PBMCs, and cultured naı̈ve T cellsexposed to 48/F plasma revealed random single nucleo-tide polymorphisms, but no lymphoid cell–specific vari-ants were identified.

Physically Distinct HCV RNA–Reactive ParticlesOccur in Plasma and Culture Supernatants from TCells Exposed to That Plasma. To gain insight into thegeneral biophysical properties of HCV RNA–reactiveparticles occurring in the plasma of individuals with SVRand those released into culture medium by T cells exposedto this plasma, samples of plasma and supernatants from

Fig. 1. Detection of HCV RNA–positive and HCV RNA–negative strandsin T cell cultures after exposure to plasma from patients followed for upto 60 months after apparently complete clinical clearance of HCV. (A)HCV RNA–positive strand and (B) HCV RNA–negative (replicative) strand.HCV sRNA–positive and HCV sRNA–negative strands were used to confirmthe specificity of the detections. Water instead of complementary DNAamplified in direct (D/W) and nested (N/W) reactions and a mockextraction (M) treated as test RNA were included as contaminationcontrols. Positive samples showed the expected 244-bp amplicons. *Ahybridization signal for 48/F with appropriate controls was overexposedto visualize its presence. Abbreviations: HCV, hepatitis C virus; HCV sRNA,synthetic hepatitis C virus RNA; rHCV UTR-E2, recombinant hepatitis Cvirus 5�-untranslated region E2 fragment.

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Figure 2.

1436 MACPARLAND ET AL. HEPATOLOGY, May 2009

the infected T cells were ultracentrifuged over sucrose,and the eight bottom fractions were collected. The anal-ysis of 44/F plasma showed that HCV RNA–reactive par-ticles occurred in fractions 1, 4, and 7, whichcorresponded to sucrose densities of 1.092, 1.064, and1.027 g/mL, respectively, with apparent HCV RNA peakreactivity in fraction 4 (Fig. 5A). In the culture superna-tant of T cells exposed to 44/F plasma, HCV RNA–posi-tive particles were found in fractions 5 to 8 at densitiesbetween 1.024 and 1.011 g/mL with the peak RNA pos-itivity in fraction 5 at a density of 1.024 g/mL (Fig. 5B).HCV RNA–reactive particles after centrifugation of 48/Fplasma banded in fraction 4 at a sucrose density of 1.047and in fractions 6 and 7 at densities of 1.018 to 1.013g/mL (Fig. 5C), whereas those occurring in the culturesupernatant of T cells exposed to this plasma were foundin fractions 5 to 8, having a sucrose density of 1.027 to1.019 g/mL, with the HCV RNA peak reactivity in frac-tion 5 at a density of 1.027 g/mL (Fig. 5D).

Infectivity of HCV Released by PBMCs After Clin-ically Apparent SVR. To assess the infectivity of HCVfound in in vivo infected PBMCs of individuals with clin-ically apparent SVR, PBMCs from four such patients fol-lowed for up to 5 years (Table 1) were stimulated withPHA and IL-2 for 72 hours, and the resulting superna-tants were used as inocula to infect T cells. The datarevealed that all cell cultures exposed to the PBMC super-natants acquired HCV RNA–positive strand reactivity,whereas HCV RNA–negative strand was detected in oneof the cultures (Fig. 6).

Ultrastructural Identification of HCV Particles inSVR Plasma and Culture Supernatants of De NovoInfected T Cells. HCV particles were visualized withanti-E2 mAb by IEM (Fig. 7). Figure 7A-C depicts HCV

virions detected in unfractionated (total) plasma obtainedfrom patient 48/F 24 months after SVR was achieved.Figure 7D-F shows HCV particles found in HCV RNA–reactive fractions 4 (panel D) and 7 (panels E and F) afterfractionation of 44/F plasma collected 42 months afterSVR. As shown in Fig. 7G-K, HCV virion particles, ei-ther singly or as aggregates, were also detected in culturesupernatants collected from T cells in vitro infected withvirus carried in 44/F plasma. Particle sizes ranged from 50to 75 nm in diameter.

DiscussionThese findings provide in vitro evidence that trace

quantities of HCV persisting in the circulation for a longtime after therapeutically induced resolution of CHC canremain infectious. The transmission of HCV infectionwas exemplified by the detection of HCV RNA–negative

Fig. 3. Effect of HCV neutralization with anti-E2 mAb, pre-incubation oftarget cells with anti-CD81 mAb, or their treatment with IFN� on thereplication of HCV in cultured T cells. (A) HCV contained in 48/F plasma waspretreated with anti-HCV E2 mAb in duplicate (ex 1 and ex 2) or an isotypeimmunoglobulin control mAb and then incubated with T cells, or T cell targetswere first incubated with anti-CD81 mAb or an appropriate isotype controland then exposed to 44/F plasma (ex 1) or 48/F plasma in duplicate (ex 1and ex 2). (B) T cell targets were exposed to 44/F or 48/F plasma in thepresence or absence (UT) of 1000 U/mL recombinant human IFN� 2b induplicate (ex 1 and ex 2). RNA was analyzed for HCV RNA–positive and HCVRNA–negative strands after 14 days of culture. Specificity and contaminationcontrols were as outlined in the legend to Fig. 1. Positive samples showedthe expected 244-bp amplicons. Abbreviations: HCV, hepatitis C virus; HCVsRNA, synthetic hepatitis C virus RNA; IFN�, interferon alpha; mAb, mono-clonal antibody; rHCV UTR-E2, recombinant hepatitis C virus 5�-untranslatedregion E2 fragment.

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Fig. 2. Expression of HCV NS5a protein in cultured T cells exposed to

plasma from individuals with SVR. (A) Cultured T cells after exposure to(A,B) 43/M plasma, (C-E) 48/F plasma, and (F) plasma from a patientwith chronic hepatitis C (positive control) were double-stained withanti-HCV NS5a and anti-tubulin mAbs and analyzed by confocal micros-copy. (G) Cultured T cells exposed to HCV and stained with an isotypecontrol antibody served as a negative control. (H) Huh7 cells transfectedwith HCV AB12-A2FL full-length HCV replicon served as an additionalpositive control. Images were captured at 60� magnification. (B) Flowcytometric quantification of HCV NS5a–positive T cells exposed to plasmafrom individuals with therapy-induced SVR. Prestimulated cells wereincubated with 44/F or 48/F plasma and cultured as described in thePatients and Methods section. T cells were stained with anti-NS5a mAbor isotype control antibody. Gates were set on the basis of isotypecontrols. Stimulated, uninfected T cells similarly stained with anti-NS5amAb served as a negative control. Huh7 cells transfected with HCVAB12-A2FL replicon were used as a positive control. Percentages indi-cate positive cells. Abbreviations: HCV, hepatitis C virus; mAb, monoclo-nal antibody; NS5A, nonstructural protein 5a; SVR, sustained virologicalresponse.

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strand and NS5a protein and the emergence of uniqueHCV variants in cultured T cells exposed to plasma fromindividuals with long-term follow-up after SVR. Further-more, HCV replication in T cells was prevented followingthe neutralization of virus with anti-E2 mAb, blockingwith anti-CD81 mAb, and treatment of the cells withrecombinant human IFN� 2b. In addition, HCV resid-ing in PBMCs after clinical resolution of infection wasalso found to be infectious. HCV virion particles specifi-cally recognized by anti-E2 mAb were uncovered inplasma of these individuals and in the supernatant derivedfrom de novo infected T cell cultures exposed to theplasma.

Similarly to CHC,7 circulating lymphomononuclear cellshave been found to be the sites of active HCV replication inlow-level infection continuing after resolution of hepatitis C,

although ex vivo activation of the cells is usually required touncover the virus presence.2,8 Among circulating immunecells, CD4� and CD8� T lymphocytes, B cells, and mono-cytes have been identified to be infected to varying degreeswith HCV, but with overall viral loads greater in CHC thanin occult infection.7 As with PBMCs from occult infection,ex vivo stimulation of T cells affinity-purified from patientswith low-level infection also significantly augments HCVreplication, allowing for more ready detection of the residingvirus, as a recent study showed.7

It was also previously uncovered that mitogen activa-tion of normal human T lymphocytes predisposes thecells to infection by wild-type HCV of different geno-types (MacParland et al., unpublished observations,2008).9 Productive replication of HCV in such treatedcells, after exposure to either plasma from patients with

Fig. 4. Nucleotide sequence alignment of clones derived from HCV 5�-UTR fragments amplified from 44/F and 48/F plasma, peripheral bloodmononuclear cells, and cultured T cells exposed to that plasma. The 5�-UTR amplicons derived from each sample were cloned, and 10 randomlyselected clones were sequenced bidirectionally. As a reference, the HCV genotype 1a sequence (GenBank accession number M67463) is shown onthe top line. Nucleotides in the sequences identical to those of the reference are shown as dots, deletions are shown as dashes, and differencesare shown as letters. Numbering of the nucleotides is according to the M67463 HCV genotype 1a sequence. Abbreviations: 5�-UTR, 5�-untranslatedregion; HCV, hepatitis C virus.

1438 MACPARLAND ET AL. HEPATOLOGY, May 2009

CHC or culture supernatants from serial passage of wild-type HCV in T cell– enriched cultures, was shown bymethods comparable to those used in the current study,that is, by detection of HCV RNA–negative strand, virusproteins (NS5a and E2), and HCV variants distinct fromthose occurring in respective inocula and by identificationof secreted complete virions, as evidenced by isopycnicbanding and ultrastructural examinations.9 In the presentwork, with the same HCV replication system and similarevaluation criteria, it became evident that the virus, oc-

curring at low levels in three of eight plasma samplescollected 2 to 5 years after SVR (cases 43/F, 44/F, and48/F; Table 1), was able to establish active HCV replica-tion in vitro. In addition, HCV derived from in vivo in-fected lymphoid cells obtained from one (case 44/F) offour patients induced de novo infection in the culturesystem. Taken together, these findings indicate that HCVcarried by three of the nine individuals investigated in thisstudy established infection in vitro, and this was con-firmed by at least one criterion of active HCV replication,

Fig. 5. Sedimentation velocity of HCV RNA–reactive particles in sucrose. Plasma samples from individuals 44/F and 48/F with clinically apparentSVR and supernatants from cultured T cells exposed to this plasma were separately layered onto 1-mL sucrose cushions and ultracentrifuged. Eight300-�L fractions collected from the bottom of each tube were assayed for HCV RNA–positive strands by reverse-transcription polymerase chainreaction/nucleic acid hybridization and for sucrose density. (A) 44/F plasma collected 3.5 years after SVR was achieved, (B) culture supernatantfrom T cells infected with HCV contained in 44/F plasma, (C) 48/F plasma collected 2 years after clinical SVR was achieved, and (D) culturesupernatant from T cells infected with HCV contained in 48/F plasma. On the left side of each panel, the HCV RNA level is expressed in relative densityunits given by hybridization signals shown on the blot under each panel. On the blots, contamination and specificity controls are marked as in thelegend to Fig. 1. Abbreviations: HCV, hepatitis C virus; rHCV UTR-E2, recombinant hepatitis C virus 5�-untranslated region E2 fragment; SVR, sustainedvirological response.

HEPATOLOGY, Vol. 49, No. 5, 2009 MACPARLAND ET AL. 1439

that is, the appearance of HCV RNA–negative strand.However, the infection initiated by the virus originatingfrom two (cases 44/F and 48/F) of these three convales-cent individuals was also confirmed by detection of viralprotein, secretion of HCV RNA–reactive particles physi-cally distinct from those occurring in inocula, and iden-tification of complete virions by IEM.

HCV RNA–reactive particles occurring in 44/F and48/F plasma and those secreted by T cells exposed to thatplasma displayed different sedimentation profiles after ul-tracentrifugation over sucrose, and this implied distinctbiophysical properties. This further supported the con-clusion that the released virus originated from the de novoinfection process. In general, although the plasma virions,as confirmed by IEM, predominantly banded at highersucrose densities (1.047-1.064 g/mL), those released fromin vitro infected T cells tended to sediment at densities notexceeding 1.027 g/mL. Because (1) HCV virions in theplasma of patients with CHC have been shown to haveheterogeneous densities, particularly when associatedwith immunoglobulins and lipids,19-21 (2) the majority ofplasma virions should or do originate from infected hepa-tocytes, and (3) the viral particles found in culture super-natants in the current study had low densities and wereexclusively produced by T cells, a possibility exists thatvirions assembled in hepatocytes and lymphoid cellscould be biophysically distinct because of association withdifferent host proteins and/or lipids, giving, as a result,different sedimentation profiles. Studies have yet to com-pare the biochemical properties of plasma virus in CHCand in persistent low-level HCV infection and of virionsproduced by lymphoid cells in CHC and occult infection.

The infectivity of HCV traces persisting during a nat-urally acquired occult HCV infection has not yet been

investigated. The present study, to our knowledge, is thefirst attempt in this regard. However, in early studies inchimpanzees, diluted plasma from a patient with acuteposttransfusion hepatitis containing approximately 10virions was capable of inducing infection, which was char-acterized by elevated serum alanine aminotransferase andliver inflammation.22 More recently, as few as 20 copies ofHCV RNA prepared by the dilution of serum obtainedduring the pre-acute phase of hepatitis C of an infectedchimpanzee has been demonstrated to cause HCV RNA–positive infection in the absence of alanine aminotrans-ferase elevation.1 However, because the sensitivity of thePCR assay used for the detection of serum HCV RNA inthe latter study appeared to be between 100 and 250copies,1 the possibility remains that lower doses of HCVmay also transmit infection in this model. Our present

Fig. 6. Detection of HCV RNA–positive and HCV RNA–negative strandsin cultured T cells exposed to culture supernatants derived from in vivoHCV-infected, ex vivo stimulated peripheral blood mononuclear cellsobtained from individuals with follow-up of up to 60 months after clinicalsustained virological response was achieved. Contamination and speci-ficity controls are marked as outlined in the legend to Fig. 1. Positivesamples show the expected 244-bp amplicons. Abbreviations: HCV,hepatitis C virus; HCV sRNA, synthetic hepatitis C virus RNA; rHCVUTR-E2, recombinant hepatitis C virus 5�-untranslated region E2 frag-ment.

Fig. 7. Ultrastructural features of HCV RNA–reactive particles in theplasma of individuals with clinical sustained virological response and inthe culture supernatant obtained from T cells exposed to one of theplasma samples as visualized by immunogold staining with anti-E2 mAb.(A-C) HCV virion particles in unfractionated plasma of patient 48/F. HCVvirions in (D) fraction 4 (sucrose density, 1.064 g/mL) and (E,F) fraction7 (sucrose density, 1.027 g/mL) of 44/F plasma. (G-K) HCV particles inthe supernatant of cultured T cells infected with HCV contained in 44/Fplasma. (L) The same culture supernatant pool shown in panels G to Kexposed to the isotype control instead of anti-E2 mAb. Preparations werecounterstained with 1% phosphotungstic acid. Bars indicate 100 nm.Abbreviations: HCV, hepatitis C virus; mAb, monoclonal antibody.

1440 MACPARLAND ET AL. HEPATOLOGY, May 2009

findings reveal that HCV circulating in some individualswith resolved hepatitis C is capable of inducing produc-tive infection in vitro at doses of 20 to 50 copies. This canbe interpreted as a strong indication of potential virusinfectivity in vivo. In future studies, it would be of interestto determine the molecular mechanisms explaining whyHCV circulating in some individuals, but not in others, isinfectious to T cells despite comparable levels of virusbeing present.

In summary, the current study provides the first exper-imental evidence that HCV RNA detectable at low quan-tities for years after apparently complete resolution ofCHC reflects the existence of traces of biologically com-petent virus that in some situations can retain infectivity.

Acknowledgment: The authors thank Dr. S. B.Reddy and D. King, a hepatology nurse specialist, fromthe Gastroenterology Clinic and Dr. J. S. McGrath fromthe General Hospital of the Health Science Centre (St.John’s, Canada) for providing clinical samples. They alsothank Dr. D. Richardson and J. Wilson from the OntarioCancer Institute (Toronto, Canada) for supplying theHCV AB12-A2FL replicon and Dr. A. Patel from theInstitute of Virology of the University of Glasgow (Glas-gow, United Kingdom) for AP33 mAb against the HCVE2 protein.

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