Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro

Sylvie Rumin1, Pascale Berthillon1, Eiji Tanaka2, Kendo Kiyosawa2, Mary-Anne Trabaud1, Thierry Bizollon3, Christian Gouillat3, Philippe Gripon4, Christiane Guguen-Guillouzo4, Geneviève Inchauspé1 and Christian Trépo 1

INSERM U271, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France1
Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan2
Service d'Hépato-Gastroent érologie and Département de Chirurgie, Hô tel Dieu, 1 place de l'Hôpital, 69288 Lyon Cedex 02, France3
INSERM U522, Hôpital Pontchaillou, 35033 Rennes Cedex, France4

Author for correspondence: Christian Tr épo.Fax +33 4 72 68 19 71. e-mail trepo{at}lyon151.inserm.fr


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Primary human hepatocytes were used to develop a culture model for in vitro propagation of hepatitis C virus (HCV). Production of positive- strand full-length viral RNA in cells and culture supernatants was monitored by PCR methods targeting three regions of the viral genome: the 5' NCR, the 3' X-tail and the envelope glycoprotein E2. De novo synthesis of negative-strand RNA was also demonstrated. Evidence for a gradual increase in viral components over a 3 month period was obtained by two quantitative assays: one for evaluation of genomic titre (quantitative PCR) and one for detection of the core antigen. Production of infectious viral particles was indicated by passage of infection to naive hepatocyte cultures. Reproducibility of the experiments was assessed using cultures from three liver donors and eleven sera. Neither the genotype, nor the genomic titre, nor the anti-HCV antibody content, were reliable predictive factors of serum infectivity, while the liver donor appeared to play a role in the establishment of HCV replication. Quasispecies present in hepatocyte cultures established from three different liver donors were analysed by sequencing hypervariable region 1 of the E2 protein. In all three cases, the complexity of viral quasispecies decreased after in vitro infection, but the major sequences recovered were different. These data strongly suggest that human primary hepatocytes are a valuable model for study of persistent and complete HCV replication in vitro and for identification of the factors (viral and/or cellular) associated with successful infection.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis C virus (HCV) was the first hepatotropic virus to be isolated by means of molecular biology techniques (Choo et al ., 1989 ). Viral genomic sequences were cloned before the viral particle itself could be visualized (Kaito et al., 1994 ). HCV is a single-stranded positive-sense RNA virus of about 9·5 kb in length, translated in a unique polyprotein precursor of approximately 3000 amino acids. Because of its genomic organization and its presumed mode of replication, HCV was classified in a new genus within the family Flaviviridae (Miller & Purcell, 1990 ). Since its discovery in 1989, considerable progress has been made in the identification of functional regions and interactions between viral products, as well as in the associated pathogenesis. However, the development of a efficient in vitro culture system is still awaited, in particular for study of the HCV replication cycle as well as for development of neutralization assays.

Attempts at establishing such a culture system have exploited the apparent double tropism of HCV for hepatocytes and lymphocytic cells. In vivo, evidence of HCV replication in cells from the immune system has been documented (Lerat et al., 1996 , 1998 ; Sansonno et al., 1996 ; Kao et al., 1997 ) but remains a matter of controversy (Lanford et al., 1995 ; Mihm et al., 1996 ; Laskus et al ., 1997 ). HCV is also able to replicate in vitro both in lymphoid cell lines and peripheral blood mononuclear cells but at very low levels and/or for short periods of time (Shimizu et al., 1992 ; Cribier et al., 1995 ; Kato et al., 1995 ; Mizutani et al ., 1996 ; Nakajima et al., 1996 ; Shimizu et al., 1996 ). In vitro propagation of HCV in liver cells has been at least as difficult to implement in hepatic cell lines as in primary hepatocyte cultures (Lanford et al., 1994 ; Kato et al., 1996 ; Ito et al., 1996 ; Iacovacci et al ., 1997 ; Seipp et al., 1997 ; Fournier et al., 1998 ). Data collected in these studies indicated (i) low levels of replication and selection of a particular quasispecies in lymphoid cell lines (Nakajima et al ., 1996 ), (ii) greater replication in hepatocytes (Lanford et al., 1994 ; Iacovacci et al., 1997 ) and (iii) maintenance of all quasispecies present in vivo in cultured hepatocytes (Ito et al., 1996 ).

Despite these attempts at culturing HCV in vitro, its life- cycle remains poorly understood. Technical difficulties, such as the susceptibility of its genome to RNase degradation, difficulty in establishing a sensitive and specific method for the detection of the putative replicative intermediate (the negative-strand RNA) and artefacts due to the use of very sensitive methods of analysis like PCR must be overcome.

In an attempt to delineate critical factors, viral and cellular, that may allow establishment of a successful propagation assay for HCV, we used the principal target of the virus, the adult human hepatocyte, in primary culture. The aim of the present work was: (i) to establish culture conditions that allow long-term HCV replication in these cells; (ii) to define the characteristics of infectious inocula and (iii) to analyse HCV quasispecies selection in long-term cultures established from different liver donors.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Patients.
Liver resection samples were obtained from three patients undergoing partial hepatectomy for metastatic liver tumour at our Center. They were negative for HIV, HBV and HCV infections by serological assays (ELISA HIV1 HIV2.3 from Abbott and HIV1-2-3 Vironostika assay, E1A assays from Abbott for detection of HBsAg, anti-HBc and anti-HBs antibodies, and detection of anti-HCV antibodies with the Abbott HCV 3.0 kit).Experimental procedures were performed in accordance with French Laws and Regulations, with the informed patient's consent.

{blacksquare} Primary hepatocyte cultures.
Cells were isolated by a two-step collagenase (Boehringer Mannheim) perfusion procedure as previously described (Guguen-Guillouzo, 1992 ); dead cells were removed on a 30% Percoll (Sigma) gradient. Freshly isolated hepatocytes were seeded in six-well dishes at a density of 1·5 to 2x105 viable cells/cm 2. This high density is essential to obtain long-term survival of primary hepatocytes (Rumin et al., 1996 ). Adhesion was performed overnight in William's E medium (with Glutamax I; Sigma) complemented with 100 UI/ml penicillin and streptomycin (Gibco), 5 g/l bovine insulin (Sigma) and 10% heat inactivated foetal calf serum (FCS) (USDA batches, Eurobio). After seeding, medium was replaced by William's E supplemented with antibiotics and insulin as described above, plus either 2% dimethyl sulfoxide (DMSO; Sigma), 2% normal human serum and 3·5x10 -7 M hydrocortisone hemisuccinate (Roussel, France), or 10% FCS and 3·5x10-5 M hydrocortisone hemisuccinate. Normal human serum was obtained from bleeds of non-infected patients suffering from haemochromatosis, with their informed consent. Hepatocytes do not grow in this medium, and therefore cultures were not passaged during the indicated culture period.

{blacksquare} In vitro infection with HCV.
Three days after seeding, cultures were incubated overnight at 37 °C with 50 µl of the indicated serum diluted in 1·5 ml of medium without normal human serum. Mock-infected cells were incubated with 50 µl of human non-infectious serum. The inoculum was removed and cell monolayers were washed three times with PBS and re-fed with the medium described above. In passage experiments, supernatants collected from original infection experiments (from days 19 to 21 post-infection) were clarified by spinning at 4000 g for 5 min at 4 °C in order to remove cell debris, and inoculated to naive cultures obtained from a different liver donor. Viral particles were not concentrated. Medium was changed every 2 to 3 days until harvest.

{blacksquare} Extraction of nucleic acids and cDNA synthesis.
At the time of cell harvest, the medium was collected and several aliquots were stored at -70 °C. Cells were washed in PBS, treated with 0·5 g/l trypsin–0·2 g/l EDTA (Gibco) in order to remove adsorbed virions, and pelleted at 4 °C. Paired cell pellets were stored at -80 °C until used. Intracellular total RNA as well as viral RNA contained in serum and supernatants were isolated using the High Pure RNA Isolation kit, according to the manufacturer's (Boehringer Mannheim) instructions. A DNase digestion of the sample is included in this procedure. Approximately 0·75x106 hepatocytes (half a dish) or 500 µl of supernatant (one-third) or 150 µl of serum were extracted and collected in a final volume of 50 µl of water. The integrity of intracellular RNA and homogeneity were checked by submitting 5 µl of total cellular RNA to electrophoresis on 1% agarose gel containing ethidium bromide.

During all the extraction steps and RT–PCR amplification, the guidelines of Kwok & Higuchi (1989) for the prevention of PCR contamination were carefully followed. Standard procedure was performed as follows: 10 µl of RNA (corresponding to approximately 1 µg of total RNA or 100 µl of supernatant) was preheated at 70 °C with 0·1 µM of primer and reverse transcription was performed for 1 h at 42 °C in a reaction mixture containing RT-buffer 1x (Gibco BRL), 10 mM dithiothreitol, 1 mM dNTP, 20 U RNasin (Promega) and 200 U MMLV RT (Gibco BRL). For 3' X-tail detection, cDNA synthesis was performed at 52 °C for 50 min in a reaction mixture containing 40 nmol dithiothreitol, 10 nmol dNTP, 20 pmol of lower primer RLO, 16 U RNasin, 80 U Superscript II and RT-buffer 1x (Gibco BRL), in a final volume of 20 µl, as described by Umlauft et al. (1996) . Sensitivity and specificity of detection of minus-strand RNA was evaluated using synthetic RNA, encompassing nt 37–901, 45–884, 37–900, 37–904 for genotype 1b-, 2a/c-, 3a- and 4-derived sequences, respectively. Detailed procedures for synthetic RNA synthesis are described in Lerat et al. (1998) . The effect of the formation of duplexes between positive- and negative- strand HCV RNA on RT–PCR sensitivity was evaluated by mixing equimolar concentrations of positive- and negative-strand synthetic RNA of genotype 1b. Synthetic RNA was mixed with 0·1 µM of primer and either water or desionized formamide, at a final concentration of 33%. The mixture was heated at 70  °C for 10 min or at 95 °C for 5 min, as indicated in the legend to Fig. 2. RT–PCR was then performed as described above. When used, the final concentration of formamide was 4·4% during the RT reaction.



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Fig. 2. Effect of duplex formation between positive- and negative-strand HCV RNA on the sensitivity of detection of synthetic negative-strand RNA. Synthetic HCV RNA encompassing the near full- length 5' NCR and CAP sequences for genotype 1b sequences were synthesized as described in Methods and in Lerat et al. (1998) , and used in serial dilution in water. Assays included amplification of 10 to 106 copies per reaction. Ethidium bromide staining of PCR products obtained after single rounds of amplification and Southern blot hybridization of this gel are shown.

 
{blacksquare} PCR amplification of cDNA.
One-tenth of the cDNA obtained was submitted to PCR amplification, after denaturation at 97 °C for either 10 or 30 min for detection of positive- and negative-strand RNA, respectively. Briefly, the reaction mixture consisted of 1xTaq buffer, 1·5 mM MgCl2, 0·1 mM dNTP, 1 µM of each primer (Table 1) and 1·25 U Taq DNA polymerase (Perkin Elmer). Two different techniques were used for amplification of the positive-strand RNA. PCR amplification of the 5' NCR was adapted from Li et al. (1995) , using single-round PCR. PCR conditions consisted of an initial step of 95 °C for 5 min, 40 cycles of 95 °C for 1 min, 64 °C for 1 min, 72 °C for 2 min, and a final extension step of 72 °C for 10 min. Amplification of the 3' X-tail was performed according to a single-step PCR method described by Umlauft et al. (1996) . Specificity of amplification products obtained by both techniques of detection for the positive-strand RNA was demonstrated using Southern blot hybridization with oligonucleotide internal probes (data not shown). Specific detection of the negative-strand RNA was performed using a method described by Lanford et al. (1994) . Two nucleotides were added to the 5' end of the primer initially described by Lanford et al. for enhanced amplification and specificity (see Table 1). After denaturation of reverse transcriptase and destruction of residual RNA in the cDNA preparation, PCR amplification was performed as follows: 95 °C for 5 min, 35 cycles of 94 °C for 1 min 20 s, 64 °C for 2 min, 72 °C for 3 min, and a final extension step at 72 °C for 10 min. Typically, one-quarter of the PCR product was analysed on agarose gel. Southern blot hybridization with a DIG-labelled probe internal to the PCR primers was performed by the Tag method according to the manufacturer's instructions (DIG-High prime DNA labelling and detection starter kit II, Boehringer).


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Table 1. Primers used in this study

 
Primers for amplification of hypervariable region 1 (HVR1) of the HCV genome were synthesized on the basis of the published genotype 1a sequence (Table 1) (Inchauspé et al ., 1991 ). Single-round PCR and DNA polymerases with high fidelity were used instead of Taq DNA polymerase in this experiment to avoid introduction of artefactual mutations (Smith et al., 1997 ). Two different enzymes with 3' exonuclease activities were compared for their efficacy of amplification and for their fidelity. Thirty-five cycles were performed at 94 °C, 30 s, 65 °C, 30 s, 68 °C, 1 min 30 s with ELONGase (Gibco) or at 94 °C, 1 min, 65 °C, 1 min, 72 °C, 2 min with Pfu (Stratagene), in both cases after initial denaturation of the sample at 95 °C. Sequences amplified from the serum by single-round PCR amplification with either Pfu or ELONGase DNA polymerases gave similar results (data not shown). Therefore, subsequent experiments were performed using ELONGase, because this mix of enzymes has a better efficacy than Pfu polymerase.

{blacksquare} Cloning of HVR1.
RT–PCR products from serum K03 or culture supernatants infected with this serum were purified with Wizard PCR Prep (Promega) and subcloned into the cloning vector PCR-Script Amp SK(+) (Stratagene). A minimum of 15 independent colonies was selected, and the cDNA clones were sequenced using an Applied Biosystems 373A sequencer. Sequence alignments were performed with Sequence Navigator software (Applied Biosystems).

{blacksquare} Virological assays.
The amount of HCV RNA was quantified by the Amplicor HCV Monitor test (Roche Diagnostic Systems), based on a simplified RNA extraction and a single, combined reverse transcription and amplification reaction, done with Thermus thermophilus DNA polymerase. Supernatants were processed directly. HCV RNA titres were also determined in some supernatants and sera by the Quantiplex HCV RNA 2.0 assay (bDNA, Chiron). Genotypes were determined by the Inno-Lipa HCV II test (Innogenetics), GB virus C co-infection by the LCx test (Roche), and anti-HCV antibodies [anti-capsid (C), envelope (E), non-structural proteins 3, 4 and 5 (NS3, NS4, and NS5)] by the Inno-Lia HCV ABIII test (Innogenetics). Anti-E1 and -E2 antibody titres were kindly determined by E. Depla (Innogenetics). Neutralization of binding (NOB) titres were kindly determined by D. Rosa (Rosa et al., 1996 ).

{blacksquare} Quantitative analysis of HCV core protein concentration.
The concentration of HCV core protein in cellular extracts and supernatants of the cell cultures was measured by sandwich fluorescent enzyme immunoassay (FEIA), as in the procedure described by Tanaka et al. (1995b ,1996 ), with modifications. Briefly, the FEIA is based on two high-affinity monoclonal antibodies directed against the HCV core protein and recognizing amino acids 21 to 40 and 41 to 60, which are relatively well-conserved across HCV genotypes. Cell pellets (corresponding to 0·75x106 hepatocytes) were resuspended in 100 µl PBS with 0·1% SDS and supernatants were processed directly, without a prior protein precipitation step. Fifty µl 10 M urea and 50 µl 0·5 M sodium hydroxide was added to 50 µl of sample. After incubation for 10 min at room temperature, 50 µl 0·5 M sodium dihydrogen phosphate with 5% Triton X-100 was added. The detection assay was then performed as reported in Tanaka et al . (1995b ). The concentration of HCV core protein was expressed in pg/ml using as standard the c11 recombinant HCV core protein. The limit of detection of this immunoassay is between 104 and 105 copies/ml HCV RNA equivalent in serum samples, and was estimated at 40 pg/ml for supernatants and 5·3 pg per 106 cells for cell extracts in the modified procedure used in this paper.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Selection of culture conditions for infection of human hepatocytes
DMSO and corticoids both favour the maintenance of hepatocyte- specific RNA transcription in primary cultures (Isom et al., 1985 ; Guguen-Guillouzo, 1986 ). Additional compounds such as growth factors and hormones are also required to maintain life-span and differentiation of these cells (Guguen- Guillouzo, 1992 ). They can be provided separately as supplements to serum-free medium (Lanford et al., 1989 ) or by addition of bovine, porcine or human serum to the culture medium (Rumin et al., 1996 ).

We determined which of two different culture media favoured infection of human hepatocytes by HCV. Serum K03 was used as the inoculum in these experiments. Fig. 1 shows detection of intra- and extracellular viral RNA in cultures grown in the presence of 2% DMSO, 3·5x10-7 M hydrocortisone and 2% human serum or 3·5x10-5 M hydrocortisone and 10% FCS. In cultures containing DMSO and human serum, positive-strand RNA was detected (by PCR with primers specific to the 5' NCR) from day 3 to day 26 post-infection in the cells and from day 5 to day 26 post-infection in the culture supernatant (Fig. 1a). At earlier time-points, starting 6 h post-infection, positive-strand RNA was already detectable in the infected cells (data not shown). However, negative-strand RNA could not be detected at these times, indicating that detection of positive- strand RNA might not result from early replication but rather from adsorption and/or partial penetration of the virus into the cells. Negative-strand RNA was present from day 3 to day 19 post-infection. In contrast to positive-strand RNA, the level of negative-strand RNA appeared to fluctuate when using this technique, showing three peaks of detection at days 5, 13 and 19 post-infection. Analysis of duplicate cultures gave similar results (data not shown). Addition of a nested- PCR to this reaction provided a continuous signal.



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Fig. 1. Kinetics of detection of positive- and negative- strand HCV RNA in cells and supernatants of primary hepatocytes cultured in two different media. Infections were performed as described in Methods using serum K03 (genotype 1a) and cultures established from liver donor A. Cultures were maintained either in the presence of 2% human serum, 2% DMSO and 3·5x10-7 M hydrocortisone (a) or in the presence of 10% FCS and 3·5x10-5 M hydrocortisone (b ). Staining of 28S RNA with ethidium bromide demonstrates the integrity of total cellular RNA after the extraction procedure. Ethidium bromide staining of single-round PCR products (269 bp) obtained by amplification of the 5' NCR are shown for the positive-strand HCV RNA. Agarose gel electrophoresis followed by Southern blot hybridization was done for the negative-strand according to the `Tagged PCR' method described by Lanford et al. (1994) (product size 392 bp). * Supernatants used for the passage of infection shown in Fig. 4 and for the quasispecies analysis documented in Fig. 6. Negative controls included PCR amplification from culture medium before infection (To), non-infected cells (NI) and water (H2O). RNA isolated from HCV-positive serum was used as a positive control for positive-strand RNA detection and as a negative control for negative-strand RNA detection.

 


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Fig. 4. Kinetics of positive- and negative-strand viral RNA in the cells and supernatants of hepatocytes infected with supernatants of cultures infected with serum K03. Cells were from liver donor B. 28S RNA and positive-strand as well as negative-strand RNA amplification products were obtained as in Fig. 1. * Supernatant used for quasispecies analysis.

 


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Fig. 6. HVR1 nucleotide sequences of HCV and numbers of clones recovered from serum K03 (inoculum) and supernatants of primary cultures of human hepatocytes from three different liver donors at various times post-infection. Cloning and sequencing were performed as described in Methods. Nucleotide sequences shown in (a) are numbered (1490 to 1583) according to strain H (Inchauspé et al., 1991 ). The corresponding amino acid sequences are shown in (b). Horizontal bars indicate nucleotides or amino acids identical to those of the consensus sequence. Nucleotide sequences have been deposited in the DDBJ/EMBL/GenBank DNA databases, accession numbers AF045896 to AF045924.

 
We evaluated the sensitivity of this technique by using synthetic RNA of different genotypes (synthetic RNA synthesis is described in detail in Lerat et al., 1998 ). To our surprise, the sensitivity of this assay was 103 copies of negative- strand RNA for genotypes 1b, 2 and 4, 104 for genotypes 1a and 105 for genotype 3. However, when performing the assay with equimolar mixtures of positive- and negative-strand synthetic RNA, one further log of sensitivity was lost (Fig. 2). We were unable to detect fewer than 104 copies of synthetic negative-strand RNA in the absence of cellular RNA, with the loss of an additional half a log in a cellular context (Lerat et al., 1996 ). We tried several methods to dissociate the putative hybrid RNAs (denaturation of the RNA preparation at 70 to 95 °C prior to reverse transcription, or addition of 33% formamide to the reaction) without any re-establishment of sensitivity (Fig. 2). If we consider that the ratio of positive-/negative-strand RNA is about 10:1 in an infected cell, the threshold of detection of negative-strand RNA might therefore be dramatically high. These data indicate that the transient detection of negative-strand RNA is probably due to inadequate sensitivity rather than disappearance of the putative replication intermediate.

Both the appearance of negative-strand RNA in the cells and secretion of positive-strand RNA into the supernatants of hepatocytes cultured in the presence of normal human serum and DMSO suggested that virus replication had occurred and was maintained throughout the duration of the experiment. In contrast, positive-strand RNA could not be maintained in cultures in the presence of hydrocortisone and FCS (loss of signal after day 3) and was never released into the culture supernatants, as indicated in Fig. 1(b). Negative-strand RNA detection was delayed, very transient and weak.

Various controls were included in these experiments to show that cross-contamination with cDNA or PCR products did not occur. Mock- infected cultures were analysed concomitantly and were always found negative for amplification products, demonstrating the absence of cross contamination during RT–PCR procedures. Moreover, PCR conditions used in our study were unable to detect viral sequences from other members of the Flaviviridae, such as bovine viral diarrhoea virus, which has been shown to contaminate most batches of FCS (Yanagi et al., 1996 ). Two different techniques were used to avoid artefactual detection of negative-strand RNA. Specificity of detection of the negative-strand RNA was confirmed with synthetic positive- and negative-strand RNA using the Tagged method (Lanford et al., 1994 ). Specificity of the RT–PCR for negative-strand detection was also confirmed using biological samples (serum and cellular extracts) with the following controls: omission of RNA (H2O), RT enzyme and specific primer in the RT reaction. Additional controls included the use of RNA extracted from non-infected cells and serum. As expected, negative-strand RNA was never detected in serum samples, including those with a very high virus titre. These controls were done systematically in each experiment (see Figs 1 to 5).



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Fig. 5. Kinetics of detection of positive- and negative- strand HCV RNA in cells and supernatants of primary hepatocyte cultures infected with serum S05 (genotype 1b) (a) and L02 (genotype 2a/2c) (b) in culture medium containing 2% human serum, 2% DMSO and 3·5x10-7 M hydrocortisone. Cultures were established from liver donor A. Viral positive- and negative-strand RNA were analysed by PCR as described in Fig. 1. Controls included PCR amplification performed on culture medium before infection (To), non-infected cells (NI), water (H2O) and serum from a chronic carrier of HCV.

 
Overall, these results indicated that HCV replication is strongly dependent on the medium used for in vitro infection. Culture medium containing 2% DMSO, 3·5x10-7  M hydrocortisone and 2% normal human serum was therefore used in subsequent experiments.

Maintenance of HCV replication in long-term cultures of infected hepatocytes
Under our culture conditions, hepatocytes can survive in a differentiated state for at least 4 months without morphological change, as assessed by phase-contrast microscopy (S. Rumin, unpublished data). Hepatocyte cultures infected with HCV (serum K03) were analysed for up to 3 months post-infection. Results are presented in Fig. 3 . In this experiment, positive-strand RNA was clearly detectable after day 8 and day 12 post-infection in the cells (Fig. 3a) and in the supernatants (Fig. 3b), respectively. Detection of negative-strand RNA also occurred later in this experiment than in previous ones (day 21 post-infection). Kinetics of RNA replication seemed therefore to vary from donor to donor. Quantitative analysis of the secreted viral RNA showed that the high titre observed at day 3 post-infection in Fig. 3(b) represented an initial release of viral sequences from the inoculum in all experiments performed. This carry-over never exceeded 3 days, consistent with the poor stability of viral RNA in culture media (Ito et al., 1996 ). At day 10 post-infection, viral RNA became clearly detectable by single-round PCR and genomic titres in the supernatants of infected cells gradually increased during the 3 months of culture, ranging from 955 at day 12 post-infection to about 60000 genomic equivalents/ml at day 90. Remarkably, an enhancement of approximately one log occurred after 2 months of culture.



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Fig. 3. Kinetics of HCV replication in long-term cultures of human hepatocytes maintained in the presence of 2% human serum, 2% DMSO and 3·5x10-7 M hydrocortisone. Time post-infection is given in days. Infections were performed as described in Methods on cultures established from liver donor C. Cellular extracts (a) and culture supernatants (b) were analysed. 28S RNA and positive-strand as well as negative-strand RNA amplification products were obtained as in Fig. 1. 3' X-tail amplification (92 bp product) was performed as described by Umlauft et al. (1996) . Viral titres obtained by the Monitor test (Roche) and core protein concentration are shown. Negative controls were RNA isolated from mock-infected cells and supernatants (NI). -, Core protein concentration below the threshold of detection; nd, these titres were not determined; * supernatants used for quasispecies analysis.

 
To further assess the presence of HCV RNA in the supernatants of long-term cultures of infected hepatocytes, we amplified the positive- strand HCV RNA using primers located in the extreme 3' NCR of the HCV genome: the highly conserved 98 nucleotide tail extending the HCV genome after the poly(U) stretch, referred to as the 3' X-tail (Tanaka et al., 1995a ; Kolykhalov et al., 1996 ). Detection of PCR products using this approach was weaker and less systematic, probably due to a lower sensitivity of this single-round PCR technique, as illustrated in Fig. 3(b). However, these data demonstrated the presence of the full-length HCV genome in the culture medium. This amplification product was scarcely detectable before 23 days in culture, and reached its highest level by the end of the culture period, ruling out a possible contamination by carry-over of infectious particles from the initial high-titre inoculum used.

We did not succeed in detecting HCV antigens in cultured cells by standard immunostaining procedures using monoclonal and polyclonal antibodies (data not shown). The production of capsid antigen in infected cells was, however, successfully demonstrated by the quantitative capture assay previously described (Tanaka et al., 1995b ) (Fig. 3a, b). We demonstrated, using a fluorescent enzyme immunoassay, that the amount of HCV core protein detected in a denatured serum sample correlated well with the level of HCV viraemia (Tanaka et al., 1996 ). Core protein could be detected both in the cellular extracts (the concentration ranged from 5·1 to 9·2 pg per 106 cells) and in supernatants of infected cells (40 pg/ml). HCV positive-strand RNA (5' NCR and 3' X-tail) as well as core protein were detected 3 days post-infection in the supernatants of infected cells, and were totally absent from cellular extracts, suggesting that, in this experiment, release of particles from infectious inoculum remained high during the first 3 days post- infection. It should be noted that the negative-strand RNA and core protein became detectable simultaneously in infected cells as late as 21 days post-infection, while 3' X-tail HCV RNA, core protein concentration and genomic titre increased in supernatants at day 23 post-infection, rising above the threshold of the techniques used for their detection.

Taken together, these data suggest that HCV replicates in long-term cultures of hepatocytes.

Passage of infection to naive hepatocytes
Detection of both viral RNA and capsid antigen in the supernatant of infected cells suggested the release of viral particles. To demonstrate that this was indeed the case, we tested the infectivity on naive cultures. Supernatants from cultures infected with serum K03 (liver donor A, Fig. 1a) were pooled from days 19 to 21 post- infection and incubated with cells obtained from liver donor B. Intra- and extracellular RNA were extracted at various times post-infection. Results are shown in Fig. 4.

Positive-strand RNA was detected in infected cells at days 5 and 12 post-infection, while negative-strand RNA could be detected only at day 12 post-infection. In this case, release of viral particles from the inoculum was barely detected. Newly synthesized viral RNA appeared around 10 days after infection. As a positive control, direct infection of cells from the same liver donor was performed with inoculum K03; this resulted in earlier replication (data not shown). These results indicate that infectious viral particles were secreted into the supernatants of in vitro-infected human hepatocytes.

Influence of infectious sera and liver cell donors on the susceptibility to infection
Three different sera (K03, S05 and L02, genotypes 1a, 1b and 2a/c respectively) were tested for their infectivity on hepatocyte cultures established from the livers of three donors (A, B and C). Results for liver donor A are illustrated in Fig. 1(a) (for serum K03) and Fig. 5(a, b) (for sera S05 and L02 respectively), while overall results are in Table 2. The kinetics of replication obtained with serum S05 were similar to those obtained with serum K03, (cf. Fig. 1a and Fig. 5a). In cells infected with serum S05, however, the level of positive-strand RNA seemed to decrease after day 17 post-infection, both intra- and extracellularly, while it remained high at least up to 26 days post-infection in cells infected with serum K03. It should also be noted that with this serum (S05 genomic titre 17·8 MEq/ml), no release of viral RNA from the inoculum was observed at day 3 post-infection. Results obtained for cells infected with serum L02 are shown in Fig. 5(b). In this case, HCV replication was not maintained after 8 days post-infection, positive- strand RNA was scarcely detectable at later times and negative-strand RNA was never detected. Overall, serum K03 was infectious for cells obtained from all three livers, serum S05 was infectious in 2/3 experiments, while replication could never be established from serum L02 (Table 2). The results, although preliminary, suggest that infectivity of a serum could be related to its genomic titre, and possibly inversely correlated with the anti-envelope antibody content. The liver donor appears to be an independent factor.


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Table 2. Characteristics of the sera used for inoculation

 
We therefore attempted to confirm characteristics of sera with in vitro infectious potential in our model. The analysis was extended to 11 sera, characterized for their genotype, genomic titre, anti-HCV antibodies and NOB titres (Table 2). An infection was considered successful when positive-strand RNA was found both in the culture supernatant and in the cells and when negative-strand RNA was detected in the cells.

Although additional experiments are required to give statistical significance, these results (Table 2) indicated that the virus titre of an inoculum is not a reliable predictive factor for in vitro infectivity. For instance, serum L02 had a particularly high virus titre (39·1 MEq/ml) but was not infectious in three independent assays. In contrast, cells infected with serum TP00114 (viral titre only 0·04 genomic equivalents per cell) gave a very strong PCR signal 12 days post-infection, while positive-strand RNA was not detected in the cells at day 1 post-infection (data not shown).

Infectivity was found across all genotypes tested: 1a (K03) and 1b (S05) as well as 2b (TP00114) and 3a (L10). In addition, our data suggest that low anti-E2 antibody-containing sera (in particular sera displaying low or no NOB antibodies) may be more infectious.

Determination of the quasispecies present in serum K03 and in infected cultures established from three different liver donors
Quasispecies present in inoculum K03 and in the supernatants of in vitro-infected cultures established from three different livers were analysed by cloning and sequencing the HCV HVR1. Quasispecies distribution was analysed at both the nucleotide and the amino acid level. Results are shown in Fig. 6(a) and 6(b), respectively. Twenty-four different nucleotide sequences (from 36 clones analysed) were found in serum K03. One nucleotide sequence (A.3) was dominant in this serum, since 11/36 clones were identical (30%), while the remaining ones were minor (representing 2·7 to 5·5% of the total clones obtained), differing from each other in 1 to 5 nucleotides. Interestingly, this major quasispecies detected in the inoculum (A.3) was only poorly represented in supernatants of infected cells and cultures derived from all three livers which preferentially supported replication of minor variants found in the inoculum (sequences B.3 for liver donor A, A.1 for donor C and C.13 for donor B). In all three cases, such variants represented less than 5·5% of circulating virions from serum K03. Major quasispecies recovered from the cultures from donors A and B were not found in the inoculum, indicating that they represented less than 2·7% of the circulating virions. When supernatant collected from donor A (21 days post-infection) was used to infect naive cells from donor B (passage), the major quasispecies recovered after passage of the infection (sequence C.13) was different from the one found in hepatocytes from donor A (sequence B.3), therefore representing less than 6·6% of clones analysed at day 21 in donor A. This could be one explanation for the observed delay in the establishment of virus replication after passage (Fig. 4). Overall, these results showed that different quasispecies were selected in cultures established from different livers, and that in all three cases reported here the quasispecies selected in the culture were minor components of the inoculum.

Since an increase in viral RNA secretion was observed after 2 months in culture (infection of hepatocyte culture from donor C; see Fig. 3), we wondered whether this was due to the adaptation of certain quasispecies to in vitro replication. To answer this question, we analysed the HVR1 sequence of viral RNA released into the supernatant of this long-term culture at days 65 and 90 post-infection, i.e. before and after the increase in virus titre. This analysis showed that at least three distinct quasispecies were maintained after 2 months of culture (sequences A.1, D.2 and D.48). In contrast, only one HVR1 sequence was detected at the end of this culture period (A.1). This could suggest that a selection of quasispecies adapted to this donor occurred before day 65 and amplification of this minor subgroup went on thereafter.

Most mutations (90%), mainly transitions, were silent, occurring in the third codon position. Thus the quasispecies distribution, when analysed at the amino acid level, indicated the existence of one dominant sequence, representing 78% of the population, and several minor forms (2·7% each) (Fig. 6b). Therefore, at the amino acid level, HVR1 sequences selected in cell cultures from donors A and C were identical, whatever the duration of the culture (sequence #1). In contrast, the predicted amino acid sequence encoded by the master sequence of the passaged sample (sequence #10) differed from the predominant amino acid sequence of the inoculum used for infection (sequence #1) in one amino acid (Gly->Ala). In that particular case, the HVR1 amino acid sequence could therefore have been involved in the selection of this variant in liver donor B, while in other cases HVR1 nucleotide heterogeneity only reflected the overall genetic heterogeneity of the variants, with possible important mutations occurring in other regions of the genome.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In recent years, knowledge of the function of viral genes and the physiopathology of HCV has progressed rapidly as a result of in vitro analysis of viral gene products in transfected cell lines and clinical studies on HCV carriers (for recent reviews see Clarke, 1997 ; Major & Feinstone, 1997 ). However, little is yet known about the replication cycle of HCV, its putative receptor and its relations with the permissive host cells, as well as the mechanisms involved in quasispecies evolution. Since experimental in vivo HCV infection is limited to the chimpanzee, development of an efficient in vitro culture model for HCV propagation is a high priority for our understanding of these essential biological characteristics of the virus.

In this study, demonstration of long-term infection of human hepatocytes with HCV was based on: (i) kinetic and quantitative analysis of the de novo production of positive-strand RNA, (ii) kinetic analysis of negative-strand RNA production, (iii) measurement of core antigen release into the supernatants of long-term cultures, (iv) passage of infection to naive cultures, and (v) analysis of quasispecies evolution in vitro. The level of HCV replication remained low despite the use of culture conditions adequate for the maintenance of highly differentiated hepatocytes. Dependence of HCV replication on culture conditions, and therefore on the hepatocyte phenotype, was suggested by Iacovacci et al. (1997) . Such dependence suggests that HCV replication is tightly regulated and linked to the presence of specific hepatic factors. Our study supports this notion since HCV replication was achieved only in the presence of DMSO and normal human serum, both favouring long-term maintenance of highly differentiated hepatocytes (Rumin et al ., 1996 ). It is important to note that in this system, negative-strand RNA is detected only intermittently. Intermittent detection of HCV genes has been reported in the chimpanzee and human model, during experimental and natural infection. Since we also observed fluctuations in the detection of intracellular core antigen during our cultures, we believe that HCV replication might have intrinsic cycles.

Distinct donor-cell susceptibility to virus infection was observed, some cultures being successfully infected by only one of the inocula tested. At the present time, we do not understand the biological reason for such variation. However, quasispecies analysis suggested that different virus quasispecies were selected by all three liver donors used for the preparation of adult hepatocytes in this study. Because there is no influence of immune pressure in our model, the data suggest that different virus variants can replicate in primary hepatocytes. It is important to note that the selected HVR1 amino acid sequence was identical in two cases, whereas nucleotide sequences differed. This could mean that this region may not be the only one involved in quasispecies selection. In vitro infection of adult human hepatocytes closely mimics the in vivo infection of HCV- negative allografts, for which several reports have shown a decreased complexity of virus quasispecies following liver transplantation (Martell et al., 1994 ; Laskus et al., 1996 ; Yun et al., 1997 ). Our results further support the idea that immune pressure is not necessarily involved in such selection. Selection of quasispecies in hepatocytes, both in vivo and in vitro, may result from the replication of a small subset of viruses due either to random sampling or to selection of only a few fit variants. Alternatively, selection might depend on unknown host factors. Direct competition between virus strains, resulting in interference preventing simultaneous continuous infection by closely related variants, could also be possible.

Previous published studies on the use of primary hepatocytes provided very little information about the characteristics of infectious inocula (Lanford et al., 1994 ; Iacovacci et al., 1997 ; Fournier et al., 1998 ). In this study, we performed extensive characterization of the sera used for in vitro infection. We found that no relevant characteristics of the sera analysed (genotype, genomic titre, anti-envelope E1 and E2 antibody titre and NOB titre) were reliable predictive factors of serum infectivity, when considered separately. Interestingly however, we obtained reproducibility of infection of cultures with a genotype 1 serum of very high genomic titre containing no detectable anti-envelope proteins and no neutralizing antibodies in the NOB assay.

Although the reproducibility of our system has yet to be established, we believe that it provides an important tool to progress understanding of the biology of HCV and to help to optimize a productive in vitro culture model. This model may be particularly well- suited for studying new antiviral agents, because (i) the level of replication permits easy detection of viral genomic sequences by single- step PCR methods or with commercial quantitative kits, (ii) replication persists sufficiently long for evaluation of potential resistance, (iii) hepatocytes are the site for metabolism of xenobiotics, and therefore of antiviral compounds, and (iv) the use of normal hepatocytes of human origin will allow assessment of the cytotoxicity and pharmacological properties of the drugs under test.


   Acknowledgments
 
This work was supported by a grant from the Association Française pour l'Etude du Foie (AFEF) to S. Rumin and by Schering-Plough Research Institute.

We thank O. Yanagihara for core protein measurements, Drs Abrignani and Rosa for the determination of NOB titres and Dr E. Depla for the measurement of anti-E1 and -E2 antibody titres. We are indebted to Dr A. Kay for help in establishing PCR protocols, to Dr D. B. Smith for helpful discussion on the genetic analysis of quasispecies and to F. Berby for technical assistance. We also thank Dr F. Bailly and S. Radenne for providing us with some sera and for clinical information about patients, and L. Garnier for reviewing the manuscript.


   Footnotes
 
Nucleotide sequences have been deposited in the DDBJ/EMBL/GenBank DNA databases, accession nos AF045896AF045924.


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Received 8 April 1999; accepted 21 July 1999.