Robust Hepatitis C Genotype 3a Cell Culture Releasing Adapted Intergenotypic 3a/2a (S52/JFH1) Viruses
2007; Elsevier BV; Volume: 133; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.08.005
ISSN1528-0012
AutoresJudith M. Gottwein, Troels K. H. Scheel, Anne Mette Hoegh, Jacob B. Lademann, Jesper Eugen‐Olsen, Gorm Lisby, Jens Bukh,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoBackground & Aims: Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide. Methods: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies. Results: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids. Conclusions: We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics. Background & Aims: Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide. Methods: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies. Results: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids. Conclusions: We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics. About 180 million people are infected with hepatitis C virus (HCV) worldwide, and are at increased risk of developing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Thus, HCV infection is a major contributor to end-stage liver disease and in developed countries to liver transplantation. Research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as for testing of new therapeutics and vaccines. In 2005, such a system was developed based on transfection of Huh7 cells with RNA transcripts from a complementary DNA (cDNA) clone of the genotype 2a isolate JFH1.1Wakita T. Pietschmann T. Kato T. et al.Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2460) Google Scholar, 2Zhong J. Gastaminza P. Cheng G. et al.Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci U S A. 2005; 102: 9294-9299Crossref PubMed Scopus (1537) Google Scholar At the same time, an intragenotypic 2a/2a recombinant genome (J6/JFH) was shown to produce infectious HCV in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic.3Lindenbach B.D. Evans M.J. Syder A.J. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1988) Google Scholar Cell-culture–derived J6/JFH viruses were apparently fully viable in vivo.4Lindenbach B.D. Meuleman P. Ploss A. et al.Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.Proc Natl Acad Sci U S A. 2006; 103: 3805-3809Crossref PubMed Scopus (389) Google Scholar It is important to develop cell culture systems for representative strains of all 6 major HCV genotypes because neutralizing antibodies are not expected to cross-neutralize all genotypes5Meunier J.C. Engle R.E. Faulk K. et al.Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1.Proc Natl Acad Sci U S A. 2005; 102: 4560-4565Crossref PubMed Scopus (229) Google Scholar and new specific antiviral compounds might have differential efficiencies against different genotypes.6De Francesco R. Migliaccio G. Challenges and successes in developing new therapies for hepatitis C.Nature. 2005; 436: 953-960Crossref PubMed Scopus (410) Google Scholar For the genotype-specific study of the function of the structural proteins, p7 and NS2, as well as related therapeutics such as neutralizing antibodies,5Meunier J.C. Engle R.E. Faulk K. et al.Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1.Proc Natl Acad Sci U S A. 2005; 102: 4560-4565Crossref PubMed Scopus (229) Google Scholar fusion inhibitors, ion-channel blockers,7Patargias G. Zitzmann N. Dwek R. et al.Protein-protein interactions: modeling the hepatitis C virus ion channel p7.J Med Chem. 2006; 49: 648-655Crossref PubMed Scopus (93) Google Scholar and protease inhibitors, it would be sufficient to construct intergenotypic recombinant viruses in analogy to J6/JFH. Recently, JFH1-based intergenotypic recombinants of genotype 1a and 1b with similar growth characteristics as seen in the genotype 2a reference systems were reported.8Yi M. Ma Y. Yates J. et al.Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus.J Virol. 2007; 81: 629-638Crossref PubMed Scopus (252) Google Scholar, 9Pietschmann T. Kaul A. Koutsoudakis G. et al.Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.Proc Natl Acad Sci U S A. 2006; 103: 7408-7413Crossref PubMed Scopus (626) Google Scholar Genotype 3a has a high prevalence worldwide, infecting up to 50% of patients in several European countries as well as a high percentage of HCV-infected individuals in many highly populated countries in Asia (eg, India) and the former Union of Soviet Socialist Republics. In HCV-infected patients, genotype 3 was associated with more pronounced hepatic steatosis10Rubbia-Brandt L. Quadri R. Abid K. et al.Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3.J Hepatol. 2000; 33: 106-115Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar and a relatively high sensitivity to combination therapy with interferon and ribavirin compared with other genotypes. In this study, we constructed a viable, JFH1-based genome of the genotype 3a reference isolate S52.11Bukh J. Purcell R.H. Miller R.H. At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.Proc Natl Acad Sci U S A. 1993; 90: 8234-8238Crossref PubMed Scopus (400) Google Scholar After passage in Huh7.5 cells we obtained HCV RNA and infectivity titers as high as those observed in the J6/JFH culture system, and identified adaptive mutations required for efficient growth. We generated several adapted genetically stable genomes suitable for efficient and sustainable growth of S52/JFH1 in Huh7.5 cells. Finally, we proved the applicability of the developed 3a/JFH1 culture system by studying the role of CD81 in HCV genotype 3a entry and the localization and amount of lipid droplets in HCV-positive cells. Strain S52, genotype 3a was recovered from a challenge plasma pool collected from a chimpanzee experimentally infected with serum from a chronically infected patient.11Bukh J. Purcell R.H. Miller R.H. At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.Proc Natl Acad Sci U S A. 1993; 90: 8234-8238Crossref PubMed Scopus (400) Google Scholar Plasmids pFL-J6/JFH (genotype 2a) and pFL-J6/JFH(GND)3Lindenbach B.D. Evans M.J. Syder A.J. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1988) Google Scholar were provided by Charles M. Rice (Rockefeller University, New York, NY). For construction of pS52/JFH1, S52 core-NS2 fused to JFH1 5′ untranslated region (UTR) and NS3 was assembled in pGEM-9Zf(−) (Promega, Madison, WI) using fusion polymerase chain reaction (PCR) with Pfu DNA polymerase (Stratagene, La Jolla, CA) and standard cloning procedures with appropriate restriction sites. S52 fragments were amplified from clones derived from the plasma pool. The 2 JFH1 fragments used for fusion of JFH1-5′UTR/S52-core and S52-NS2/JFH1-NS3 were amplified from plasmid pFL-J6/JFH, including the EcoRI (in vector upstream of JFH1 5′UTR) and AvrII (in NS3 of JFH1) sites. The EcoRI/AvrII fragment of the resulting pGEM-9Zf(−) clone finally was inserted into pFL-J6/JFH. pS52/JFH1(GND), with a point mutation in NS5B (G8624A), was created by transferring the EcoRI/AvrII fragment of pS52/JFH1 into pFL-J6/JFH(GND). For construction of adapted S52/JFH1 genomes, mutations were introduced in pS52/JFH1 by fusion PCR and cloning. Each plasmid HCV sequence was verified by sequencing of the final DNA preparation (EndoFree Maxi Kit; Qiagen, Hilden, Germany). Plasmid DNA was linearized with XbaI (New England BioLabs, Beverly, MA),12Yanagi M. Purcell R.H. Emerson S.U. et al.Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.Virology. 1999; 262: 250-263Crossref PubMed Scopus (169) Google Scholar gel purified (Wizard SV Gel and PCR Clean-Up System; Promega), and in vitro transcribed with T7 RNA Polymerase (Promega) for 2 hours at 37°C. The amount of RNA transcripts was estimated by standard agarose gel electrophoresis. The human hepatoma cell line Huh7.5 (a gift from Charles M. Rice)3Lindenbach B.D. Evans M.J. Syder A.J. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1988) Google Scholar was cultured in Dulbecco's modified Eagle medium + 4500 mg/L glucose + GlutaMAX-I + Pyruvate (Gibco/Invitrogen Corporation, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (Sigma, St. Louis, MO), penicillin 100 U/mL and streptomycin 100 μg/mL (Gibco/Invitrogen Corporation), at 5% CO2 and 37°C. Cells were split every second to third day at a ratio of 1:2 to 1:3. Cells were washed with phosphate-buffered saline (PBS) (Dulbecco's phosphate buffered saline; Sigma) and trypsinized (trypsin/ethylenediaminetetraacetic acid, Invitrogen). Cells were plated 4 × 105 per well of a 6-well plate in Dulbecco's modified Eagle medium (10% fetal bovine serum, without antibiotics) and cultured for 24 hours. For transfection, cells were incubated with lipofection complexes (2.5 μg RNA transcripts and 5 μL Lipofectamine 2000 [Invitrogen]) in serum-free medium (Opti-MEM; Invitrogen) for approximately 12 hours. For infection, cells were incubated with filtrated cell culture supernatants. The incubation times for different experiments are given in the figure legends. Supernatants of virus-infected cell cultures or controls were rescued every 2–3 days. Cell-free supernatants were aliquoted and stored at −80°C. NS5A antigen staining was performed as previously described3Lindenbach B.D. Evans M.J. Syder A.J. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1988) Google Scholar using primary antibody anti-NS5A, 9E10 (a gift from Charles M. Rice) at 1:200 in PBS/Tween, secondary antibody ECL anti-mouse immunoglobulin (Ig)G, horseradish-peroxidase–linked whole antibody (GE Healthcare Amersham, Buckinghamshire, UK) at 1:300 in PBS/Tween, and horseradish-peroxidase substrate (DAB substrate kit, DAKO, Glostrup, Denmark). For core antigen staining13Sakai A. Takikawa S. Thimme R. et al.In vivo study of the HC-TN strain of hepatitis C virus recovered from a patient with fulminant hepatitis: RNA transcripts of a molecular clone (pHC-TN) are infectious in chimpanzees but not in Huh7.5 cells.J Virol. 2007; 81: 7208-7219Crossref PubMed Scopus (47) Google Scholar we used the primary antibody mouse anti-HCV core protein monoclonal antibody (B2) (Anogen, Yes Biotech Laboratories, Mississauga, ON) at 1:200 in PBS containing 5% bovine serum albumin, and the secondary antibody Alexa Fluor 594 goat anti-mouse IgG (H+L) (Invitrogen) at 1:500 in PBS/Tween; cell nuclei were counterstained with Hoechst 33342 (Invitrogen). The percentage of HCV-positive cells was evaluated by microscopy, assigning values of 0% (no cells infected), 1%, 5%, 10%–90% (in steps of 10%), 95%, and 100% (all cells infected). Cells were fixed as described previously14Rouille Y. Helle F. Delgrange D. et al.Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus.J Virol. 2006; 80: 2832-2841Crossref PubMed Scopus (166) Google Scholar and incubated with anti-core (as described previously), followed by incubation with secondary antibody Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen) at 1:300 in PBS containing 5% bovine serum albumin. Lipid droplets were stained with oil red O (Sigma) as described previously.14Rouille Y. Helle F. Delgrange D. et al.Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus.J Virol. 2006; 80: 2832-2841Crossref PubMed Scopus (166) Google Scholar Cell nuclei were counterstained with Hoechst 33342. Images were obtained by confocal microscopy. For quantification of intracellular lipid contents pixel saturation of the oil red O signal was avoided and images were analyzed using MetaMorph (MDS Analytical Technologies, Downingtown, PA). The relative amount of intracellular lipid per cell was determined as the intensity of oil red O signal per nucleus. Eight randomized areas were analyzed per infected/mock-infected culture, each area representing 50 cells on average. The 50% tissue culture infectious dose (TCID50) of culture supernatants was determined as previously described.3Lindenbach B.D. Evans M.J. Syder A.J. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1988) Google Scholar Huh7.5 cells were plated 6 × 103 per well of a poly-D-lysine–coated 96-well plate (Nunc, Rochester, NY). After approximately 24 hours cells were incubated with 10-fold serial dilutions of viral stock cell culture supernatant in replicates of 6. After approximately 48 hours cells were stained for NS5A as described previously. Wells were scored positive if at least 1 positive cell was detected. Huh7.5 cells were plated 6 × 103 per well of a poly-D-lysine–coated 96-well plate. After approximately 24 hours cells were incubated with anti-CD81 (JS-81; BD Biosciences Pharmingen, Franklin Lakes, NJ) or isotype-matched control antibody (anti–human immunodeficiency virus, p24, clone Kal-1; DAKO) for 1 hour. Subsequently, cells were infected with 100 TCID50 of S52/JFH1(T728C;T2718G;T7160C) for 3 hours and washed with PBS. Supernatants were collected after 1, 2, and 3 days. Cells were stained for HCV NS5A after 3 days to determine the number of focus forming units (FFU) per well. RNA was purified from 200 μL heat-inactivated (56°C for 30 min) cell culture supernatant using the Total Nucleic Acid Isolation Kit (Roche Applied Science, Mannheim, Germany) with the Total NA Variable Elution Volume protocol on a MagNA Pure LC Instrument (Roche Applied Science). As an internal control, phocine distemper virus was added. In parallel to RNA purified from culture supernatants, a quantitative HCV standard panel covering concentrations of 0–5 × 106 IU/mL in 1-log increments (OptiQuant HCV Panel; AcroMetrix Europe, Alkmaar, The Netherlands) was analyzed. Real-time PCR analyses of HCV and phocine distemper virus RNA were performed in separate reactions using TaqMan EZ reverse-transcription PCR Kit (Applied Biosystems, Foster City, CA). For HCV, primers and a 6-carboxyfluorescein-labeled minor groove binder-probe were specific for the 5′UTR and were shown previously to perform similarly against a panel of the 6 HCV genotypes in a different TaqMan assay.15Engle RE, Russell RS, Purcell RH, et al. Development of a TaqMan Assay for the six major genotypes of hepatitis C virus: comparison with commercial assays. J Med Virol (in press).Google Scholar For phocine distemper virus, a ready-to-use primer/probe mix was used (Dr H. G. M. Niesters, Erasmus Medical Centre, Rotterdam, the Netherlands). PCR analysis was performed on a 7500 real-time PCR System (Applied Biosystems) using the following cycle parameters: 2 minutes at 50°C, 30 minutes at 60°C, and 5 minutes at 95°C, followed by 45 cycles of 20 seconds at 94°C and 1 minute at 62°C. HCV RNA titers (IU/mL) were calculated using a standard curve from the standard panel and corresponding cycle threshold (Ct) values (cycle number, at which the normalized fluorescence signal increases to greater than a fixed 0.2 threshold). The reproducible detection limit was 500 IU/mL. The Ct of the phocine distemper virus reaction was compared with the expected Ct (based on a mean of previous runs; n >9) using the MedLab QC freeware program. Results of samples with a Ct within ±2 SD of the expected value were accepted. The details of direct sequencing, clonal analysis, and 5′ rapid amplification of cDNA ends of viruses recovered from culture supernatant are described in the supplementary materials and methods (see supplementary material online at www.gastrojournal.org). The intragenotypic 2a/2a recombinant J6/JFH, in which core through NS2 of JFH1 was replaced by the corresponding sequence of the infectious clone pJ6CF,12Yanagi M. Purcell R.H. Emerson S.U. et al.Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.Virology. 1999; 262: 250-263Crossref PubMed Scopus (169) Google Scholar produced infectious virus in the human hepatoma cell line Huh7.5.3Lindenbach B.D. Evans M.J. Syder A.J. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1988) Google Scholar In our hands, transfection of J6/JFH RNA transcripts into Huh7.5 cells resulted in positive HCV NS5A immunostaining of most cells within 5 days (Figure 1A and B), and J6/JFH virus from supernatant infected naive Huh7.5 cells (data not shown). In supernatant from day 8 of a J6/JFH first passage we recorded an HCV infectivity titer of 104.6 TCID50/mL and an HCV RNA titer of 107.2 IU/mL, yielding a specific infectivity of 1:398 (Table 1). These results are comparable with those obtained by Lindenbach et al.3Lindenbach B.D. Evans M.J. Syder A.J. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1988) Google Scholar, 4Lindenbach B.D. Meuleman P. Ploss A. et al.Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.Proc Natl Acad Sci U S A. 2006; 103: 3805-3809Crossref PubMed Scopus (389) Google Scholar Because we did not detect a single mutation in the open reading frame consensus sequence of day 8 first-passage virus compared with the original J6/JFH plasmid, J6/JFH apparently did not require mutations for efficient growth.Table 1Representative Infectivity and HCV RNA Titers of J6/JFH and S52/JFH1 CulturesViral isolateOrigin of supernatantExperimental dayTCID50/mL% infected cellsHCV RNA titer (IU/mL)Specific infectivityJ6/JFHFirst viral passage (used for kinetic experiment)8104.6aaverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.95107.21:398J6/JFHSecond viral passage (inoculated with 104 TCID50)3104.7baverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.80106.01:20S52/JFH1First experimentS52/JFH1 Transfection11102,5caverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.40S52/JFH1 Second viral passage (used for kinetic experiment)11104.2caverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.95106.91:501S52/JFH1 Third viral passage (inoculated with 104 TCID50)3104.6baverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.90106.01:25S52/JFH1 Third viral passage (inoculated with 102,5 TCID50)12104.7baverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.95106.61:79S52/JFH1Second experimentS52/JFH1 Transfection33103.2baverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.70S52/JFH1 First viral passage8104.5baverage of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D.95107.21:501NOTE. TCID50 infectivity titers:a/b/c average of 3, 1, or 2 independent determinations, respectively. HCV-RNA titers were determined by TaqMan; supernatants from transfections were not tested because of DNA/RNA input. Specific infectivity was calculated as infectious units (TCID50/mL) per genome number (IU/mL). First experiment, see Figure 1A and C; Supplementary Figure 1, and Figures 2A and B. Second experiment, see Figure 1B and D. Open table in a new tab NOTE. TCID50 infectivity titers: We constructed pS52/JFH1, which contains (1) the 5′UTR of the JFH1 isolate (nts 1–340), differing from the sequence provided for JFH1 (accession number AB047639) at 1 position (C301T); (2) core through NS2 of S52 (nts 341–3436); and (3) NS3 through 3′UTR of JFH1 (nts 3437–9684). Compared with the S52 consensus sequence, pS52/JFH1 contains 8 noncoding nucleotide changes (data not shown). After 2 independent transfections of Huh7.5 cells with RNA transcripts of pS52/JFH1, we found evidence of replication with NS5A antigen–positive cells on day 1 (Figure 1A and B). However, the percentage of NS5A-positive cells decreased in the S52/JFH1 cultures and evidence of viral spread was first detected at day 8 (Figure 1A) and day 26 posttransfection (Figure 1B). We recorded HCV infectivity titers of 102.5 TCID50/mL on day 11 (Figure 1A) and 103.2 TCID50/mL on day 33 (Figure 1B) (Table 1). In contrast, almost all Huh7.5 cells in the J6/JFH reference culture became HCV-NS5A positive within 5 days. We did not detect NS5A-positive cells in the negative control cultures transfected with RNA transcripts of pFL-J6/JFH(GND) (Figure 1A) and pS52/JFH1(GND) (Figure 1B). The delayed viral spread of S52/JFH1 compared with J6/JFH indicated selection of adaptive mutations. To further characterize the cell culture–derived S52/JFH1 we inoculated naive Huh7.5 cells with filtered cell-free supernatant from the transfection experiments (Figure 1C and D), and in 1 case performed additional passages (Supplementary Figure 1; see supplementary material online at www.gastrojournal.org). The infectivity titers of selected culture supernatants were determined (Table 1). On day 11, the second-passage S52/JFH1 culture had an infectivity and HCV RNA titer of 104.2 TCID50/mL and 106.9 IU/mL (specific infectivity, 1:501). After inoculation of naive Huh7.5 cells with approximately 104 TCID50 of S52/JFH1 (second passage) and J6/JFH (first passage) viruses (Table 1), more than 90% of cells were HCV positive after 6 days in both cultures, and HCV-RNA titers and infectivity titers were similar at each time point analyzed (Figure 2A). HCV RNA and infectivity titers peaked at approximately 107 IU/mL and approximately 104.6 TCID50/mL, respectively (Table 1). We recorded the highest specific infectivity on day 3 (1:25 for S52/JFH1 and 1:20 for J6/JFH) and the lowest values on day 1 (1:794 for S52/JFH1 and 1:398 for J6/JFH). The kinetics of S52/JFH1 infection depended on the virus dose. After infection of naive Huh7.5 cells with 104, 103, and 102.5 TCID50, the viruses spread to most cells after 3, 9, and 12 days, respectively, with differences reflected also in HCV-RNA titers (Figure 2B). Specific infectivities of S52/JFH1 viruses from these cultures at peak infection were comparable (Table 1). We determined the consensus sequence of viral genomes recovered from cell culture supernatants from the first transfection experiment and consecutive first, second, and third viral passages by direct sequencing of overlapping PCR fragments spanning the entire open reading frame (Table 2). In genomes recovered during transfection we detected mutations in p7 (A2721G) and NS3 (A4845T), whereas T728C, A1553G, A1907C, T2718G, and T7160C in core, E2, p7, and NS5A became dominant during the consecutive passages. For second-passage virus the potential for mutations outside the open reading frame was examined by sequencing of the entire 5′UTR and partial 3′UTR (see Materials and Methods section); no mutations were found. Interestingly, the nucleotide changes at all 7 positions with clear evidence of mutations also resulted in changes of the deduced amino acid sequence (Table 3). Nucleotide changes at the 7 positions described were all occurring as quasispecies with the pS52/JFH1 sequence (Table 2). This could be explained by the original pS52/JFH1 sequence still being present and/or by infection with different viral genomes, which had the detected mutations in various combinations. Thus, we performed clonal analysis of a long PCR fragment amplified from second-passage viral genomes, which contained all nucleotide positions, at which clear evidence of mutations had been observed. Interestingly, the 9 clones analyzed all contained specific combinations of the mutations identified by direct sequencing (Table 2) and we could not identify any genomes with the original pS52/JFH1 sequence at all of these positions.Table 2Nucleotide Changes of S52/JFH1 Viruses Recovered From CultureCoreE2p7NS2NS3NS4ANS5A Nucleotide position S52/JFH17281527155319072297271827202721302337484464455045524845540771547160 Nucleotide position H77 (AF009606)ant/aa position in analogy to the H77 reference genome (accession number AF009606) determined as described by Kuiken et al.23 For aa pos
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