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Hepatitis C virus experimental model systems and antiviral drug research

  • Published:
Virologica Sinica

Abstract

An estimated 130 million people worldwide are chronically infected with hepatitis C virus (HCV) making it a leading cause of liver disease worldwide. Because the currently available therapy of pegylated interferon-alpha and ribavirin is only effective in a subset of patients, the development of new HCV antivirals is a healthcare imperative. This review discusses the experimental models available for HCV antiviral drug research, recent advances in HCV antiviral drug development, as well as active research being pursued to facilitate development of new HCV-specific therapeutics.

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References

  1. Abe K, Kurata T, Teramoto Y C. 1993. Lack of susceptibility of various primates and woodchucks to hepatitis C virus. J Med Primatol, 22(7–8): 433–434.

    PubMed  CAS  Google Scholar 

  2. Afdhal N H. 2004. The natural history of hepatitis C. Semin Liver Dis, 24Suppl 2: 3–8.

    Article  PubMed  Google Scholar 

  3. Ahmed A, Keeffe E B. 1999. Treatment strategies for chronic hepatitis C: update since the 1997 National Institutes of Health Consensus Development Conference. J Gastroenterol Hepatol, 14Suppl: S12–18.

    PubMed  Google Scholar 

  4. Alter H J, Seeff L B. 2000. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin Liver Dis, 20(1): 17–35.

    Article  PubMed  CAS  Google Scholar 

  5. Alter M J, Margolis H S, Krawczynski K, et al. 1992. The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. N Engl J Med, 327(27): 1899–1905.

    Article  PubMed  CAS  Google Scholar 

  6. Aly H H, Shimotohno K, Hijikata M. 2009. 3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV. Biochem Biophys Res Commun, 379(2): 330–334.

    Article  PubMed  CAS  Google Scholar 

  7. Azuma H, Paulk N, Ranade A, et al. 2007. Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/-mice. Nat Biotechnol, 25(8): 903–910.

    Article  PubMed  CAS  Google Scholar 

  8. Bartenschlager R. 2006. Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture. Curr Opin Microbiol, 9(4): 416–422.

    Article  PubMed  CAS  Google Scholar 

  9. Bartenschlager R, Lohmann V. 2000. Replication of hepatitis C virus. J Gen Virol, 81Pt 7: 1631–1648.

    PubMed  CAS  Google Scholar 

  10. Bartosch B, Dubuisson J, Cosset F L. 2003. Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J Exp Med, 197(5): 633–642.

    Article  PubMed  CAS  Google Scholar 

  11. Bartosch B, Vitelli A, Granier C, et al. 2003. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem, 278(43): 41624–41630.

    Article  PubMed  CAS  Google Scholar 

  12. Beames B, Chavez D, Lanford R E. 2001. GB virus B as a model for hepatitis C virus. Ilar J, 42(2): 152–160.

    PubMed  CAS  Google Scholar 

  13. Beard M R, Abell G, Honda M, et al. 1999. An infectious molecular clone of a Japanese genotype 1b hepatitis C virus. Hepatology, 30(1): 316–324.

    Article  PubMed  CAS  Google Scholar 

  14. Behrens S E, Grassmann C W, Thiel H J, et al. 1998. Characterization of an autonomous subgenomic pestivirus RNA replicon. J Virol, 72(3): 2364–2372.

    PubMed  CAS  Google Scholar 

  15. Benedicto I, Molina-Jimenez F, Bartosch B, et al. 2009. The tight junction-associated protein occludin is required for a postbinding step in hepatitis C virus entry and infection. J Virol, 83(16): 8012–8020.

    Article  PubMed  CAS  Google Scholar 

  16. Berger K L, Cooper J D, Heaton N S, et al. 2009. Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication. Proc Natl Acad Sci USA, 106(18): 7577–7582.

    Article  PubMed  Google Scholar 

  17. Berger K L, Randall G. 2009. Potential roles for cellular cofactors in hepatitis C virus replication complex formation. Commun Integr Biol, 2(6): 471–473.

    Article  PubMed  Google Scholar 

  18. Bissig K D, Le T T, Woods N B, et al. 2007. Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model. Proc Natl Acad Sci U S A, 104(51): 20507–20511.

    Article  PubMed  Google Scholar 

  19. Bissig K D, Wieland S F, Tran P, et al. 2010. Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J Clin Invest.

  20. Blanchard E, Belouzard S, Goueslain L, et al. 2006. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol, 80(14): 6964–6972.

    Article  PubMed  CAS  Google Scholar 

  21. Blight K J, Kolykhalov A A, Rice C M. 2000. Efficient initiation of HCV RNA replication in cell culture. Science, 290(5498): 1972–1975.

    Article  PubMed  CAS  Google Scholar 

  22. Blight K J, McKeating J A, Marcotrigiano J, et al. 2003. Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture. J Virol, 77(5): 3181–3190.

    Article  PubMed  CAS  Google Scholar 

  23. Bode J G, Brenndorfer E D, Karthe J, et al. 2009. Interplay between host cell and hepatitis C virus in regulating viral replication. Biol Chem, 390(10): 1013–1032.

    Article  PubMed  CAS  Google Scholar 

  24. Boriskin Y S, Leneva I A, Pecheur E I, et al. 2008. Arbidol: a broad-spectrum antiviral compound that blocks viral fusion. Curr Med Chem, 15(10): 997–1005.

    Article  PubMed  CAS  Google Scholar 

  25. Boriskin Y S, Pecheur E I, Polyak S J. 2006. Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection. Virol J, 3: 56.

    Article  PubMed  CAS  Google Scholar 

  26. Broering T J, Garrity K A, Boatright N K, et al. 2009. Identification and characterization of broadly neutralizing human monoclonal antibodies directed against the E2 envelope glycoprotein of hepatitis C virus. J Virol, 83(23): 12473–12482.

    Article  PubMed  CAS  Google Scholar 

  27. Buck M. 2008. Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures. PLoS One, 3(7): e2660.

    Article  PubMed  CAS  Google Scholar 

  28. Buckwold V E, Beer B E, Donis R O. 2003. Bovine viral diarrhea virus as a surrogate model of hepatitis C virus for the evaluation of antiviral agents. Antiviral Res, 60(1): 1–15.

    Article  PubMed  CAS  Google Scholar 

  29. Bukh J. 2004. A critical role for the chimpanzee model in the study of hepatitis C. Hepatology, 39(6): 1469–1475.

    Article  PubMed  CAS  Google Scholar 

  30. Bukh J, Pietschmann T, Lohmann V, et al. 2002. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci USA, 99(22): 14416–14421.

    Article  PubMed  CAS  Google Scholar 

  31. Burioni R, Perotti M, Mancini N, et al. 2008. Perspectives for the utilization of neutralizing human monoclonal antibodies as anti-HCV drugs. J Hepatol, 49(2): 299–300.

    Article  PubMed  CAS  Google Scholar 

  32. Cai Z, Cai L, Jiang J, et al. 2007. Human serum amyloid A protein inhibits hepatitis C virus entry into cells. J Virol, 81(11): 6128–6133.

    Article  PubMed  CAS  Google Scholar 

  33. Chang K, Wang T, Luo G. 2009. Proteomics study of the hepatitis C virus replication complex. Methods Mol Biol, 510: 185–193.

    Article  PubMed  CAS  Google Scholar 

  34. Chang K S, Cai Z, Zhang C, et al. 2006. Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities. J Virol, 80(15): 7364–7374.

    Article  PubMed  CAS  Google Scholar 

  35. Chockalingam K, Simeon R L, Rice C M, et al. 2010. A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle. Proc Natl Acad Sci U S A, 107(8): 3764–3769.

    Article  PubMed  Google Scholar 

  36. Choi S, Sainz B Jr., Corcoran P, et al. 2009. Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells. Xenobiotica, 39(3): 205–217.

    Article  PubMed  CAS  Google Scholar 

  37. Choo Q-L, Kuo G, Weiner A J, et al. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science, 244: 359–362.

    Article  PubMed  CAS  Google Scholar 

  38. Chung R, He W, Saquib A, et al. 2001. Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system. Proc Natl Acad Sci USA, 98(17): 9847–9852.

    Article  PubMed  CAS  Google Scholar 

  39. Davis G L, Nelson D R, Terrault N, et al. 2005. A randomized, open-label study to evaluate the safety and pharmacokinetics of human hepatitis C immune globulin (Civacir) in liver transplant recipients. Liver Transpl, 11(8): 941–949.

    Article  PubMed  Google Scholar 

  40. Durantel D. 2009. Celgosivir, an alpha-glucosidase I inhibitor for the potential treatment of HCV infection. Curr Opin Investig Drugs, 10(8): 860–870.

    PubMed  CAS  Google Scholar 

  41. Einav S, Gerber D, Bryson P D, et al. 2008. Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nat Biotechnol, 26(9): 1019–1027.

    Article  PubMed  CAS  Google Scholar 

  42. Evans M J, von Hahn T, Tscherne D M, et al. 2007. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature, 446(7137): 801–805.

    Article  PubMed  CAS  Google Scholar 

  43. Fartoux L, Poujol-Robert A, Guechot J, et al. 2005. Insulin resistance is a cause of steatosis and fibrosis progression in chronic hepatitis C. Gut, 54(7): 1003–1008.

    Article  PubMed  CAS  Google Scholar 

  44. Feinstone S, Alter H, Dienes H, et al. 1982. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis, 144(6): 588–598.

    Google Scholar 

  45. Feld J J, Hoofnagle J H. 2005. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature, 436(7053): 967–972.

    Article  PubMed  CAS  Google Scholar 

  46. Ferreon J C, Ferreon A C, Li K, et al. 2005. Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease. J Biol Chem, 280(21): 20483–20492.

    Article  PubMed  CAS  Google Scholar 

  47. Flisiak R, Feinman S V, Jablkowski M, et al. 2009. The cyclophilin inhibitor Debio 025 combined with PEG IFNalpha2a significantly reduces viral load in treatmentnaive hepatitis C patients. Hepatology, 49(5): 1460–1468.

    Article  PubMed  CAS  Google Scholar 

  48. Foy E, Li K, Wang C, et al. 2003. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science, 300(5622): 1145–1148.

    Article  PubMed  CAS  Google Scholar 

  49. Galun E, Burakova T, Ketzinel M, et al. 1995. Hepatitis C virus viremia in SCID—>BNX mouse chimera. J Infect Dis, 172(1): 25–30.

    PubMed  CAS  Google Scholar 

  50. Garry R F, Dash S. 2003. Proteomics computational analyses suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class II fusion proteins. Virology, 307(2): 255–265.

    Article  PubMed  CAS  Google Scholar 

  51. Gastaminza P, Whitten-Bauer C, Chisari F V. 2010. Unbiased probing of the entire hepatitis C virus life cycle identifies clinical compounds that target multiple aspects of the infection. Proc Natl Acad Sci USA, 107(1): 291–296.

    Article  PubMed  Google Scholar 

  52. Glue P, Fang J W, Rouzier-Panis R, et al. 2000. Pegylated interferon-alpha2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data. Hepatitis C Intervention Therapy Group. Clin Pharmacol Ther, 68(5): 556–567.

    Article  PubMed  CAS  Google Scholar 

  53. Goergen B, Jakobs S, Symmons P, et al. 1994. Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method. J Hepatol, 21(4): 678–682.

    Article  PubMed  CAS  Google Scholar 

  54. Gosert R, Egger D, Lohmann V, et al. 2003. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol, 77(9): 5487–5492.

    Article  PubMed  CAS  Google Scholar 

  55. Gottwein J M, Scheel T K, Hoegh A M, et al. 2007. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology, 133(5): 1614–1626.

    Article  PubMed  CAS  Google Scholar 

  56. Gottwein J M, Scheel T K, Jensen T B, et al. 2009. Development and characterization of hepatitis C virus genotype 1–7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology, 49(2): 364–377.

    Article  PubMed  CAS  Google Scholar 

  57. Gouttenoire J, Penin F, Moradpour D. 2010. Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory. Rev Med Virol, 20(2): 117–129.

    Article  PubMed  CAS  Google Scholar 

  58. Griffin S. 2010. Inhibition of HCV p7 as a therapeutic target. Curr Opin Investig Drugs, 11(2): 175–181.

    PubMed  CAS  Google Scholar 

  59. Grompe M, Laconi E, Shafritz D. 1999. Principles of therapeutic liver repopulation. Semin Liver Dis, 19(1): 7–14.

    Article  PubMed  CAS  Google Scholar 

  60. Grove J, Huby T, Stamataki Z, et al. 2007. Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity. J Virol, 81(7): 3162–3169.

    Article  PubMed  CAS  Google Scholar 

  61. Haid S, Pietschmann T, Pecheur E I. 2009. Low pH-dependent Hepatitis C Virus Membrane Fusion Depends on E2 Integrity, Target Lipid Composition, and Density of Virus Particles. J Biol Chem, 284(26): 17657–17667.

    Article  PubMed  CAS  Google Scholar 

  62. Heckel J L, Sandgren E P, Degen J L, et al. 1990. Neonatal bleeding in transgenic mice expressing urokinasetype plasminogen activator. Cell, 62(3): 447–456.

    Article  PubMed  CAS  Google Scholar 

  63. Helle F, Dubuisson J. 2008. Hepatitis C virus entry into host cells. Cell Mol Life Sci, 65(1): 100–112.

    Article  PubMed  CAS  Google Scholar 

  64. Hezode C, Forestier N, Dusheiko G, et al. 2009. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med, 360(18): 1839–1850.

    Article  PubMed  CAS  Google Scholar 

  65. Hiraga N, Imamura M, Tsuge M, et al. 2007. Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon. FEBS Lett, 581(10): 1983–1987.

    Article  PubMed  CAS  Google Scholar 

  66. Hiramatsu N, Dash S, Gerber M. 1997. HCV cDNA transfection to HepG2 cells. J Viral Hepat, 4(Suppl 1): 61–67.

    Article  PubMed  Google Scholar 

  67. Hirowatari Y, Hijikata M, Shimotohno K. 1995. A novel method for analysis of viral proteinase activity encoded by hepatitis C virus in cultured cells. Anal Biochem, 225(1): 113–120.

    Article  PubMed  CAS  Google Scholar 

  68. Holzer M, Ziegler S, Albrecht B, et al. 2008. Identification of terfenadine as an inhibitor of human CD81-receptor HCV-E2 interaction: synthesis and structure optimization. Molecules, 13(5): 1081–1110.

    Article  PubMed  CAS  Google Scholar 

  69. Honda A, Arai Y, Hirota N, et al. 1999. Hepatitis C virus structural proteins induce liver cell injury in transgenic mice. J Med Virol, 59(3): 281–289.

    Article  PubMed  CAS  Google Scholar 

  70. Hong Z, Beaudet-Miller M, Lanford R E, et al. 1999. Generation of transmissible hepatitis C virions from a molecular clone in chimpanzees. Virology, 256(1): 36–44.

    Article  PubMed  CAS  Google Scholar 

  71. Hoofnagle J H. 2002. Course and outcome of hepatitis C. Hepatology, 36(5 Suppl 1): S21–29.

    Article  PubMed  Google Scholar 

  72. Hsu M, Zhang J, Flint M, et al. 2003. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA, 100(12): 7271–7276.

    Article  PubMed  CAS  Google Scholar 

  73. Huang Y, Staschke K, De Francesco R, et al. 2007. Phosphorylation of hepatitis C virus NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA replication? Virology, 364(1): 1–9.

    Article  PubMed  CAS  Google Scholar 

  74. Ikeda M, Abe K, Yamada M, et al.2006. Different anti-HCV profiles of statins and their potential for combination therapy with interferon. Hepatology, 44(1): 117–125.

    Article  PubMed  CAS  Google Scholar 

  75. Ikeda M, Yi M, Li K, et al. 2002. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol, 76(6): 2997–3006.

    Article  PubMed  CAS  Google Scholar 

  76. Ilan E, Arazi J, Nussbaum O, et al. 2002. The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV. J Infect Dis, 185(2): 153–161.

    Article  PubMed  CAS  Google Scholar 

  77. Iro M, Witteveldt J, Angus A G, et al. 2009. A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication. Antiviral Res, 83(2): 148–155.

    Article  PubMed  CAS  Google Scholar 

  78. Jensen T B, Gottwein J M, Scheel T K, et al. 2008. Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. J Infect Dis, 198(12): 1756–1765.

    Article  PubMed  Google Scholar 

  79. Jirasko V, Montserret R, Appel N, et al. 2008. Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem, 283(42): 28546–28562.

    Article  PubMed  CAS  Google Scholar 

  80. Jones C T, Murray CL, Eastman D K, et al. 2007. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol, 81(16): 8374–8383.

    Article  PubMed  CAS  Google Scholar 

  81. Jopling C L, Yi M, Lancaster A M, et al. 2005. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science, 309(5740): 1577–1581.

    Article  PubMed  CAS  Google Scholar 

  82. Kapadia S B, Barth H, Baumert T, et al. 2007. Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J Virol, 81(1): 374–383.

    Article  PubMed  CAS  Google Scholar 

  83. Karayiannis P, Scheuer P J, Bamber M, et al. 1983. Experimental infection of Tamarins with human non-A, non-B hepatitis virus. JMedVirol, 11: 251–256.

    CAS  Google Scholar 

  84. Kato T, Date T, Miyamoto M, et al. 2003. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology, 125(6): 1808–1817.

    Article  PubMed  CAS  Google Scholar 

  85. Kato T, Furusaka A, Miyamoto M, et al. 2001. Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol, 64(3): 334–339.

    Article  PubMed  CAS  Google Scholar 

  86. Kawamura T, Furusaka A, Koziel M J, et al. 1997. Transgenic expression of hepatitis C virus structural proteins in the mouse. Hepatology, 25(4): 1014–1021.

    Article  PubMed  CAS  Google Scholar 

  87. Keck Z Y, Machida K, Lai M M, et al. 2008. Therapeutic control of hepatitis C virus: the role of neutralizing monoclonal antibodies. Curr Top Microbiol Immunol, 317: 1–38.

    Article  PubMed  CAS  Google Scholar 

  88. Khromykh A A, Westaway E G. 1997. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol, 71(2): 1497–1505.

    PubMed  CAS  Google Scholar 

  89. Kneteman N M, Mercer D F. 2005. Mice with chimeric human livers: who says supermodels have to be tall? Hepatology, 41(4): 703–706.

    Article  PubMed  CAS  Google Scholar 

  90. Kneteman N M, Toso C. 2009. In vivo study of HCV in mice with chimeric human livers. Methods Mol Biol, 510: 383–399.

    Article  PubMed  CAS  Google Scholar 

  91. Kneteman N M, Weiner A J, O’Connell J, et al. 2006. Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application. Hepatology, 43(6): 1346–1353.

    Article  PubMed  CAS  Google Scholar 

  92. Koike K, Moriya K, Ishibashi K, et al. 1995. Expression of hepatitis C virus envelope proteins in transgenic mice. JGenVirol, 76: 3031–3038.

    CAS  Google Scholar 

  93. Kolykhalov A, Mihalik K, Feinstone S, et al. 2000. Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo. J Virol, 74(4): 2046–2051.

    Article  PubMed  CAS  Google Scholar 

  94. Kolykhalov A A, Agapov E V, Blight K J, et al. 1997. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science, 277(5325): 570–574.

    Article  PubMed  CAS  Google Scholar 

  95. Kota S, Scampavia L, Spicer T, et al. 2009. A Time-Resolved Fluorescence-Resonance Energy Transfer Assay for Identifying Inhibitors of Hepatitis C Virus Core Dimerization. Assay Drug Dev Technol.

  96. Koutsoudakis G, Herrmann E, Kallis S, et al. 2007. The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells. J Virol, 81(2): 588–598.

    Article  PubMed  CAS  Google Scholar 

  97. Koutsoudakis G, Kaul A, Steinmann E, et al. 2006. Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J Virol, 80(11): 5308–5320.

    Article  PubMed  CAS  Google Scholar 

  98. Krieger N, Lohmann V, Bartenschlager R. 2001. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 2001 May; 75(10): 4614–24, 75 (10): 4614–4624.

    Article  PubMed  CAS  Google Scholar 

  99. Kwo P, Lawitz E J, McCone J, et al. 2008. HCV SPRINT-1: Boceprevir plus Peginterferon alfa-2b/Ribavirin for Treatment of Genotype 1 Chronic Hepatitis C in Previously Untreated Patients: 59th Annual Meeting of the American Association for the Study of Liver Diseases, San Francisco, CA. Hepatology, 48,Suppl 1 (4): 1027A.

    Google Scholar 

  100. Lanford R E, Bigger C, Bassett S, et al. 2001. The chimpanzee model of hepatitis C virus infections. Ilar J, 42(2): 117–126.

    PubMed  CAS  Google Scholar 

  101. Lanford R E, Hildebrandt-Eriksen E S, Petri A, et al. 2010. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science, 327(5962): 198–201.

    Article  PubMed  CAS  Google Scholar 

  102. Lavie M, Voisset C, Vu-Dac N, et al. 2006. Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system. Hepatology, 44(6): 1626–1634.

    Article  PubMed  CAS  Google Scholar 

  103. Lavillette D, Bartosch B, Nourrisson D, et al. 2006. Hepatitis C virus glycoproteins mediate low pHdependent membrane fusion with liposomes. J Biol Chem, 281(7): 3909–3917.

    Article  PubMed  CAS  Google Scholar 

  104. Lavillette D, Pecheur E I, Donot P, et al. 2007. Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol, 81(16): 8752–8765.

    Article  PubMed  CAS  Google Scholar 

  105. Law M, Maruyama T, Lewis J, et al. 2008. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med, 14(1): 25–27.

    Article  PubMed  CAS  Google Scholar 

  106. Lemm J A, O’Boyle D, 2nd, Liu M, et al. 2010. Identification of hepatitis C virus NS5A inhibitors. J Virol, 84(1): 482–491.

    Article  PubMed  CAS  Google Scholar 

  107. Lerat H, Honda M, Beard M R, et al. 2002. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology, 122(2): 352–365.

    Article  PubMed  CAS  Google Scholar 

  108. Li K, Foy E, Ferreon J C, et al. 2005. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA, 102(8): 2992–2997.

    Article  PubMed  CAS  Google Scholar 

  109. Li Q, Brass A L, Ng A, et al. 2009. A genome-wide genetic screen for host factors required for hepatitis C virus propagation. Proc Natl Acad Sci USA, 106(38): 16410–16415.

    Article  PubMed  Google Scholar 

  110. Li X D, Sun L, Seth R B, et al. 2005. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci U S A, 102(49): 17717–17722.

    Article  PubMed  CAS  Google Scholar 

  111. Liang T J, Rehermann B, Seeff L B, et al. 2000. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med, 132(4): 296–305.

    PubMed  CAS  Google Scholar 

  112. Lim S P, Soo H M, Tan Y H, et al. 2002. Inducible system in human hepatoma cell lines for hepatitis C virus production. Virology, 303(1): 79–99.

    Article  PubMed  CAS  Google Scholar 

  113. Lindenbach B D, Evans M J, Syder A J, et al. 2005. Complete replication of hepatitis C virus in cell culture. Science, 309(5734): 623–626.

    Article  PubMed  CAS  Google Scholar 

  114. Lindenbach B D, Meuleman P, Ploss A, et al. 2006. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci USA.

  115. Liu S, Yang W, Shen L, et al. 2009. Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J Virol, 83(4): 2011–2014.

    Article  PubMed  CAS  Google Scholar 

  116. Lohmann V, Hoffmann S, Herian U, et al. 2003. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol, 77(5): 3007–3019.

    Article  PubMed  CAS  Google Scholar 

  117. Lohmann V, Korner F, Dobierzewska A, et al. 2001. Mutations in Hepatitis C Virus RNAs Conferring Cell Culture Adaptation. J Virol, 75(3): 1437–1449.

    Article  PubMed  CAS  Google Scholar 

  118. Lohmann V, Korner F, Koch J, et al. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science, 285(5424): 110–113.

    Article  PubMed  CAS  Google Scholar 

  119. Maeda N, Watanabe M, Okamoto S, et al. 2004. Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone. Liver Int, 24(3): 259–267.

    Article  PubMed  CAS  Google Scholar 

  120. Masson D, Koseki M, Ishibashi M, et al. 2009. Increased HDL cholesterol and apoA-I in humans and mice treated with a novel SR-BI inhibitor. Arterioscler Thromb Vasc Biol, 29(12): 2054–2060.

    Article  PubMed  CAS  Google Scholar 

  121. Matsumura T, Hu Z, Kato T, et al. 2009. Amphipathic DNA polymers inhibit hepatitis C virus infection by blocking viral entry. Gastroenterology, 137(2): 673–681.

    Article  PubMed  CAS  Google Scholar 

  122. McHutchison J G, Everson G T, Gordon S C, et al. 2009. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N Engl J Med, 360(18): 1827–1838.

    Article  PubMed  CAS  Google Scholar 

  123. Meertens L, Bertaux C, Dragic T. 2006. Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles. J Virol, 80(23): 11571–11578.

    Article  PubMed  CAS  Google Scholar 

  124. Mercer D, Schiller D, Elliott J, et al. 2001. Hepatitis C virus replication in mice with chimeric human livers. Nat Med, 7(8): 927–933.

    Article  PubMed  CAS  Google Scholar 

  125. Meunier J C, Russell R S, Goossens V, et al. 2008. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus. J Virol, 82(2): 966–973.

    Article  PubMed  CAS  Google Scholar 

  126. Moradpour D, Penin F, Rice C M. 2007. Replication of hepatitis C virus. Nat Rev Microbiol, 5(6): 453–463.

    Article  PubMed  CAS  Google Scholar 

  127. Moriya K, Yotsuyanagi H, Shintani Y, et al. 1997. Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol, 78(Pt 7): 1527–1531.

    PubMed  CAS  Google Scholar 

  128. Murakami Y, Noguchi K, Yamagoe S, et al. 2009. Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR. Antiviral Res, 83(2): 112–117.

    Article  PubMed  CAS  Google Scholar 

  129. Murray E M, Grobler J A, Markel E J, et al. 2003. Persistent replication of hepatitis C virus replicons expressing the betalactamase reporter in subpopulations of highly permissive Huh7 cells. J Virol, 77(5): 2928–2935.

    Article  PubMed  CAS  Google Scholar 

  130. Myung J, Khalap N, Kalkeri G, et al. 2001. Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone. J Virol Methods, 94(1–2): 55–67.

    Article  PubMed  CAS  Google Scholar 

  131. Nahmias Y, Goldwasser J, Casali M, et al. 2008. Apolipoprotein B-dependent hepatitis C virus secretion is inhibited by the grapefruit flavonoid naringenin. Hepatology, 47(5): 1437–1445.

    Article  PubMed  CAS  Google Scholar 

  132. Nakabayashi H, Taketa K, Miyano K, et al. 1982. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res, 42(9): 3858–3863.

    PubMed  CAS  Google Scholar 

  133. Noto H, Raskin P. 2006. Hepatitis C infection and diabetes. J Diabetes Complications, 20(2): 113–120.

    Article  PubMed  Google Scholar 

  134. Paeshuyse J, Kaul A, De Clercq E, et al. 2006. The nonimmunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replication in vitro. Hepatology, 43(4): 761–770.

    Article  PubMed  CAS  Google Scholar 

  135. Pan K L, Lee J C, Sung H W, et al. 2009. Development of NS3/4A protease-based reporter assay suitable for efficiently assessing hepatitis C virus infection. Antimicrob Agents Chemother, 53(11): 4825–4834.

    Article  PubMed  CAS  Google Scholar 

  136. Pasquinelli C, Shoenberger J M, Chung J, et al. 1997. Hepatitis C virus core and E2 protein expression in transgenic mice. Hepatology, 25(3): 719–727.

    Article  PubMed  CAS  Google Scholar 

  137. Pecheur E I, Lavillette D, Alcaras F, et al. 2007. Biochemical mechanism of hepatitis C virus inhibition by the broad-spectrum antiviral arbidol. Biochemistry, 46(20): 6050–6059.

    Article  PubMed  CAS  Google Scholar 

  138. Pietschmann T, Kaul A, Koutsoudakis G, et al. 2006. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci USA, 103(19): 7408–7413.

    Article  PubMed  CAS  Google Scholar 

  139. Pietschmann T, Zayas M, Meuleman P, et al. 2009. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog, 5(6): e1000475.

    Article  PubMed  CAS  Google Scholar 

  140. Pileri P, Uematsu Y, Campagnoli S, et al. 1998. Binding of hepatitis C virus to CD81. Science, 282(5390): 938–941.

    Article  PubMed  CAS  Google Scholar 

  141. Ploss A, Evans M J, Gaysinskaya V A, et al. 2009. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature, 457(7231): 882–886.

    Article  PubMed  CAS  Google Scholar 

  142. Ploss A, Khetani S R, Jones C T, et al. 2010. Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures. Proc Natl Acad Sci USA, 107(7): 3141–3145.

    Article  PubMed  Google Scholar 

  143. Ploss A, Rice C M. 2009. Towards a small animal model for hepatitis C. EMBO Rep, 10(11): 1220–1227.

    Article  PubMed  CAS  Google Scholar 

  144. Popescu C I, Dubuisson J. 2009. Role of lipid metabolism in hepatitis C virus assembly and entry. Biol Cell, 102(1): 63–74.

    Article  PubMed  Google Scholar 

  145. Poynard T, Yuen M F, Ratziu V, et al. 2003. Viral hepatitis C. Lancet, 362(9401): 2095–2100.

    Article  PubMed  CAS  Google Scholar 

  146. Prabhu R, Joshi V, Garry R F, et al. 2004. Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone. Exp Mol Pathol, 76(3): 242–252.

    Article  PubMed  CAS  Google Scholar 

  147. Reisner Y, Dagan S. 1998. The Trimera mouse: generating human monoclonal antibodies and an animal model for human diseases. Trends Biotechnol, 16(6): 242–246.

    Article  PubMed  CAS  Google Scholar 

  148. Romero-Gomez M. 2006. Hepatitis C and insulin resistance: steatosis, fibrosis and non-response. Rev Esp Enferm Dig, 98(8): 605–615.

    Article  PubMed  CAS  Google Scholar 

  149. Romero-Gomez M. 2006. Insulin resistance and hepatitis C. World J Gastroenterol, 12(44): 7075–7080.

    PubMed  CAS  Google Scholar 

  150. Sainz B Jr., Barretto N, Uprichard S L. 2009. Hepatitis C Virus infection in phenotypically distinct Huh7 cell lines. PLoS ONE, 4(8): e6561.

    Article  PubMed  CAS  Google Scholar 

  151. Sainz B, Jr., Chisari F V. 2006. Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells. J Virol, 80(20): 10253–10257.

    Article  PubMed  CAS  Google Scholar 

  152. Sainz B, Jr., TenCate V, Uprichard S L. 2009. Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol J, 6: 103.

    Article  PubMed  CAS  Google Scholar 

  153. Sarrazin C, Rouzier R, Wagner F, et al. 2007. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology, 132(4): 1270–1278.

    Article  PubMed  CAS  Google Scholar 

  154. Scarselli E, Ansuini H, Cerino R, et al. 2002. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. Embo J, 21(19): 5017–5025.

    Article  PubMed  CAS  Google Scholar 

  155. Schaller T, Appel N, Koutsoudakis G, et al. 2007. Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J Virol, 81(9): 4591–4603.

    Article  PubMed  CAS  Google Scholar 

  156. Scheel T K, Gottwein J M, Jensen T B, et al. 2008. Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc Natl Acad Sci USA, 105(3): 997–1002.

    Article  PubMed  Google Scholar 

  157. Schinazi R, Ilan E, Black P, et al. 1999. Cell-based and animal models for hepatitis B and C viruses. Antivir Chem Chemother, 10(3): 99–114.

    PubMed  CAS  Google Scholar 

  158. Steinmann E, Whitfield T, Kallis S, et al. 2007. Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus. Hepatology, 46(2): 330–338.

    Article  PubMed  CAS  Google Scholar 

  159. Sulkowski M S. 2007. Specific targeted antiviral therapy for hepatitis C. Curr Gastroenterol Rep, 9(1): 5–13.

    Article  PubMed  Google Scholar 

  160. Suzuki T, Aizaki H, Murakami K, et al. 2007. Molecular biology of hepatitis C virus. J Gastroenterol, 42(6): 411–423.

    Article  PubMed  CAS  Google Scholar 

  161. Tai A W, Benita Y, Peng L F, et al. 2009. A functional genomic screen identifies cellular cofactors of hepatitis C virus replication. Cell Host Microbe, 5(3): 298–307.

    Article  PubMed  CAS  Google Scholar 

  162. Tencate V, Sainz B J, Cotler S, et al. 2010. Potential treatment options and future research to increase hepatitis C virus treatment response rate. Hepatic Medicine: Evidence and Research, in press.

  163. Trotard M, Lepere-Douard C, Regeard M, et al. 2009. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening. Faseb J, 23(11): 3780–3789.

    Article  PubMed  CAS  Google Scholar 

  164. Tscherne D M, Jones C T, Evans M J, et al. 2006. Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol, 80(4): 1734–1741.

    Article  PubMed  CAS  Google Scholar 

  165. Uprichard S L, Chung J, Chisari F V, et al. 2006. Replication of a hepatitis C virus replicon clone in mouse cells. Virol J, 3: 89.

    Article  PubMed  CAS  Google Scholar 

  166. von Hahn T, Rice C M. 2008. Hepatitis C virus entry. J Biol Chem, 283(7): 3689–3693.

    Article  Google Scholar 

  167. Wakita T, Pietschmann T, Kato T, et al. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med, 11(7): 791–796.

    Article  PubMed  CAS  Google Scholar 

  168. Wang C, Gale M Jr., Keller B C, et al. 2005. Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication. Mol Cell, 18(4): 425–434.

    Article  PubMed  CAS  Google Scholar 

  169. Watanabe T, Katagiri J, Kojima H, et al. 1987. Studies on transmission of human non-A, non-B hepatitis to marmosets. J Med Virol, 22: 143–156.

    Article  PubMed  CAS  Google Scholar 

  170. Welsch S, Miller S, Romero-Brey I, et al. 2009. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe, 5(4): 365–375.

    Article  PubMed  CAS  Google Scholar 

  171. Williams R. 2006. Global challenges in liver disease. Hepatology, 44(3): 521–526.

    Article  PubMed  Google Scholar 

  172. Wolk B, Buchele B, Blum H E, et al. A dynamic view of hepatitis C virus replication complexes. Presented at the 11th International Symposium on HCV and related viruses; 2004, Heidelberg, Germany.

  173. Wu G Y, Konishi M, Walton C M, et al. 2005. A novel immunocompetent rat model of HCV infection and hepatitis. Gastroenterology, 128(5): 1416–1423.

    Article  PubMed  CAS  Google Scholar 

  174. Wunschmann S, Medh J D, Klinzmann D, et al. 2000. Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor. J Virol, 74(21): 10055–10062.

    Article  PubMed  CAS  Google Scholar 

  175. Xie Z, Riezu-Boj J, Lasarte J, et al. 1998. Transmission of hepatitis C virus infection to tree shrews. Virology, 244(2): 513–520.

    Article  PubMed  CAS  Google Scholar 

  176. Yanagi M, Purcell R H, Emerson S U, et al. 1999. Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology, 262(1): 250–263.

    Article  PubMed  CAS  Google Scholar 

  177. Yanagi M, Purcell R H, Emerson S U, et al. 1997. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA, 94(16): 8738–8743.

    Article  PubMed  CAS  Google Scholar 

  178. Yanagi M, St Claire M, Emerson S U, et al. 1999. In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone. Proc Natl Acad Sci USA, 96(5): 2291–2295.

    Article  PubMed  CAS  Google Scholar 

  179. Yanagi M, St Claire M, Shapiro M, et al. 1998. Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology, 244(1): 161–172.

    Article  PubMed  CAS  Google Scholar 

  180. Yang J P, Zhou D, Wong-Staal F. 2009. Screening of small-molecule compounds as inhibitors of HCV entry. Methods Mol Biol, 510: 295–304.

    Article  PubMed  CAS  Google Scholar 

  181. Ye J, Wang C, Sumpter R Jr., et al. 2003. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci U S A, 100(26): 15865–15870.

    Article  PubMed  CAS  Google Scholar 

  182. Yi M, Bodola F, Lemon S M. 2002. Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology, 304(2): 197–210.

    Article  PubMed  CAS  Google Scholar 

  183. Yi M, Lemon S. 2002. Replication of subgenomic hepatitis A virus RNAs expressing firefly luciferase is enhanced by mutations associated with adaptation of virus to growth in cultured cells. J Virol, 76(3): 1171–1180.

    PubMed  CAS  Google Scholar 

  184. Yi M, Villanueva R A, Thomas D L, et al. 2006. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA, 103(7): 2310–2315.

    Article  PubMed  CAS  Google Scholar 

  185. Yoo B J, Selby M J, Choe J, et al. 1995. Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol, 69(1): 32–38.

    PubMed  CAS  Google Scholar 

  186. Yu X, Sainz B Jr., Uprichard S L. 2009. Development of a cell-based hepatitis C virus infection fluorescent resonance energy transfer assay for high-throughput antiviral compound screening. Antimicrob Agents Chemother, 53(10): 4311–4319.

    Article  PubMed  CAS  Google Scholar 

  187. Zeisel M B, Barth H, Schuster C, et al. 2009. Hepatitis C virus entry: molecular mechanisms and targets for antiviral therapy. Front Biosci, 14: 3274–3285.

    Article  PubMed  CAS  Google Scholar 

  188. Zeisel M B, Koutsoudakis G, Schnober E K, et al. 2007. Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology, 46(6): 1722–1731.

    Article  PubMed  CAS  Google Scholar 

  189. Zhang Y, Weady P, Duggal R, et al. 2008. Novel chimeric genotype 1b/2a hepatitis C virus suitable for high-throughput screening. Antimicrob Agents Chemother, 52(2): 666–674.

    Article  PubMed  CAS  Google Scholar 

  190. Zhao X, Tang Z, Klumpp B, et al. 2002. Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection. J Clin Invest, 109(2): 221–232.

    PubMed  CAS  Google Scholar 

  191. Zhong J, Gastaminza P, Cheng G, et al. 2005. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA, 102(26): 9294–9299.

    Article  PubMed  CAS  Google Scholar 

  192. Zhu H, Elyar J, Foss R, et al. 2009. Primary human hepatocyte culture for HCV study. Methods Mol Biol, 510: 373–382.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Susan L. Uprichard.

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Foundation items: The author was supported by National Institutes of Health grants AI070827 and CA33266, American Cancer Society grant RSG-09-076-01 and the UIC Walter Payton Center GUILD.

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Uprichard, S.L. Hepatitis C virus experimental model systems and antiviral drug research. Virol. Sin. 25, 227–245 (2010). https://doi.org/10.1007/s12250-010-3134-0

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