title: Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains.
abstract: Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5' end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation of the genetically divergent isolate J8 (genotype 2b), which differed from the J6 nucleotide sequence by 24%. The most efficient recombinant, J8cc, had nine adaptive mutations and was genetically stable after viral passage. The availability of these robust JFH1-independent genotype 2a and 2b culture systems represents an important advance, and the approach used might permit culture development of other isolates, with implications for improved individualized treatments of HCV patients and for development of broadly efficient vaccines.
authors.affiliation: Copenhagen Hepatitis C Program, CO-HEP, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, DK-2650 Hvidovre, Denmark.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Li
authors.forename: Santseharay
authors.initials: S
authors.lastname: Ramirez
authors.forename: Judith M
authors.initials: JM
authors.lastname: Gottwein
authors.forename: Troels K H
authors.initials: TK
authors.lastname: Scheel
authors.forename: Lotte
authors.initials: L
authors.lastname: Mikkelsen
authors.forename: Robert H
authors.initials: RH
authors.lastname: Purcell
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D020413:3' Untranslated Regions; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D020413:3' Untranslated Regions; D002352:Carrier Proteins; D004279:DNA, Viral; D047908:Intracellular Signaling Peptides and Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C080107:NS4B protein, flavivirus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase