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.
Six major hepatitis C virus (HCV) genotypes and numerous subtypes have been described, and recently a seventh major genotype was discovered. Genotypes show significant molecular and clinical differences, such as differential response to combination therapy with interferon-alpha and ribavirin. Recently, HCV research has been accelerated by cell culture systems based on the unique growth capacity of strain JFH1 (genotype 2a). By development of JFH1-based intergenotypic recombinants containing Core, envelope protein 1 and 2 (E1, E2), p7, and nonstructural protein 2 (NS2) of genotype 6a and 7a strains, as well as subtype 1b and 2b strains, we have completed a panel of culture systems for all major HCV genotypes. Efficient growth in Huh7.5 cells depended on adaptive mutations for HK6a/JFH1 (6a/2a, in E1 and E2) and J4/JFH1 (1b/2a, in NS2 and NS3); viability of J8/JFH1 (2b/2a) and QC69/JFH1 (7a/2a) did not require adaptation. To facilitate comparative studies, we generated virus stocks of genotype 1-7 recombinants with infectivity titers of 10(3.7) to 10(5.2) 50% tissue culture infectious dose/mL and HCV RNA titers of 10(7.0) to 10(7.9) IU/mL. Huh7.5 cultures infected with genotype 1-6 viruses had similar spread kinetics, intracellular Core, NS5A, and lipid amounts, and colocalization of Core and NS5A with lipids. Treatment with interferon-alpha2b but not ribavirin or amantadine showed a significant antiviral effect. Infection with all genotypes could be blocked by specific antibodies against the putative coreceptors CD81 and scavenger receptor class B type I in a dose-dependent manner. Finally, neutralizing antibodies in selected chronic phase HCV sera had differential effects against genotype 1-7 viruses.


CONCLUSIONS
We completed and characterized a panel of JFH1-based cell culture systems of all seven major HCV genotypes and important subtypes and used these viruses in comparative studies of antivirals, HCV receptor interaction, and neutralizing antibodies.
Gottwein
Judith M
JM
Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark.
Scheel
Troels K H
TK
Jensen
Tanja B
TB
Lademann
Jacob B
JB
Prentoe
Jannick C
JC
Knudsen
Maria L
ML
Hoegh
Anne M
AM
Bukh
Jens
J
D020121:5' Untranslated Regions / Q000235:genetics; D015703:Antigens, CD / Q000502:physiology*; D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D014644:Genetic Variation; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy* / Q000503:physiopathology*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D051122:Scavenger Receptors, Class B / Q000502:physiology*; D060225:Tetraspanin 28
D020121:5' Untranslated Regions; D015703:Antigens, CD; D000998:Antiviral Agents; C554798:CD81 protein, human; D012367:RNA, Viral; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; D060225:Tetraspanin 28