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-␣ 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-␣2b 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. Conclusion: 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. (HEPATOLOGY 2009;49:364-377.) A bout 180 million people are infected with hepatitis C virus (HCV) worldwide, and HCV is a main contributor to chronic liver disease. The positive-stranded RNA genome of HCV has significant heterogeneity with six major genotypes and numerous subtypes. In the Americas, Europe and Japan genotypes 1a, 1b, and 3a are the most common, but 2a and 2b also show a significant presence. Genotypes 4 and 5 are found primarily in the Middle East and Africa, while genotype 6 predominates in Southeast Asia, a region with a high prevalence of HCV. 1 Recently, genotype 7a was discovered in Canadian and Belgian patients, who were presumably infected in Central Africa. 2

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To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (⌬40) or 25-aa (⌬25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. ⌬40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log 10 focus-forming units (FFU)/ml. 2a(J6) with ⌬40 or ⌬25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP⌬40 acquired various deletions in EGFP, while 2a(J6)⌬40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP-or RLuc-NS5A⌬40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log 10 FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence-and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.Hepatitis C virus (HCV) is a small, enveloped virus classified as a member of the family Flaviviridae. Its single-stranded RNA genome contains one long open reading frame (ORF) encoding structural proteins (core and envelope glycoproteins E1 and E2), p7, forming a putative ion channel, and nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (8). The NS5A phosphoprotein consists of 3 domains (47). While NS5A domains I and II are important for replication (46,47), domain III has been shown to be important for viral assembly (5,28,45). Due to significant genetic heterogeneity, HCV was classified into 7 major genotypes and numerous subtypes that differ in ϳ30% and ϳ20% of their sequences, respectively (42). Genotypes also differ biologically (34) and in their rates of response to interferon (IFN)-based combination therapy and specific antivirals (27, 36).HCV poses a major socioeconomic burden, with approximately 180 million chronically infected individuals worldwide who are at risk of developing liver cirrhosis and hepatocellular carcinoma (15, 41). Treatment options are limited; combination therapy with IFN-alfa-2 and ri...