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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Efficient in vitro systems to study the life cycle of hepatitis C virus (HCV) were recently developed for JFH1 (genotype 2a), which has unique replication capacity in Huh7 cells. We developed 4a/JFH1 intergenotypic recombinants containing the structural genes (Core, E1, and E2), p7, and all or part of NS2 of the 4a prototype strain ED43 that, after transfection of Huh7.5 cells with RNA transcripts, produced infectious viruses. Compared with the J6/JFH control virus, production of viruses was delayed. However, efficient spread of infection and high HCV RNA and infectivity titers were obtained in serial passages. Sequence analysis of recovered viruses and subsequent reverse genetic studies revealed a vital dependence on one or two NS2 mutations, depending on the 4a/2a junction. Infectivity of ED43/JFH1 viruses was CD81 dependent. The genotype 4 cell culture systems permit functional analyses as well as drug and vaccine research on an increasingly important genotype in the Middle East, Africa, and Europe. We also developed genotype 1a intergenotypic recombinants from H77C with vital mutations in NS3. Using H77C/JFH1 and ED43/JFH1 viruses, we demonstrated high homologous neutralizing antibody titers in 1a and 4a patient sera, respectively. Furthermore, availability of JFH1 viruses with envelope proteins of the six major HCV genotypes permitted cross-neutralization studies; 1a and 4a serum crossneutralized 1a, 4a, 5a, and 6a but not 2a and 3a viruses. Thus, the JFH1 intergenotypic recombinants will be of importance for future studies of HCV neutralization and accelerate the development of passive and active immunoprophylaxis.Egypt ͉ hepatitis C virus strain ED43 ͉ in vitro infection ͉ intergenotypic recombinant ͉ vaccine A pproximately 180 million people are infected with hepatitis C virus (HCV) and are at increased risk of developing severe liver disease. HCV isolates from around the world cluster into six major genotypes, which differ by Ϸ30% at the nucleotide (nt) and deduced amino acid level (1). Genotype 4 is primarily found in the Middle East and Africa (2, 3). In Egypt, Ϸ15% of the population is HCV-infected, with genotype 4a comprising Ϸ90% of cases (2, 3). This particularly high prevalence, presumably caused by an unintended transmission through parenteral treatment for schistosomiasis (3, 4), is at least partly responsible for a still rather high incidence (5), making Egypt a potential region for vaccine trials. Additionally, genotype 4 has been spreading in Europe, resulting in a prevalence of 10% in certain regions (2). Although the only approved treatment for chronic HCV infection, combination therapy with IFN-␣ and ribavirin, leads to a sustained virologic response in most of genotype 2 or 3 patients, viral clearance is obtained for only approximately half of patients with genotype 1 or 4. There is no vaccine against HCV.Research on specific antiviral drugs and vaccines has been hampered by the absence of a full viral life cycle cell culture system. However, development of such a system came with cDNA c...

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein has been implicated in virus neutralization and persistence. We deleted HVR1 from JFH1-based HCV recombinants expressing Core/E1/E2/p7/NS2 of genotypes 1 to 6, previously found to grow efficiently in human hepatoma Huh7.5 cells. The 2a ⌬HVR1 , 5a ⌬HVR1 , and 6a ⌬HVR1 Core-NS2 recombinants retained viability in Huh7.5 cells, whereas 1a ⌬HVR1 , 1b ⌬HVR1 , 2b ⌬HVR1 , 3a ⌬HVR1 , and 4a ⌬HVR1 recombinants were severely attenuated. However, except for recombinant 4a ⌬HVR1 , viruses eventually spread, and reverse genetics studies revealed adaptive envelope mutations that rescued the infectivity of 1a ⌬HVR1 , 1b ⌬HVR1 , 2b ⌬HVR1 , and 3a ⌬HVR1 recombinants. Thus, HVR1 might have distinct functional roles for different HCV isolates. Ultracentrifugation studies showed that deletion of HVR1 did not alter HCV RNA density distribution, whereas infectious particle density changed from a range of 1.0 to 1.1 g/ml to a single peak at ϳ1.1 g/ml, suggesting that HVR1 was critical for low-density HCV particle infectivity. Using chronic-phase HCV patient sera, we found three distinct neutralization profiles for the original viruses with these genotypes. In contrast, all HVR1-deleted viruses were highly sensitive with similar neutralization profiles. In vivo relevance for the role of HVR1 in protecting HCV from neutralization was demonstrated by ex vivo neutralization of 2a and 2a ⌬HVR1 produced in human liver chimeric mice. Due to the high density and neutralization susceptibility of HVR1-deleted viruses, we investigated whether a correlation existed between density and neutralization susceptibility for the original viruses with genotypes 1 to 6. Only the 2a virus displayed such a correlation. Our findings indicate that HVR1 of HCV shields important conserved neutralization epitopes with implications for viral persistence, immunotherapy, and vaccine development.

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MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′ UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5′ UTR of different HCV genotypes have not been available for testing 5′ UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5′ UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5′ UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 5′ UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.cell culture infection | locked nucleic acid therapy | miRNA | RNA recombination H epatitis C virus (HCV) infects ∼180 million people worldwide, often causing fatal chronic liver diseases. HCV is a ∼9.6-kb positive-sense RNA virus belonging to the genus Hepacivirus in the Flaviviridae family (1), which has been classified into seven major genotypes and numerous subtypes (2, 3). The viral genome consists of a single ORF flanked by 5′ and 3′ UTRs. The 5′ UTR consists of ∼341 nucleotides forming four major domains. Domain I [nucleotides 1-43, numbering according to reference isolate H77 (GenBank accession no. AF009606)] contains one stem-loop structure (SLI), essential for RNA replication (1), and two microRNA-122 (miR-122) binding sites (Fig. S1), through which liver-abundant miR-122 stimulates HCV replication and translation (4-6). Although the 5′ UTR is a conserved region, a number of genotype-specific nucleotides naturally exist, which have been used for HCV genotyping (3). To date, genotype-specific functional analysis of the HCV 5′ UTR has been hampered by lack of suitable culture systems.Current therapy with pegylated IFN-α and ribavirin has severe side effects and the outcome is dependent on the infecting HCV genotype. Overall, ∼50% of patients completing treatment are cured. Thus, therapeutic drugs that are more effective against diverse HCV genotypes are urgently needed. R...

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.epatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The outcome of infection is associated with genetic variability of HCV and host factors (1). No vaccine is available, and current IFNbased treatment is suboptimal, with many side effects, with low efficacy against the most prevalent HCV variants (2-4), and with differential influence from host factors (5). Directly acting antivirals (DAA) might improve treatment outcome but also have differential efficacy in treatment of patients with different HCV genotypes (6). The HCV positive sense single-strand RNA genome (∼9.6 kb) contains a single ORF flanked by 5′ and 3′ untranslated regions (UTRs). The ORF encodes virus structural proteins (Core, E1, and E2), p7, and six nonstructural (NS) proteins (7). HCV isolates are classified into seven major genotypes and numerous subtypes differing by 31-33% and 20-25%, respectively (8).The high heterogeneity of HCV and the lack of representative culture systems have hampered HCV vaccine development, preclinical drug testing, assessment of neutralizing antibodies, and basic HCV research. Although a number of HCV full-length genomes were shown to be infectious in chimpanzees (9-15), to date only the JFH1 strain (genotype 2a) could replicate auton...