Abstract: The present inventors used the previously developed H77/JFH 1T27OOC,A4O8OT (1a/2a), J4/JFH 1T2996C,A4827T,?HVRI (1b/2a), J6/JFH 1?HVRI (2a/2a), J8/JFH 1?HVRI (2b/2a), S52/JFH 1T27i8G,?7i6oc (3a/2a), SA13/JFH 1C34O5G,A3696G (5a/2a) and HK6a/JFH 1T1389c,A1590G (6a/2a) constructs for the deletion of Hypervariable Region 1 (HVR1) to construct viable, JFH 1 (genotype 2a) based, genomes. The present inventors serially passaged the viruses in cell culture obtaining relatively high HCV RNA titers and infectivity titers. Sequence analysis of the viruses identified mutations adapting H77/JFH 1T27OOC,A4O8OT,?HVR1 (1a/2a), J8/JFH 1?HVR1 (2b/2a), S52/JFH 1T2718G,T716OC,?HVR1 (3a/2a) and J4/JFH 1T2996C,A4827T,?HVR1 (1b/2a) to the HVR1 deletion.

Abstract: Hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A were developed. A deletion upstream of the inserted reporter gene sequence conferred favorable growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. JFH1-based intergenotypic recombinants with genotype specific homotypic 5?UTR, or heterotypic 5?UTR (either of genotype 1a (strain H77) or of genotype 3a (strain S52)) were also developed. The present inventors additionally developed J6/JFH1 recombinants with the 5?UTR of genotypes 1-6. These recombinants with different 5?UTRs are a useful to study the function of the 5?UTR in a genotype specific manner.

Abstract: The present inventors developed hepatitis C virus 1a/2a and 1b/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and NS2 were replaced by the corresponding genes of the genotype Ia reference strain H77C or TN or the corresponding genes of the genotype Ib reference strain J4. Sequence analysis of recovered 1a/2a and 1b/2a recombinants from 2 serial passages and subsequent reverse genetic studies revealed adaptive mutations in e.g. p7, NS2 and/or NS3. In addition, the inventors demonstrate the possibility of using adaptive mutations identified for one HCV isolate in generating efficient cell culture systems for other isolates by transfer of mutations across isolates, subtypes or major genotypes. Furthermore neutralization studies showed that viruses of e.g. genotype 1 were efficiently neutralized by genotype Ia, 4a and 5a serum, an effect that could be utilized e.g. in vaccine development and immunological prophylaxis.

Abstract: The present inventors used the previously developed H77/JFH1T27OOC,A4O8OT (1a/2a), J4/JFH1T2996C,A4827T,?HVRI (1b/2a), J6/JFH1?HVRI (2a/2a), J8/JFH1?HVRI (2b/2a), S52/JFH1T27i8G,?7i6oc (3a/2a), SA13/JFH1C34O5G,A3696G (5a/2a) and HK6a/JFH1T1389c,A1590G (6a/2a) constructs for the deletion of Hypervariable Region 1 (HVR1) to construct viable, JFH1 (geno-type 2a) based, genomes. The present inventors serially passaged the viruses in cell culture obtaining relatively high HCV RNA titers and infectivity titers. Sequence analysis of the viruses identified mutations adapting H77/JFH1T27OOC,A4O8OT,?HVR1 (1a/2a), J8/JFH1?HVR1 (2b/2a), S52/JFH1T2718G,T716OC,?HVR1 (3a/2a) and J4/JFH1T2996C,A4827T,?HVR1 (1b/2a) to the HVR1 deletion.

Abstract: The present inventors developed hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A. The inventors have identified a deletion upstream of the inserted reporter gene sequence, which conferred favourable growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. Drugs could be evaluated for their potential to prevent infection or cure infected cells. The neutralizing capacity of antibodies induced by vaccine candidates could be evaluated in order to define successful vaccination strategies. Broadly neutralizing antibodies could be identified testing engineered antibodies and antibodies derived from serum of HCV infected individuals; thus this technique could contribute to the development of immunotherapy.

Abstract: The present inventors developed hepatitis C virus 1a/2a and 1b/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and NS2 were replaced by the corresponding genes of the genotype Ia reference strain H77C or TN or the corresponding genes of the genotype Ib reference strain J4. Sequence analysis of recovered 1a/2a and 1b/2a recombinants from 2 serial passages and subsequent reverse genetic studies revealed adaptive mutations in e.g. p7, NS2 and/or NS3. In addition, the inventors demonstrate the possibility of using adaptive mutations identified for one HCV isolate in generating efficient cell culture systems for other isolates by transfer of mutations across isolates, subtypes or major genotypes. Furthermore neutralization studies showed that viruses of e.g. genotype 1 were efficiently neutralized by genotype Ia, 4a and 5a serum, an effect that could be utilized e.g. in vaccine development and immunological prophylaxis.