Abstract: The present inventors developed three 4a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and all of or part of NS2 were replaced by the corresponding genes of the genotype 4a reference strain ED43. The 4a/2a junction in NS2 was placed after the first transmembrane domain (?), in the cytoplasmic part (?) or at the NS2/NS3 cleavage site (y). Following transfection of Huh7.5 cells with RNA transcripts, infectious viruses were produced in the ED43/JFH1-? and -y cultures only. Compared to the 2a control virus, production of infectious viruses was significantly delayed. However, in subsequent passages efficient spread of infection and high HCV RNA titers were obtained. Infectivity titers were approximately 10-fold lower than for the 2a control virus. Sequence analysis of recovered 4a/2a recombinants from 3 serial passages and subsequent reverse genetic studies revealed a vital dependence on a mutation in the NS2 4a part. ED43/JFH1-? further depended on a second NS2 mutation.

Abstract: The present inventors developed hepatitis C virus recombinants expressing NS5A from genotype 1a, 1b, 2a, 3a, 4a, 5a, 6a or 7a in the context of a genotype 2a backbone. Additional recombinants express NS5A and the structural proteins (Core, E1 and E2), p7 and NS2 from genotype 1a, 1b, 3a, 4a, 5a, 6a or 7a in the genotype 2a backbone. Sequence analysis of the recombinants recovered after viral passage in Huh7.5 cells revealed adaptive mutations in NS5A and/or NS3. The importance of these mutations for improved growth kinetics was shown in reverse genetic studies.

Abstract: The present invention relates to molecular approaches to the production of nucleic acid sequences, which comprises the genome of infectious hepatitis C virus. In particular, invention provides nucleic acid sequences which comprise the genomes of infectious hepatitis C viruses of either genotype 3a (strain S52) or genotype 4a (strain ED43). The invention therefore relates to the use of the nucleic acid sequences and polypeptides encoded by all or part of the sequences in the development of vaccines and diagnostic assays for HCV and in the development of screening assays for the identification of antiviral agents for HCV. The invention therefore also relates to the use of viral particles derived from laboratory animals infected with S52 and ED43 viruses.

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: Genotype 7a has been identified recently, thus not much is known about the biology of this new, major HCV genotype. The present inventors developed hepatitis C virus 7a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 7a strain QC69 and characterized them in Huh7.5 cells. Sequence analysis of 7a/JFH1 recombinants recovered after viral passage in Huh7.5 cells following 4 independent transfection experiments revealed adaptive mutations in Core, E2, NS2, NS5A and NS5B. In reverse genetic studies the importance of these mutations for improved growth kinetics was shown. Adapted 7a/JFH1 viruses showed growth kinetics, infectivity and RNA titers comparable to a previously developed 3a/JFH1 reference virus. Conclusion: The developed 7a/JFH1 viruses provide a robust in vitro tool for research in HCV genotype 7, including vaccine studies and functional analyses.

Abstract: The present inventors developed hepatitis C virus 2b/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 2b reference strain J8. Sequence analysis of recovered 2b/2a recombinants from 2 transfection experiments revealed that 2b/2a was genetically stable. Conclusion: The developed 2b/2a viruses provide a robust in vitro tool for research in HCV genotype 2b, including vaccine studies and functional analyses.

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 developed hepatitis C virus 6a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 6a reference strain HK6a. Sequence analysis of recovered 6a/2a recombinants from 2 transfection experiments and subsequent reverse genetic studies revealed adaptive mutations in E1 and E2. Conclusion: The developed 6a/2a viruses provide a robust in vitro tool for research in HCV genotype 6, including vaccine studies and functional analyses.

Abstract: The present inventors developed 5a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and all of or part of NS2 were replaced by the corresponding genes of the genotype 5a reference strain SA13. Compared to the J6/JFH control virus, after transfection of in vitro transcripts in Huh7.5 cells, production of infectious viruses was delayed. However, in subsequent viral passages efficient spread of infection and HCV RNA titers as high as for J6/JFH were obtained. Infectivity titers were at all time points analyzed comparable to J6/JFH control virus. Sequence analysis of recovered 5a/2a recombinants from 2 serial passages and subsequent reverse genetic studies revealed adaptive mutations in p7, NS2 and/or NS3. Infectivity of the 5a/2a viruses was CD81 and SR-BI dependant, and the recombinant viruses could be neutralized by chronic phase sera from patients infected with genotype 5a.

Abstract: The present inventors developed three 4a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and all of or part of NS2 were replaced by the corresponding genes of the genotype 4a reference strain ED43. The 4a/2a junction in NS2 was placed after the first transmembrane domain (a), in the cytoplasmic part (?) or at the NS2/NS3 cleavage site (y). Following transfection of Huh7.5 cells with RNA transcripts, infectious viruses were produced in the ED43/JFH1-? and -y cultures only. Compared to the 2a control virus, production of infectious viruses was significantly delayed. However, in subsequent passages efficient spread of infection and high HCV RNA titers were obtained. Infectivity titers were approximately 10-fold lower than for the 2a control virus. Sequence analysis of recovered 4a/2a recombinants from 3 serial passages and subsequent reverse genetic studies revealed a vital dependence on a mutation in the NS2 4a part. ED43/JFH1-? further depended on a second NS2 mutation.

Abstract: The present inventors have developed a culture system for genotype 3a, which has a high prevalence worldwide. Since intergenotypic recombinant genomes exploiting the replication characteristics of JFH1 will be a valuable tool for the genotype specific study of the replaced genes and related therapeutics, the present inventors constructed a genotype 3a/2a (S52/JFH1) recombinant containing the structural genes (Core, E1, E2), p7 and NS2 of strain S52 and characterized it in Huh7.5 cells. S52/JFH1 and J6/JFH viruses passaged in cell culture had comparable growth kinetics and yielded similar peak HCV RNA titers and infectivity titers. Direct genome sequencing of cell culture derived S52/JFH1 viruses identified putative adaptive mutations in Core, E2, p7, NS3 and NS5A; clonal analysis revealed, that all genomes analyzed exhibited different combinations of these mutations.