# US 8,569,472 B2 · Efficient cell culture system for hepatitis C virus genotype 6A > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,569,472 B2 - Document ID: US8569472B2 - Status: Granted - Kind: B2 - Application number: 12/808,565 - Filed: 2008-12-19 - Published: 2011-03-10 - Granted: 2013-10-29 - Earliest claimed: 2007-12-20 - Assignees: Hvidovre Hospital - Applicants: Judith M. Gottwein; Troels Kasper Hoyer Scheel; Tanja Bertelsen Jensen; Jens Bukh - Inventors: Judith M. Gottwein; Troels Kasper Hoyer Scheel; Tanja Bertelsen Jensen; Jens Bukh - Web page: https://patentgrep.com/patent/US8569472B2 ## CPC - A61K 39/29: Hepatitis virus - A61K 39/12: Viral antigens - C07K 14/005: from viruses - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - A61K 2039/525: {Virus} - A61K 2039/53: {DNA (RNA) vaccination} - C12N 2770/24221: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2770/24222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2770/24234: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## 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 analysis. ## Claims ### Claim 1 A nucleic acid molecule comprising a nucleic acid which encodes a recombinant human hepatitis C virus of genotype 6a/JFH1 that is capable of expressing said virus when transfected into cells, wherein said molecule encodes an amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 2, wherein said molecule encodes an adaptive mutation F350S in the E1 protein and a T416A or N417T adaptive mutation in the E2 protein of SEQ ID NO: 2, and wherein said adaptive mutations provide an improved ability to generate infectious viral particles in cell culture compared to an HCV genotype 6a/JFH1 virus without said adaptive mutations. ### Claim 2 (depends on 1) The nucleic acid molecule according to claim 1, wherein said nucleic acid molecule is further capable of infectivity in vivo. ### Claim 3 (depends on 1) The nucleic acid molecule according to claim 1, wherein said molecule comprises the nucleic acid sequence with a sequence identity of at least 95% to that of SEQ ID NO:1. ### Claim 4 (depends on 1) The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises T1389C and A1590C adaptive mutations in SEQ ID NO:1. ### Claim 5 (depends on 1) The nucleic acid molecule according to claim 1, wherein said molecule encodes adaptive mutations F350S in the E1 gene and T416A in the E2 gene of SEQ ID NO:2. ### Claim 6 A nucleic acid molecule comprising a nucleic acid which encodes a recombinant human hepatitis C virus of genotype 6a/JFH1 that is capable of expressing said virus when transfected into cells, wherein said molecule encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 2, wherein said molecule comprises a T1389C adaptive mutation in the E1 gene and a T1581C, T1581G, or A1586G adaptive mutation in the E2 gene of SEQ ID NO:1, and wherein said adaptive mutations provide an improved ability to generate infectious viral particles in cell culture compared to an HCV genotype 6a/JFH1 virus without said adaptive mutations. ### Claim 7 (depends on 1) The nucleic acid molecule according to claim 1, wherein said molecule encodes adaptive mutations an F350S in the E1 protein and a N417T in the E2 protein of SEQ ID NO:2. ### Claim 8 (depends on 7) The nucleic acid molecule according to claim 7, wherein said molecule is capable of generating a HCV RNA titer of 104 IU/ml or above following transfection and/or subsequent viral passage. ### Claim 9 (depends on 7) The nucleic acid molecule according to claim 7, wherein said molecule is capable of generating a HCV infectivity titer of 102 TCID50/ml (50% tissue culture infectious doses)/ml or above following transfection and/or subsequent viral passage. ### Claim 10 A method for producing a hepatitis C virus particle, comprising: (a) culturing a cell comprising a nucleic acid molecule which encodes human hepatitis C virus of genotype 6a/JFH1 that is capable of expressing said virus when transfected into cells and wherein said nucleic acid molecule encodes an amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO:2, wherein said molecule encodes adaptive mutations F350S in the E1 protein and T416A or N417T in the E2 protein of SEQ ID NO:2, said adaptive mutations providing an improved ability to generate infectious viral particles in cell culture compared to an HCV genotype 6a/JFH1 virus without said adaptive mutations; and, (b) allowing the cell to produce the virus. ### Claim 11 A hepatitis C virus particle comprising a nucleic acid molecule which encodes human hepatitis C virus of genotype 6a/JFH1 that is capable of expressing said virus when transfected into cells and wherein said nucleic acid molecule encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO:2, wherein said molecule encodes adaptive mutations F350S in the E1 protein and a T416A or a N417T in the E2 protein of SEQ ID NO:2, and wherein said adaptive mutation provides an improved ability to generate infectious viral particles in cell culture compared to an HCV genotype 6a/JFH1 virus without said adaptive mutations. ### Claim 12 (depends on 11) The hepatitis C virus particle according to claim 11, wherein said hepatitis C virus particle is further capable of infectivity in vivo. ### Claim 13 (depends on 11) The hepatitis C virus particle according to claim 11, wherein said hepatitis C virus particle comprises the nucleic acid sequence with a sequence identity of at least 95% to that of SEQ ID NO 1. ### Claim 14 (depends on 11) The hepatitis C virus particle according to claim 11, wherein the nucleic acid molecule comprises a T1389C and an A1590C adaptive mutation in SEQ ID NO 1. ### Claim 15 (depends on 11) The hepatitis C virus particle according to claim 11, wherein the nucleic acid molecule encodes-adaptive mutations an F350S in the E1 protein and T416A in the E2 protein of SEQ ID NO:2. ### Claim 16 A hepatitis C virus particle, comprising a nucleic acid which encodes a recombinant human hepatitis C virus of genotype 6a/JFH1 that is capable of expressing said virus when transfected into cells and wherein said nucleic acid encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO 2, wherein said molecule comprises a T1389C adaptive mutation in the E1 gene and a T1581C, T1581G, or A1586G adaptive mutation in the E2 gene of SEQ ID NO:1, and wherein said adaptive mutations provide an improved ability to generate infectious viral particles in cell culture compared to an HCV genotype 6a/JFH1 virus without said adaptive mutations. ### Claim 17 (depends on 15) The hepatitis C virus particle according to claim 15, wherein said nucleic acid molecule is capable of generating a HCV RNA titer of 104 IU/ml or above following transfection and/or subsequent viral passage. ### Claim 18 (depends on 15) The hepatitis C virus particle according to claim 15, wherein said nucleic acid molecule is capable of generating a HCV infectivity titer of 102 TCID50/ml (50% tissue culture infectious doses)/ml or above following transfection and/or subsequent viral passage.