Claims
- 1. An isolated nucleic acid molecule encoding a replication competent recombinant Hepatitis C Virus (HCV) genome, which nucleic acid comprises all or part of an HCV genome and is able to replicate efficiently when transfected into a susceptible cell line without reducing the growth rate of said cell line by more than 10 fold.
- 2. The isolated nucleic acid molecule encoding a recombinant HCV genome of claim 1, which nucleic acid comprises from 5′ to 3′ on the positive-sense nucleic acid
(a) a functional 5′ HCV non-translated region (NTR) comprising an extreme 5′-terminal conserved sequence; (b) at least one open reading frame (ORF) encoding a heterologous gene operatively associated with an expression control sequence, wherein the heterologous gene and expression control sequence are oriented on the positive-strand nucleic acid molecule; (c) an ORF encoding at least a portion of an HCV polyprotein whose cleavage products form functional components of HCV virus particles and RNA replication machinery, and (d) an HCV 3′ NTR comprising an extreme 3′-terminal conserved sequence, and wherein said nucleic acid is able to replicate efficiently in a susceptible cell line without reducing the growth rate of said cell line by more than 10 fold.
- 3. The isolated nucleic acid of claim 1, wherein the susceptible cell line is selected from the group consisting of human hepatoma cell line Huh-7, human hepatoma cell line HepG2, hepatoma cell line PH5CH, T. belangeri liver cell line MBTL, human diploid fibroblast cell line VERO, secondary monkey kidney cell line CV-1, T cell line MT-2, T cell line HPBMa10-2, T cell line MOLT-4, and B cell line Daudi.
- 4. The susceptible cell line of claim 4, which is human hepatoma cell line Huh-7.
- 5. The isolated nucleic acid molecule according to claim 1, which is selected from the group consisting of double stranded DNA, single stranded DNA, double stranded RNA, and single stranded RNA.
- 6. An isolated nucleic acid molecule which is not more than 99.9% identical and is at least 95% identical to SEQ ID NO: 1.
- 7. The isolated nucleic acid molecule of claim 6 comprising nucleotide sequence of HCVR 2 (SEQ ID NO: 2).
- 8. The isolated nucleic acid molecule of claim 6 comprising nucleotide sequence of HCVR 8 (SEQ ID NO: 3).
- 9. The isolated nucleic acid molecule of claim 6 comprising nucleotide sequence of HCVR 9 (SEQ ID NO: 4).
- 10. The isolated nucleic acid molecule of claim 6 comprising nucleotide sequence of HCVR 22 (SEQ ID NO: 5).
- 11. The isolated nucleic acid molecule of claim 6 comprising nucleotide sequence of HCVR 24 (SEQ ID NO: 6).
- 12. A stable cell line transfected with the isolated nucleic acid molecule according to claim 1, wherein said cell line:
(a) has a growth rate which is not less than 10% of the growth rate of the corresponding naive cell line, and (b) is capable of supporting efficient replication of said isolated nucleic acid.
- 13. The cell line of claim 12 wherein said cell line is selected from the group consisting of human hepatoma cell line Huh-7, human hepatoma cell line HepG2, hepatoma cell line PH5CH, T. belangeri liver cell line MBTL, human diploid fibroblast cell line VERO, secondary monkey kidney cell line CV-1, T cell line MT-2, T cell line HPBMa10-2, T cell line MOLT-4, and B cell line Daudi.
- 14. The cell line of claim 12 wherein said cell line is derived from a human hepatoma cell line Huh-7.
- 15. The cell line of claim 14 designated HCVR 2 and having ATCC Accession No. PTA-2489.
- 16. The cell line of claim 14 designated HCVR 8 and having ATCC Accession No. PTA-2490.
- 17. The cell line of claim 14 designated HCVR 9 and having ATCC Accession No. PTA-2486.
- 18. The cell line of claim 14 designated HCVR 22 and having ATCC Accession No. PTA-2487.
- 19. The cell line of claim 14 designated HCVR 24 and having ATCC Accession No. PTA-2488.
- 20. A method of screening for anti-HCV therapeutics, which method comprises comparing a level of HCV subgenomic replicon RNA or replicon RNA-associated protein expression in the cell line of claim 12 contacted with a candidate therapeutic agent to the cell line not contacted with the candidate therapeutic agent, wherein a decrease in the level of HCV subgenomic replicon RNA or replicon RNA-associated protein expression is indicative of the inhibitory activity of the agent.
- 21. A method for detecting antibodies to HCV in a biological sample from a subject comprising contacting said sample with the protein fractions derived from the cell line of claim 12 under conditions that permit interaction of HCV-specific antibodies in the sample with the HCV protein(s) produced in said cell line, followed by detecting binding of the antibodies in the sample to these HCV-derived protein(s), wherein said binding is indicative of the presence of HCV infection in the subject from which the sample was derived.
- 22. The method of claim 21 wherein said biological sample is selected from the group consisting of blood, serum, plasma, blood cells, lymphocytes, and liver cells.
FIELD OF THE INVENTION
[0001] This application claims the benefit of U.S. Provisional Application No. 60/245,866 filed Nov. 7, 2000, which is hereby incorporated by reference in its entirety.
[0002] The present invention relates to recombinant hepatitis C virus (HCV)-derived nucleic acids and to stable rapidly growing cell clones supporting their efficient replication.
Provisional Applications (1)
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Number |
Date |
Country |
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60245866 |
Nov 2000 |
US |