Infectious hepatitis C viruses of genotype 3A and 4A and uses thereof

Patent metadata

Field	Value
Publication number	US-9683269-B2
Application number	US-201414514023-A
Country	US
Kind code	B2
Filing date	Oct 14, 2014
Priority date	Oct 2, 2009
Publication date	Jun 20, 2017
Grant date	Jun 20, 2017

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, the 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.

First claim

What is claimed is:

1. An isolated nucleic acid molecule comprising a mutated human hepatitis C virus genome of genotype 3a wherein said molecule encodes a nucleic acid sequence according to SEQ ID NO:3 wherein all of the sequences encoding the E1 gene, E2 gene, P7 gene, and NS2 gene sequences are deleted, a part of the Core gene sequence is deleted, and a heterologous reporter gene sequence is inserted, and wherein HCV genomic portions of the molecule with the deletions have a sequence identity of at least 98% to a reference SEQ ID NO:3 sequence wherein all of the E1 gene, E2 gene, P7 gene and NS2 gene sequences are deleted and wherein said part of said Core gene sequence is deleted.

2. The nucleic acid molecule according to claim 1 , wherein said molecule encoding the mutated human hepatitis C virus of genotype 3a encodes the amino acid sequence that has a sequence identity of at least 98% to that of a reference SEQ ID NO:1 sequence wherein all of the E1, E2, P7, and NS2 amino acid sequences are deleted and part of the Core amino acid sequence is deleted.

3. The nucleic acid molecule according to claim 1 , wherein said molecule when encoding human hepatitis C virus of genotype 3a has a sequence identity of at least 99% to the reference SEQ ID NO: 3 sequence wherein nucleic acid sequences encoding all of the E1, E2, P7, and NS2 genes are deleted and part of the Core gene sequence is deleted.

4. A DNA construct comprising a nucleic acid molecule according to claim 1 .

5. An RNA transcript of the DNA construct according to claim 4 which encodes the mutated human hepatitis C virus genome of genotype 3a.

6. A cell transfected with the DNA construct of claim 4 .

7. A cell transfected with the RNA transcript according to claim 5 .

8. A method for producing a mutated hepatitis C virus genome comprising transfecting a host cell with the RNA transcript according to claim 5 .

9. A method for assaying candidate antiviral agents for activity against HCV, comprising: a) exposing a cell containing the mutated hepatitis C virus genome according to claim 1 to the candidate antiviral agent; and b) measuring the presence or absence of hepatitis C virus replication or correlates thereof in the cell of step (a).

10. The method according to claim 9 , wherein said replication in step (b) is measured by at least one of the following: negative strand RT-PCR, quantitative RT-PCR, Western blot, immunofluorescence, reporter gene activity, or non-fluorescent immuno-staining.

11. A method for determining the susceptibility of cells in vitro to support HCV replication, comprising the steps of: a) growing animal cells in vitro; b) transfecting into said cells the nucleic acid according to claim 1 ; and c) determining if said cells show indicia of HCV replication.

12. The method according to claim 11 , wherein said cells are human cells.

13. The method according to claim 11 , wherein said replication in step (c) is measured by at least one of the following reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, or reporter gene activity.

Assignees

• Hvidovre Hospital
• Us Health

Inventors

• Gottwein Judith M
• Scheel Troels Kasper Hoyer
• Purcell Robert
• Bukh Jens

Classifications

• C12N2770/24243

  viral genome or elements thereof as genetic vector · CPC title

• C12N15/86 Primary

  Viral vectors · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2770/24221

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12Q1/18 Primary

  Testing for antimicrobial activity of a material · CPC title