WO2008147735A1

WO2008147735A1 - Identification of adaptive mutations that increase infectivity of hepatitis c virus jfh1 strain in cell culture

Info

Publication number: WO2008147735A1
Application number: PCT/US2008/063982
Authority: WO
Inventors: Rodney Russell; Jens Bukh; Suzanne U. Emerson; Robert H. Purcell
Original assignee: THE GOVERNMENT OF UNITED STATES OF AMERICA as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES
Priority date: 2007-05-21
Filing date: 2008-05-16
Publication date: 2008-12-04

Abstract

Aspects of the present invention concern hepatitis C viruses (HCVs) that exhibit enhanced infectivity in cell culture and HCV RNAs that have autonomous replication and the ability to produce viral particles. Preferred embodiments utilize an HCV genomic RNA that has an amino acid mutation such as N417S, N765D, Q1012R, or L2175V, or a corresponding nucleotide mutation such as A1590G, A2633G, A3375G, T6863G, or synonymous mutation T1681C, and chimeras thereof.

Description

IDENTIFICATION OF ADAPTIVE MUTATIONS THAT INCREASE INFECTIVITY OF HEPATITIS C VIRUS JFHl STRAIN IN CELL CULTURE

Field of the Invention

Aspects of the present invention concern hepatitis C viruses (HCVs) that exhibit enhanced infectivity in cell culture and HCV RNAs that have autonomous replication and the ability to produce viral particles. Preferred embodiments utilize an HCV genomic RNA that has an amino acid mutation such as N417S, N765D, Q1012R, or L2175V, or a corresponding nucleotide mutation such as Al 590G, A2633G, A3375G, T6863G, or synonymous mutation Tl 681C, and chimeras thereof.

Description of the Related Art

Hepatitis C virus (HCV), a small, enveloped RNA virus of the Bepacivirus genus of the Flaviviridae family, is an important cause of acute and chronic liver disease worldwide (Bowen, D.G. & Walker, CM. 2005 J Hepatol 42:408-417; Lindenbach, B.D. & Rice, CM. 2001 In : Knipe DM, Howley PM, eds. Fields Virology, 991-1042). HCV research was severely hampered by the lack of a robust in vitro cell culture system until the isolation of a unique HCV genotype 2a sequence (JFHl) allowed for the development of a useful cell culture system (Kato, T. et al. 2001 J Med Virol 64:334-339). Wakita et al. demonstrated that Huh-7 cells transfected with full-length JFHl genomes secreted infectious virus particles (HC Vcc), albeit with low efficiency [(10²-10³ focus-forming units per ml (ffu/ml)] (Wakita, T. et al. 2005 Nat Med 11:791-796). Increased virion production was achieved by propagating virus in especially permissive subclones of Huh-7 cells (Zhong, J. et al. 2005 Pr oc Natl Acad Sci USA 102:9294-9299), by serially passaging JFHl to select adaptive mutations (Zhong, J. et al. 2006 J Virol 80:11082-11093), or by creating a chimeric virus (Lindenbach, B.D. et al. 2005 Science 309:623-626) between JFHl and another 2a strain, J6 (Yanagi, M. et al. 1999 Virology 262:250-263). Such manipulations resulted in infectious virus titers of 10⁴-10⁵ ffu/ml.

A number of recent studies have identified adaptive or compensatory mutations that enhance infectious virus production from either wild-type JFHl (Zhong, J. et al. 2006 J Virol 80: 11082-11093; Delgrange, D. et al. 2007 J Gen Virol 88:2495-2503; Kaul, A. et al. 2007 J Virol 81:13168-13179) or from inter-genotypic chimeras (Gottwein, J₁M, et al. 2007 Gastroenterology 133:1614-1626; Yi, M. et al. 2007 J Virol 81:629-638; Yi, M. et al. 2006 Proc Natl Acad Sci USA 103:2310-2315). To date, cell culture- selected mutations have been found in nearly all of the structural and non-structural proteins, but the majority of these have mapped to the core-NS2 coding region, with a noticeable preference for the p7 and NS2 proteins (Gottwein, J.M. et al. 2007 Gastroenterology 133:1614-1626; Yi, M. et al. 2007 J Virol 81:629-638). The functions of p7 and NS2 are not well defined, but accumulating evidence suggests that these proteins may be involved in assembly and/or release of infectious virus particles (Jones, CT. et al. 2007 J Virol 81:8374-8383; Murray, CL. et al. 2007 J Virol 81: 10220-10231; Steinmann, E. et al. 2007 PLoS Pathog 3:elO3), and that adaptive mutations in p7 and NS2 improve these putative functions in virus assembly and/or release (. Yi, M. et al. 2007 J Virol 81:629-638). Significant enhancements in virus expansion have also been attributed to adaptive mutations in the E2 glycoprotein (Zhong, J. et al. 2006 J Virol 80:11082-11093; Delgrange, D. et al. 2007 J Gen Virol 88:2495-2503). There remains a need for a better understanding of the impact that certain mutations have on HCV.

Summary of the Invention

Some aspects of invention concern the identification of new mutations in HCV that confer enhanced viral replication. By culturing JFHl-transfected Huh-7.5 cells under conditions that select for enhanced replication capacity, several unique adaptive mutations were identified. In some approaches, mutant HCV RNA genomes were transfected into a virtually non-infectable CD81 -deficient subclone of Huh-7.5 cells, which allowed for the identification of mutations that conferred enhanced virus entry and production. Several embodiments, for example, concern an isolated nucleic acid that comprises a genomic or subgenomic region of hepatitis C virus of genotype 2 a, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, wherein the hepatitis C virus of genotype 2a is of JFHl strain. Some embodiments include a viral RNA that comprises a nucleotide sequence having a 5' untranslated region, which contains an IRES sequence, a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, an NS3 protein coding sequence, an NS4A protein coding sequence, an NS4B protein coding sequence, an NS5A protein coding sequence, an NS5B protein coding sequence, and a 3' untranslated region of genomic RNA of hepatitis C virus of genotype 2a, which has at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation H618C, or at least one nucleotide mutation encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V_} wherein the genomic RNA of hepatitis C virus of genotype 2a is an RNA of JFHl strain. In some embodiments, the viral RNA comprises genomic RNA of hepatitis C virus of genotype 2a with a nucleotide sequence shown in SEQ ID NO: 1.

In more embodiments, the 5¹ untranslated region comprises a nucleotide sequence shown from nucleotide 1 to nucleotide 340 in SEQ ID NO: 1, the core protein coding sequence comprises a nucleotide sequence shown from nucleotide 341 to nucleotide 913 in SEQ ID NO: 1, the El protein coding sequence comprises a nucleotide sequence shown from nucleotide 914 to nucleotide 1489 in SEQ ID NO: 1, the E2 protein coding sequence comprises a nucleotide sequence shown from nucleotide 1490 to nucleotide 2590 in SEQ ID NO: 1, the p7 protein coding sequence comprises a nucleotide sequence shown from nucleotide 2591 to nucleotide 2779 in SEQ ID NO: 1, the NS2 protein coding sequence comprises a nucleotide sequence shown from nucleotide 2780 to nucleotide 3430 in SEQ ID NO: 1, the NS 3 protein coding sequence comprises a nucleotide sequence shown from nucleotide 3431 to nucleotide 5323 in SEQ ID NO: 1, the NS4A protein coding sequence comprises a nucleotide sequence shown from nucleotide 5324 to nucleotide 5485 in SEQ ID NO: 1, the NS4B protein coding sequence comprises a nucleotide sequence shown from nucleotide 5486 to nucleotide 6268 in SEQ ID NO: 1, the NS5A protein coding sequence comprises a nucleotide sequence shown from nucleotide 6269 to nucleotide 7666 in SEQ ID NO: 1, the NS 5 B protein coding sequence comprises a nucleotide sequence shown from nucleotide 7667 to nucleotide 9439 in SEQ ID NO: 1, and the 3' untranslated region comprises a nucleotide sequence shown from nucleotide 9440 to nucleotide 9678 in SEQ ID NO: 1.

Some embodiments comprise a viral RNA₃ having the following (a) or (b):

(a) an RNA comprising a nucleotide sequence shown in SEQ ID NO: I₅ having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V;

(b) an RNA comprising a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 1 by deletion, substitution or addition of 1 to 100 nucleotides, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation Ω618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and having autonomous replication ability and virus particle production ability.

More embodiments concern methods for producing a cell, which replicates an HCV RNA, comprising introducing one or more of the aforementioned viral RNAs into a cell. In some embodiments, the cell is a proliferative, human liver-derived cell (e.g., a Huh7 cell or a HepG2 cell). The cell culture cell, which replicates a viral RNA above may produce a viral particle. Accordingly, methods for producing hepatitis C virus particles, wherein any one or more of the aforementioned viral RNAs are introduced into a permissive cell are embodiments, as well as, the hepatitis C virus particle obtainable by said methods. By some approaches described herein, a hepatitis C virus infected cell is produced, by culturing a cell and infecting other cells with a virus particles present in the culture. Accordingly, some embodiments include a hepatitis C virus infected cell containing one or more of the nucleic acids described herein. Hepatotropic viral vector vectors that include one or more of the nucleic acids described herein and methods of making these compositions are also embodiments. Some embodiments, for example, concern a method for producing a cell, which replicates an RNA and produces a virus particle, comprising introducing into the cell the RNA comprising a nucleotide sequence shown in SEQ ID NO. 1, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation H618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V.

Another embodiment is a method for producing a hepatitis C virus particle, comprising introducing into a cell the RNA comprising a nucleotide sequence shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of A1590G, A2633G, A3375G, T6863G, and synonymous mutation H618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and culturing the cell to allow the cell to produce a virus particle,

Another embodiment is a modified hepatitis C virus genomic RNA comprising genomic RNA portions of two or more types of hepatitis C viruses, which comprises a 5' untranslated region, a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, a partial RNA sequence encoding NS3, NS4A, NS4B, NS5A, and NS5B proteins of a JFHl strain shown in SEQ ID NO: I₅ having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation Ω618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and a 3' untranslated region, and which can be autonomously replicated.

Another embodiment is a modified hepatitis C virus genomic RNA, which is produced by substituting a hepatitis C virus genomic RNA portion ranging from an NS3 protein coding sequence to an NS5B protein coding sequence with a partial RNA sequence encoding the NS3, NS4, NS5A, and NS5B proteins of a JPHl strain shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and which can be autonomously replicated.

Some embodiments include a modified hepatitis C virus genomic RNA, wherein the hepatitis C virus genotype is selected from the group consisting of Ia, Ib, Ic, 2a, 2b_; 2c, 2k, 3a, 3b, 3k, 4a, 5a, 6a, 6b, 6d, 6g, 6h and 6k. Another embodiment is a cell culture cell into which the modified hepatitis C virus genomic RNA is introduced, and which replicates the hepatitis C virus genomic RNA and can generate virus particles.

Another embodiment is a method for producing hepatitis C virus particles, the method comprising culturing the cell into which the modified hepatitis C virus genomic RNA is introduced, and which replicates the hepatitis C virus genomic RNA and can generate virus particles and recovering virus particles from the culture.

Another embodiment is a method for purifying HCV particles, the method comprising subjecting a product obtained from a homogenate of a cell or from the medium of cultured cells to column chromatography and/or density gradient centrifugation.

More embodiments concern hepatitis C vaccines or hepatitis C immunogenic compositions that comprise one or more of the nucleic acids or peptides described herein. In some embodiments, the hepatitis C immunogenic substance and/or vaccine comprises an isolated nucleic acid that comprises a genomic or subgenomic region of hepatitis C virus of genotype 2a, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, wherein the hepatitis C virus of genotype 2a is of JFHl strain. Methods for producing and formulating these compositions are also embodiments.

Another embodiment is a hepatitis C immunogenic substance and/or vaccine comprising a modified hepatitis C virus genomic RNA comprising genomic RNA portions of two or more types of hepatitis C viruses, which comprises a 5' untranslated region, a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, a partial RNA sequence encoding NS3, NS4A, NS4B, NS5A, and NS5B proteins of a JFHl strain shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and a 3' untranslated region, and which can be autonomously replicated. Another embodiment is a hepatitis C immunogenic substance and/or vaccine comprising a modified hepatitis C virus genomic RNA, which is produced by substituting a hepatitis C virus genomic RNA portion ranging from an NS3 protein coding sequence to an NS5B protein coding sequence with a partial RNA sequence encoding the NS3, NS4, NS5A, and NS5B proteins of a JFHl strain shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation T1618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and which can be autonomously replicated.

Another embodiment is a method for screening an anti-hepatitis C virus substance, the method comprising culturing a cell into which the modified hepatitis C virus genomic RNA is introduced, and which replicates the hepatitis C virus genomic RNA, in the presence of a test substance and detecting hepatitis C virus RNA or virus particles in the culture, thereby evaluating the anti-hepatitis C virus effects of the test substance. For example, methods for screening and/or identifying an anti-hepatitis C virus substance can comprise culturing, in the presence of a test substance, at lease one composition selected from the group consisting of the following (a), (b) and (c):

(a) a cell culture cell, which replicates a viral RNA and produces the virus particle, (b) a hepatitis C virus infected cell, and (c) a hepatitis C virus particle and a hepatitis C virus permissive cell; and detecting the viral RNA or the virus particles in the resulting culture.

Another aspect is a method of screening for an anti-hepatitis C virus substance comprising the steps of: a) mixing a hepatitis C virus particle that comprises a viral RNA having at least one nucleotide mutation selected from the group consisting of A 1590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V with a substance; b) exposing a hepatitis C virus-permissive cell to the virus/substance mixture of step (a); and c) measuring viral replication in the cells; wherein a reduction or increase in the replication of virus that was pre-exposed to the substance relative to the replication of control virus that were not pre-exposed to the substance demonstrates that the substance alters viral replication.

Another embodiment is a method of screening for an anti-hepatitis C virus substance comprising the steps of: a) treating hepatitis C virus-permissive cells with a substance; b) exposing the treated cells to a hepatitis C virus particle that comprises a viral RNA having at least one nucleotide mutation selected from the group consisting of A1590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and c) measuring viral replication in said treated cells; wherein a reduction or increase in the replication of the virus in the treated cells relative to the replication of virus in control cells that were not treated with the substance demonstrates that the substance alters viral replication.

Another embodiment is method of screening for an anti-hepatitis C virus substance comprising the steps of: a) infecting a hepatitis C virus -permissive cell with a hepatitis C virus particle that comprises a viral RNA having at least one nucleotide mutation selected from the group consisting of Al 59OG₅ A2633G, A3375G, T6863G, and synonymous mutation T1618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V; b) treating the infected cells with a substance; and c) measuring viral replication in said treated cells, wherein a reduction or increase in the replication of the virus in the treated cells relative to the replication of the virus in control cells that were not treated with said substance demonstrates that the substance alters viral replication.

Brief Description of the Drawings

Figure 1. Gene organization of HCV and processing of viral proteins. Dotted boxes and open boxes indicate regions for the structural and nonstructural proteins, respectively. Untranslated regions of 5' and 3' are shown on the left and the right side of the genome structure, respectively. Cleavage at the Core (C)/E1_> E1/E2, E2/p7, and p7/NS2 junctions are mediated by host signalases(s) indicated by open triangles. Processing at the NS2/NS3 junction mediated by the NS2-3 metalloprotease is indicated by a hatched triangle. NS3 serine protease cleavage sites are indicated by filled triangles.

Figure 2. Amino Acid and nucleotide sequences of hepatitis C Virus (isolate JFH-I), (A) Amino acid sequence (SEQ ID NO: 2), (B) Nucleotide sequence (SEQ ID NO: 1). The nucleotide changes selected during JFHl culture are as follows: E2 - (Al 590G), E2 - (Tl 681C), p7 - (A2633G), NS2 - (A3375G), and NS5A (T6863G).

Figure 3. The structures of the HCV RNA genome, the SGR-JFHl replicon, and full- length chimeric genomes FL-J6/JFH and FL-H77/JFH. NCR, noncoding region; C, core; light gray, J6; dark gray, H77; medium gray, JFH.

Figure 4. Mutations permit 3-4 log higher levels of virus production. One million Huh-7.5 cells were inoculated with transfection supernatants containing 100 ffu (m.o.i. = 0.0001) of indicated HCVcc. (A) HCV RNA levels in culture fluids at indicated time-points post-infection were measured by TaqMan Real Time RT-PCR. Culture supernatant from an SGR-JFHl transfection was used as a mock infection control. Data points represent the mean value obtained from duplicate TaqMan amplifications of the same sample. (B) Infectious viral titers were measured at indicated time-points post-infection by a limiting dilution assay for focus-forming units. Results are representative of at least 2 independent transfection/infection experiments. Assays of focus-forming units were performed in triplicate and the means plus standard error are plotted. The dotted line represents the cutoff of the assay, which was 10 ffu/ml.

Figure 5. Effects of individual mutations on virus replication capacity. One million Huh-7.5 cells were inoculated with RNase-treated transfection supernatants containing 500 ffu (m.o.i. = 0.0005) of indicated HCVcc (A) HCV RNA levels and (B) infectious viral titers were measured at indicated days post-infection.

Figure 6. Characterization of a cell line that can support virus replication but cannot be infected. (A) Huh-7.5 cells pre-infected with JFH-AM2 were co-cultured in 8-chamber slides with either Huh-7,5 or S29 cells at a 1:100 ratio of infected:uninfected cells. Virus spread was monitored by IF of HCV core antigen on days 1 and 3. Green fluorescence represents HCV core and blue represents DAPI- stained nuclei as observed using the 1OX objective. (B) Two hundred thousand S29 cells were transfected with either irrelevant vector (left) or human CD81 (right) in 2-chamber culture slides and inoculated with 500,000 ffu (m.o.i. = 2.5) of JFH-AM2 24 hrs later. Two days post-inoculation, cells were co-stained by direct IF for CD81 using a FITC-conjugated murine monoclonal Ab against CD81, and indirect IF for HCV proteins using serum from an HCV-infected chimpanzee in combination with anti-human AlexaFluor® 568. Green fluorescence represents CD81, red fluorescence represents HCV-infected cells, and DAPI-stained nuclei are shown in blue as observed using the 4OX objective. White boxes outline two cells that have been enlarged in order to better display the dual staining. Results are representative of 2 independent experiments.

Figure 7. Adaptive mutations enhance the accumulation of infectious virus. One million CD81 -deficient S29 cells were transfected with 4μl of a 20μl DNase-treated T7 transcription reaction containing indicated wild-type JFHl, JFHl plus culture-selected mutations, or SGR-JFHl RNA. (A) Transfected HCV core-positive cells were visualized by indirect IF with murine monoclonal anti-core followed by anti-mouse AlexaFluor® 488. Green fluorescence represents HCV core and blue represents DAPI-stained nuclei, as observed using the 1OX objective. (B) Total extracellular and intracellular infectious virus accumulated by day 2 was measured by assays for focus-forming units in triplicate and means plus standard errors are plotted. The dotted line represents the cutoff of the assay, which was lOffu/ml. (C) Levels of extracellular and intracellular infectious virus present in the cultures of JFHl mutant viruses were compared to that of wild-type JFHl and plotted. Fold increases are indicated above the respective bars. Results are representative of 2 independent transfection experiments.

Figure 8. Adaptive mutations increase the efficiency of infectious virus production. One million CD81 -deficient S29 cells were transfected, as described in Fig. 7, and total extracellular infectious virus produced each day was measured by assays for focus-forming units in triplicate and means plus standard errors are plotted. The bar representing day 2 for the p7 mutant appears not to have an error bar because the standard error of the 3 measurements taken for this sample was zero. The dotted line represents the cutoff of the assay, which was lOffu/ml. Results are representative of 3 independent experiments.

Detailed Description of the Preferred Embodiment

The JFHl strain of hepatitis C virus (HCV) is unique among HCV isolates in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. The effects of 5 mutations, which were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than wild-type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81 -deficient cells was developed in order to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released, but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g., Singleton P and Sainsbury D,, Dictionary of Microbiology and Molecular Biology, 3rd ed., J. Wiley & Sons, Chichester, New York, 2001 and Fields Virology, 5^th Ed. (D.M. Knipe, P.M. Howley, D.E. Griffin, R. A. Lamb, M.A. Martin, B. Roizman, and S.E. Straus, eds), Lippincott Williams & Wilkins, Philadelphia, PA, 2007.

The transitional term "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The transitional phrase "consisting of excludes any element, step, or ingredient not specified in the claim, but does not exclude additional components or steps that are unrelated to the invention such as impurities ordinarily associated therewith.

The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. The structure of the HCV genome is schematically illustrated in Fig. 1. HCV has a positive- stranded RNA genome of about 9.6 kb. The genome has a large open reading frame encoding a precursor polyprotein of 3008-3037 amino acids (aa). Structural proteins (Core, El, and E2) are located in the N-terminal one-third of the polyprotein, while nonstructural viral proteins occupy the rest of the C-terminal part. The genome structure of HCV is similar to that of pestiviruses and flaviviruses. The precursor polyprotein is cleaved by host cell signalase(s) and viral proteases as shown in Fig. 1.

The 5¹ untranslated region (UTR) of HCV RNA is approximately 340 nt long and is highly conserved among different strains of HCV. The 5' UTRs have been shown to contain an internal ribosome entry site (IRES), which could start translation in a cap-independent manner.

The 3' UTR of HCV consists of a short homopolymeric poly(U) stretch in addition to a 98-nt sequence designated as 3' tail. The short homopolymeric stretch is variable among genotypes, while the 3' tail is well conserved like the 5' UTR. The predicted secondary structure of the 3' tail shows a stable stem loop structure.

The core protein is located at the N terminus of the viral polyprotein and cleaved by host signal peptidases. This protein is considered to be a nucleocapsid, and its aa sequence is highly conserved among different strains of HCV in comparison with other structural and nonstructural proteins. The core protein is highly basic and not glycosylated.

The HCV genome encodes two envelope proteins, El and E2, which are possibly responsible for the binding and entry of the virus to target cells. El and E2 proteins are cleaved at aa 383/384 and 746/747 by the host cellular signalase(s) and have 5-6 and 9-11 sites for N-linked glycosylation, respectively. The E2 protein extending to aa 809 is also generated because of inefficient signalase-mediated cleavage at aa 746/747. The small protein named ρ7 is composed of the C terminus of larger E2 proteins. The biological significance of diverse E2 and p7 proteins is still unknown.

El and E2 proteins have been shown to form a heterooligomer and to associate with the core and NS2. Detailed study of the direct interaction of these viral proteins might reveal the mechanism of viral particle formation. Recently it was shown that structural proteins of HCV expressed in insect cells infected with a recombinant baculovirus appeared to assemble into virus-like particles.

Sequence analysis of different HCV isolates revealed the variability of nucleotide sequences in El and E2. This variation in the envelope proteins has been considered the cause of viral escape from the host immune response. Two highly variable regions of short aa sequences in the E2 protein are termed hypervariable region 1 (HVRl) and 2 (HVR2). HVRl, spanning from aa 386 to 410 at the N terminus of the E2 protein, is the most variable region of the HCV genome

NS2-NS5B are putative non structural proteins responsible for the processing of viral proteins and replication. They owe their name to their correspondence to the non-structural proteins of flaviviruses and pestiviruses. The NS2 protein of 23 IcD is a transmembrane protein, extending from aa 810 to aa 1,026. Processing at the C terminus of the NS2 protein is mediated by a viral protease located in most of the NS2 protein and the N-terminal part of the NS3 protein. This processing is considered to be an autocatalytic cis cleavage. The NS2- 3 protease has been proposed to be a metalloprotease.

NS3 is a 70-kD protein, containing aa sequence motifs for serine proteases in the N- terminal portion and for nucleoside triphosphatase (NTPase) and RNA helicases in the C- terminal portion. Numerous studies of various expression systems have revealed that the NS3 serine protease cleaves at the NS3/4A, 4A/4B, 4B/5A, and 5A/5B sites. The enzymatic activity of the NS3 protease is not required for NS2/3 cleavage. The cleavage of NS3/4A is an autocatalytic event which could only be mediated in cis, whereas other cleavages could be carried out in trans. In addition to NS3, the NS4A protein is required for cleavage at the NS3/4A and NS4B/5A sites and accelerates the efficiency of cleavage at the NS5A/5B site. It has been revealed that the formation of stable complexes of NS3 and NS4A is required for protease activity.

The NS4A 8-kD protein consisting of 54 aa acts as a cofactor for NS3 serine protease, as described above. NS4A is also associated with NS5A, and they play an important role in the hyperphosphorylation of NS5A. NS4B is a 26-kD hydrophobic protein with unknown function. NS5A are phosphorylated proteins of 56 kD (p56) and 58 kD (ρ58). Both proteins are phosphorylated at serine residues by serine/threonine kinase. p58 is a hyperphosphorylated form ofp56

The sequence of the NS 5 B protein is highly conserved among different strains of HCV. NS5B is a 65-kD membrane-associated phosphoprotein containing the conserved aa motif of RNA-dependent RNA polymerase (RdRp). NS5B protein has been shown to have a primer-dependent RdRp activity, which is able to copy a full-length HCV RNA without addition of other HCV proteins. RdRp is only utilized in the replication of RNA viruses and is therefore considered to play an essential role in viral replication.

The total number of HCV variants is primarily divided into 6 genetic groups, irrespective of the hugely increased numbers of subtypes or variants. The division of HCV variants into the 6 genetic groups of HCV is supported by each of the principal methods of phylogenetic analysis of the core/El, NS5B, and the complete genome sequences (Table 1). The genetic groups are oftentimes referred to as "genotypes." The term "clade" can also be used to describe an HCV genotype. Variants of HCV currently designated with genotype numbers above 6 are to be renamed according to the genotype group in which they fall, and with the next available subtype designation.

Kuiken et al. proposed a numbering system adapted from the Los Alamos HIV database (Kuiken C. et al. 2006 Hepatology 44:1355-1367). The system comprises both nucleotides and amino acid sequences and epitopes. It uses the full length genome sequence of isolate H77 (accession number AF009606) as a reference, and includes a method for numbering insertions and deletions relative to this reference sequence.

According to Kuiken et al. numbering HCV nucleotide sequences is done by analogy to H77. The next step is aligning a given sequence to H77. If there is no length variation, the numbering is straightforward; nucleotide numbers run from 1 (start of 5' UTR) to 9646 (end of 3' UTR). Insertions relative to H77 are labeled with letters. In the present disclosure, HCV nucleotide sequences are numbered according to the genomic sequence of JFHl, Genbank Accession number AB047639.

Protein numbering works like the nucleotide numbering, but starts at the start of the polyprotein. The sequence databases will support both systems, but use polyprotein numbering as a basis. Absolute numbering moves across the coding regions, relative numbering starts over at every coding region. Relative numbering is almost exclusively used for proteins, polyprotein numbering mainly in immunology, protein numbering in drug resistance research. The HCV databases are publicly available on the internet.

A HCV genotype 2a clone called JFH-I, a full-length hepatitis C virus genomic RNA, constructed by Takaji Wakita's group and isolated from a Japanese patient with fulminant hepatitis, is the first authentic HCV clone considered by the scientific community as capable of growing in cell culture. Transfection of the full-length JFH-I genome into Huh-7 cells leads to the production of HCV particles that are infectious for naive cells and for chimpanzees. Referring to Fig. 2, the consensus sequence of JFHl is registered at international DNA data bank under Genbank accession number AB 047639, substituting U (uracil) with T (thymine), herein designated SEQ ID NO: 1.

Given that full-length genomic RNA derived from other clones of the same genotype or different HCV genotypes was again not infectious in a cell culture system, chimeric viruses were constructed successfully. Referring to Fig. 3, using a genotype 2a subgenoraic replicon, SGR-JFHl, that efficiently replicates in cell culture, investigators constructed full- length chimeric genomes with the use of the core-NS2 gene regions from the infectious J6 (genotype 2a) and H77 (genotype Ia) virus strains. Hence, the sequences coding for the HCV JFH-I structural proteins were replaced by the corresponding sequences from different HCV genotypes.

Nucleic Acid Constructs

1 , Full-Length or Subgenomic HCV RNAs and DNAs

In one aspect, an isolated nucleic acid molecule disclosed herein comprises RNA molecules or DNA molecules that encode the HCV genomic or subgenomic region of hepatitis C virus of genotype 2a, having at least one nucleotide mutation selected from the group consisting of A1590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, wherein the hepatitis C virus of genotype 2a is of JFHl strain. A viral RNA having autonomous replication ability and virus particles production ability was constructed using an HCV genomic RNA having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation T161SC, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V.

In a preferred embodiment, a full-length HCV RNA of hepatitis C virus is, but not limited to, a hepatitis C virus of genotype 2a. In one embodiment, "hepatitis C virus of genotype 2a" or "HCV of genotype 2a" means a hepatitis virus identified as the genotype 2a according to the international classification by Simmonds et al. 2005 Hepatology 42:962-973. In some aspects, "hepatitis C virus of genotype 2a" or "HCV of genotype 2a" includes not only virus having naturally-occurring HCV genomic RNA but also virus having a genomic RNA in which the naturally-occurring HCV genomic sequence is modified artificially. A particular example of the HCV of genotype 2a includes JFH-I strain (see Wakita et al. 2005 Nat Med 11:791-796).

In one aspect, "the genomic RNA of hepatitis C virus" means RNA comprising the nucleotide sequence over the entire region of the single-stranded (+) sense RNA genome of hepatitis C virus. Referring to Fig. 2, the genomic RNA of hepatitis C virus of genotype 2a is, but not limited to, preferably RNA comprising the nucleotide sequence registered at international DNA data bank under Genbank accession number AB047639, substituting U (uracil) with T (thymine), herein designated SEQ ID NO: 1.

One of the embodiments of the full length HCV RNA described herein is a viral RNA comprising the nucleotide sequence comprising a 5' untranslated region, which contains an internal ribosome entry sequence (IRES), a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, an NS3 protein coding sequence, an NS4A protein coding sequence, an NS4B protein coding sequence, an NS5A protein coding sequence, an NS5B protein coding sequence, and a 3' untranslated region.

The full length HCV RNA used in some embodiments comprises: the 5' untranslated region, at least one selectable marker gene or reporter gene, at least one IRES sequence, the core protein coding sequence, the El protein coding sequence, the E2 protein coding sequence, the p7 protein coding sequence, the NS2 protein coding sequence, the NS 3 protein coding sequence, the NS4A protein coding sequence, the NS4B protein coding sequence, the NS5A protein coding sequence, the NS 5 B protein coding sequence, and the 3' untranslated region, in this order in the 5' to 3' direction.

In some contexts, the "5' untranslated region" (5' NTR or 5' UTR), "core protein coding sequence" (core region or C region), "El protein coding sequence¹¹ (El region), "E2 protein coding sequence" (E2 region), "p7 protein encoding sequence" (p7 region), "NS2 protein coding sequence" (NS2 region), "NS3 protein coding sequence" (NS3 region), "NS4A protein coding sequence" (NS4A region), "NS4B protein coding sequence" (NS4B region), "NS5A protein coding sequence" (NS5A region), "NS5B protein coding sequence" (NS5B region) and "3' untranslated region" (3' NTR or 3' UTR), and other specific regions or sites are defined based on the full length genomic RNA (SEQ ID NO: 1) comprising the entire region of the genome of the JFH-I strain (Genbank accession number AB047639), which is a HCV virus of genotype 2a.

Also, a partial region or site in the genome of hepatitis C virus (HCV) according to one embodiment may be defined based on the sequence shown in SEQ ID NO: 1 that is the partial nucleotide sequences of the genomic RNA of JFH-I strain (SEQ ID NO: 1). (1) "5¹ untranslated region" of the full length genomic RNA of JFH-I strain (derived from JFH-I clone; SEQ ID NO: 1) comprises the nucleotide sequence shown from nucleotide 1 to nucleotide 340 in SEQ ID NO: 1. (2) "Core protein coding sequence" comprises the nucleotide sequence shown from nucleotide 341 to nucleotide 913 in SEQ ID NO: 1. (3) "El protein coding sequence" comprises the nucleotide sequence shown from nucleotide 914 to nucleotide 1489 in SEQ ID NO: 1. (4) "E2 protein coding sequence¹¹ comprises the nucleotide sequence shown from nucleotide 1490 to nucleotide 2590 in SEQ ID NO: 1. (5) "p7 protein coding sequence" comprises the nucleotide sequence shown from nucleotide 2591 to nucleotide 2779 in SEQ ID NO: 1. (6) "NS2 protein coding sequence" comprises the nucleotide sequence shown from nucleotide 2780 to nucleotide 3430 in SEQ ID NO: 1. (7) "N S 3 protein coding sequence" comprises the nucleotide sequence shown from nucleotide 3431 to nucleotide 5323 in SEQ ID NO: 1. (8) "NS4A protein coding sequence" comprises the nucleotide sequence shown from nucleotide 5324 to nucleotide 5485 in SEQ ID NO: 1. (9) "NS4B protein coding sequence" comprises the nucleotide sequence shown from nucleotide 5486 to nucleotide 6268 in SEQ ID NO: 1. (10) "NS5A protein coding sequence" comprises the nucleotide sequence shown from nucleotide 6269 to nucleotide 7666 in SEQ ID NO: 1. (11) "NS5B protein coding sequence" comprises the nucleotide sequence shown from nucleotide 7667 to nucleotide 9439 in SEQ ID NO: 1. (12) "3' untranslated region" comprises the nucleotide sequence shown from nucleotide 9440 to nucleotide 9678 in SEQ ID NO: 1.

For example, a region or site in the RNA sequence derived from HCV may be defined by the nucleotide numbers within the nucleotide sequence of SEQ ID NO: 1 which is determined by alignment of the RNA sequence and the nucleotide sequence shown in SEQ ID NO. 1. In the alignment, a gap, addition, deletion, substitution and the like may be present.

In other embodiments, the 5' untranslated region, the core protein coding sequence, the El protein coding sequence, the E2 protein coding sequence, the p7 protein coding sequence, the NS2 protein coding sequence, the NS3 protein coding sequence, the NS4A protein coding sequence, the NS4B protein coding sequence, the NS 5 A protein coding sequence, the NS5B protein coding sequence, and the 3' untranslated region, which are contained in the full length HCV viral RNA, preferably comprises the nucleotide sequences shown (1) from nucleotide 1 to nucleotide 340, (2) from nucleotide 341 to nucleotide 913, (3) from nucleotide 914 to nucleotide 1489, (4) from nucleotide 1490 to nucleotide 2590, (5) from nucleotide 2591 to nucleotide 2779, (6) from nucleotide 2780 to nucleotide 3430, (7) from nucleotide 3431 to nucleotide 5323, (8) from nucleotide 5324 to nucleotide 5485, (9) from nucleotide 5486 to nucleotide 6268, (10) from nucleotide 6269 to nucleotide 7666, (11) from nucleotide 7667 to nucleotide 9439, and (12) from nucleotide 9440 to nucleotide 9678 in SEQ ID NO. 1.

Furthermore, a viral RNA comprising a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 1 by deletion, substitution or addition of 1-100, preferably 1-30, more preferably 1-10, still more preferably 1-6 and most preferably one to several (2-5) nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 and having autonomous replication ability and virus particle production ability is a preferred embodiment of the HCV RNA and is included in aspects of the embodiments described herein. Some embodiments also concern a DNA vector, preferably an expression vector, which encodes the viral RNA as disclosed herein.

In some contexts, an "autonomous replication ability" of RNA refers to an RNA that is capable of growing autonomously when introduced into the cell. The autonomous replication ability of RNA may be confirmed by the following procedure although it is not limited. Huh7 cells are transfected with the RNA of interest and cultured. RNAs are extracted from the resulting cultured cells and subjected to Northern blot hybridization or PCR using a probe capable of specifically detecting the introduced RNA. Detection of increasing amounts of the RNA of interest over time confirms the autonomous replication.

The term, "virus particle production ability" of RNA may refer to virus particles that are generated in a cell when the RNA is introduced into the cell (e.g., cultured cell such as Huh7 cells). The virus particle production ability may be confirmed, for example, by applying for detection the RT-PCR method using primers specific to the RNA to the culture supernatant of the RNA-introduced cell. It may also be confirmed by subjecting the culture supernatant to the sucrose density gradient method to separate virus particles and by detecting HCV protein. 2. Preparation of HCV particles

A recombinant cell that can replicate the HCV RNA, preferably continuously, can be obtained by introducing the HCV RNA prepared as described above into a cell. In this application, a recombinant cell that replicates the HCV RNA is referred to as an "HCV RNA- replicating cell."

The HCV RNA-replicating cell can produce virus particles. The produced virus particles contain the HCV RNA in a shell composed of HCV virus proteins. Thus, the virus particles produced by the HCV RNA-replicating cell of some embodiments are HCV particles. That is, in some embodiments, HCV particles can be prepared in a cell culture system by culturing the HCV RNA-replicating cells. Preferably, HCV particles can be obtained by culturing the HCV RNA-replicating cells and collecting the virus particles generated in the culture (preferably the culture supernatant).

Alternatively, HCV particles can be produced by a recombinant cell, which is obtained by introducing the HCV genomic RNA into a cell. The HCV genomic RNA is replicated with high efficiency in the cell, into which the HCV genomic RJSTA (preferably the HCV genomic RNA derived from JFH-I clone, and more preferably RNA having the nucleotide sequence shown in SEQ ID NO: 1) is introduced, In this specification, a cell that replicates the HCV genomic RNA is referred to as an "HCV genomic RNA-replicating cell". The HCV genomic RNA-replicating cells can produce virus particles. The virus particles produced by the HCV genomic RNA-replicating cells contain the HCV genomic RNA in a shell composed of HCV virus proteins. Thus, the virus particles produced by the cell into which the HCV genomic RNA of some embodiment s is introduced are HCV particles. Preferably, the HCV particles are prepared in a cell culture system, by culturing the cell into which the HCV genomic RNA derived from JFH-I clone (e.g., RNA having the nucleotide sequence shown in SEQ ID NO: 1) is introduced. For example, HCV particles can be obtained by culturing the cells into which the HCV genomic RNA (e.g., RNA having the nucleotide sequence shown in SEQ ID NO: 1) is introduced and collecting virus particles generated in the culture (preferably the culture supernatant).

For a cell into which the HCV viral RNA or the HCV genomic RNA described above is to be introduced, any liver-derived cell can be used, as long as it can be subcultured (e.g., Huh7 cells and HepG2 cells). Some aspects of the invention concern the particular cell line used for HCV culturing. Certain clones of the Huh-7 cell line were found to be far better than others for growing HCV. "HCV-cured clones" produced by antiviral agents and Huh-7 subclones support more efficient viral replication and production. Detailed studies have shown that some important features (such as high levels of CD81 receptor expression) could increase viral production and spreading. Furthermore, defects in innate immunity (such as deficiencies in interferon induction or production) could increase the cell line's permissivity. Identification of new cell lines of hepatic origin could improve our understanding of certain physiopathological aspects of hepatitis C.

Introduction of the HCV RNA or the HCV genomic RNA into cells can be achieved using any technique known to persons skilled in the art. Examples of such an introduction method include electroporation, particle gun method, lipofection method, calcium phosphate method, microinjection method, DEAE dextran method and the like. The method using electroporation is preferred; and the examples utilize lipofection. The HCV RNA or the HCV genomic RNA may be introduced alone, or may be introduced after being mixed with other nucleic acids. To vary the amount of the HCV RNA or the HCV genomic RNA while keeping RNA amount to be introduced at a certain level, the desired amount of the HCV RNA or the HCV genomic RNA to be introduced is mixed with total cellular RNA extracted from the cells, to which the RNA is introduced, to bring the total RNA amount up to a certain level, and then the mixture is used for introduction into cells. The amount of viral RNA to be used for introducing into cells may be determined according to the introduction method employed, and is preferably between 1 picogram and 100 micrograms, and more preferably between 10 picograms and 10 micrograms.

Cells can be cloned from the formed colonies by standard procedures. The newly obtained cell clone that replicates the HCV RNA is referred to as "an HCV RNA-replicating cell clone" in this specification. The HCV RNA-replicating cell of some embodiments includes the HCV RNA-replicating cell clone.

For the HCV RNA-replicating cell, actual replication of the HCV RNA in the cell or cell clone can be confirmed by detecting the replicated HCV RNA and/or by detecting HCV proteins.

The HCV RNA that has been replicated may be detected according to any RNA detection method known to persons skilled in the art. For example, the HCV RNA can be detected in total RNA extracted from the cell by the Northern hybridization method using a DNA fragment specific to the HCV RNA as a probe.

An HCV protein can be detected by, for example, reacting an antibody against the HCV protein to be expressed from the introduced HCV RNA with the extracted cellular proteins. This method can be carried out by any protein detection method known to persons skilled in the art. Specifically, HCV protein can be detected by, for example, blotting a protein sample extracted from the cell onto a nitrocellulose membrane, reacting an anti-HCV protein antibody (e.g., anti-NS3 specific antibody or antiserum collected from a hepatitis C patient) with the nitrocellulose membrane and detecting the anti-HCV protein antibody. If the HCV protein is detected among the extracted cellular proteins, it can be concluded that this cell replicates the HCV RNA and expresses the HCV protein. The virus particle production ability of the HCV RNA-replicating cells or the HCV genomic RNA-replicating cells may be confirmed by any virus detection method known to the persons skilled in the art. For example, the culture supernatant of cells, which are suspected of producing virus particles is fractionated through the sucrose density gradient, and the density of fraction, HCV core protein concentration, and amount of the HCV RNA or the HCV genomic RNA are determined for each fraction. As a result, if the peak of the core protein coincides with that of the HCV RNA or the HCV genomic RNA, and the density of the fraction showing the detected peaks (e.g., 1.18-1.20 mg) is lighter than the density of the equivalent fraction as obtained by fractionating the culture supernatant treated with 0.25% NP40 (polyoxyethylene(9)octylphenyl ether), the cells can be considered to have a virus particle production ability.

HCV virus particles released in the culture supernatant can be detected, for example, using antibodies to the core protein, the El protein or the E2 protein. Also, the presence of HCV virus particles can be detected indirectly by amplifying and detecting the HCV RNA in the culture supernatant by the RT-PCR method using specific primers. 3. Infection of another cell with HCV particles

The HCV virus particles prepared as described herein have an ability to infect an HCV permissive cell. Some embodiments also concern a method for producing a hepatitis C virus-infected cell comprising culturing the HCV RNA-replicating cell or the HCV genomic RNA-replicating cell, and infecting another HCV permissive cell with virus particles in the thus obtained culture (preferably culture supernatant). In some embodiments, the HCV permissive cell is a cell that is susceptible to HCV infection, and is preferably, but not limited to, a hepatic cell. In particular, the hepatic cell includes a primary hepatocyte, Huh7 cells, and HepG2 cells (Ito et al. 2001, Hepatology 34:566-572.

When an HCV permissive cell is infected with HCV particles produced by the HCV RNA-replicating cell described herein, the HCV RNA is replicated and virus particles are also formed in the infected cell. By infecting a cell with virus particles generated in the HCV RNA-replicating cell of some embodiments, the HCV RNA is replicated in the cell and the virus particles can be produced. Still further, by infecting an HCV permissive cell with the HCV particles generated in the HCV genomic RNA-replicating cell as described herein, the HCV genomic RNA is replicated and virus particles are also formed in the infected cell. By infecting a cell with virus particles generated in the HCV genomic RNA-replicating cell in accordance with the teachings herein, the HCV genomic RNA is replicated in the cell and furthermore the virus particles are produced. 4. Other embodiments

The HCV RNA can be replicated with high efficiency in the HCV RNA-replicating cell. Also the HCV genomic RNA is replicated with a high efficiency in a HCV genomic RNA-replicating cell, as described herein. Thus, the HCV RNA or the HCV genomic RNA can be produced with a high efficiency using the HCV RNA-replicating cell or the HCV genomic RNA-replicating cell in accordance with the teachings provided.

In some embodiments, the HCV RNA can be produced by culturing the HCV RNA- replicating cell, extracting RNA from the culture (cultured cells and/or culture medium), isolating and/or purifying the HCV the RNA (e.g., by an electrophoresis or HPLC method). The HCV genomic RNA can also be produced by using the HCV genomic RNA-replicating cell by a similar method. The RNA produced by such a way comprises the genomic sequence of hepatitis C virus. By this method, a more detailed analysis of hepatitis C virus genome becomes possible.

Further, the HCV RNA-replicating cell or the HCV genomic RNA-replicating cell can be suitably used for producing HCV protein. HCV protein may be produced by any method known to persons skilled in the art. For example, HCV protein may be produced by introducing the HCV RNA or the HCV genomic RNA into a cell, culturing the recombinant cell and collecting proteins from the culture thus obtained (cultured cells and/or culture medium) by the known procedure.

Further, the HCV virus particles prepared as described herein may possess hepatotropism. Thus a hepato tropic virus vector can be produced using the HCV RNA described above. This viral vector is suitably used for gene delivery. In some embodiments, a foreign gene can be introduced into a cell, replicated in the cell and expressed, by integrating an RNA encoding the foreign gene into the HCV RNA or HCV genomic RNA and introducing the integrated RNA into the cell. Further, by preparing an RNA in which the El protein coding sequence and/or the E2 protein coding sequence of the HCV RNA or HCV genomic RNA are replaced with an outer shell protein coding sequence of virus derived from other biological species, it becomes possible to infect the RNA to various biological species, In this case also, a foreign gene is integrated into the HCV RNA or HCV genomic RNA and this can be used as a hepatotropic virus vector for expressing the foreign gene in hepatocytes. Some embodiments also concern methods for producing a viral vector that contain a foreign gene. Some of these methods are practiced, for example, by inserting an RNA encoding the foreign gene into an RNA comprising the nucleotide sequence shown in SEQ ID NO: 1, introducing said RNA into a cell and culturing the cell to produce a virus particle.

Modified Human Hepatitis C Virus Genomic RNA Having Autonomous Replicative Competence

1. Modified chimeric hepatitis C virus genomic RNA

Some embodiments also concern modified hepatitis C virus genomic RNA constructed by combining genomic RNA of an HCV JFHl strain that can be autonomously replicated with genomic RNA of an HCV strain that cannot be autonomously replicated in vitro. It is contemplated that the resulting hybrid genomic RNA can be autonomously replicated in a cultured cell system. It is believed that the introduction of a genomic portion ranging from the NS 3 protein coding sequence of the JFHl strain to the 3 '-terminus thereof, having a mutation L2175V, allows for the adaptive conversion of an HCV genomic RNA that poorly replicates in vitro to a hybrid RNA that exhibits improved autonomous replication in a cultured cell system.

In some embodiments, HCV genomic RNA that can be autonomously replicated is not limited to the aforementioned known virus types (HCVIa, HCVIb, HCV2a, HCV2b, etc.), but it includes all types of HCV genomic RNA that can be autonomously replicated, that is, ability to release infectious particles out of the cell. In some contexts, the expression RNA "can be autonomously replicated" or "is autonomously replicated" is used to mean that when HCV genomic RNA is introduced into a cell, the HCV genomic RNA autonomously replicates, that is, it can release infectious particles out of the cell. The modified or hybrid hepatitis C virus genomic RNA described herein includes modified or hybrid hepatitis C virus genomic RNA, which has the nucleotide sequences of genomic RNA portions of two or more types of hepatitis C viruses, comprising a 5¹ untranslated region, a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, the protein coding sequence of each of NS3, NS4A, NS4B, NS5A, and NS5B of a JFHl strain, and a 3¹ untranslated region, and which can be autonomously replicated. Specifically, some embodiments include a modified hepatitis C virus genomic RNA, which is produced by substituting a hepatitis C virus genomic RNA portion ranging from the NS3 protein coding sequence to the NS 5 B protein coding sequence that is a genome sequence at the 3 '-terminus, with a partial RNA sequence encoding the NS3, NS4, NS5A, and NS5B proteins of the JFHl strain shown in SEQ ID NO: 1 (RNA sequence obtained by substituting T with U in a sequence corresponding to 3867-9678 of the DNA sequence deposited under Genbank Accession No. AB047639), and which can be autonomously replicated.

Preferably, aspects of the invention include modified hepatitis C virus genomic RNA obtained by using a hepatitis C virus with genotypes Ib and 2a and which has a nucleotide sequence, comprising a 5' untranslated region, a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, the protein coding sequence of each of NS3, NS4A, NS4B, NS5A, and NS5B of the JFHl strain, and a 3' untranslated region, and which can be autonomously replicated.

In some aspects, an HCV strain that can be autonomously replicated in a cultured cell system can be combined with an HCV strain that cannot be autonomously replicated in such a cultured cell system. In this way, the HCV strain that cannot be autonomously replicated can be modified to be made autonomously replicated. Alternatively, a virus strain that replicates inefficiently can be modified so that it replicates more efficiently.

The HCV genomic RNA of some embodiments has an RNA sequence portion that encodes NS3, NS4, NS5A, and NS5B proteins in the JFHl HCV genomic RNA.

In some embodiments, the "5' untranslated region (5'-NTR or 5'-UTR)," "core protein coding sequence (core region or C region)," "El protein coding sequence (El region)," "E2 protein coding sequence (E2 region)," "p7 protein encoding sequence", "NS2 protein coding sequence (NS2 region)," "NS3 protein coding sequence (NS3 region)," "NS4A protein coding sequence (NS4A region)," "NS4B protein coding sequence (NS4B region)," "NS5A protein coding sequence (NS5A region)," "NS5B protein coding sequence (NS5B region)," "3' untranslated region (3'-NTR or 3'-UTR)," and other specific regions or sites, have already been known in various genotypes. The aforementioned regions or sites of an unknown HCV strain can easily be determined by aligning the full-length genomic RNA sequence of a known HCV with that of the above HCV strain.

The thus produced HCV genomic RNA comprising an RNA sequence portion encoding the NS3, NS4, NS5A, and NS5B proteins of the JFHl strain is introduced into suitable host cells, so as to obtain recombinant cells that can autonomously replicate the HCV genomic RNA₃ and preferably can persistently autonomously replicate the HCV genomic RNA (that is, can replicate HCV genomic RNA). Hereinafter, in some embodiments, such recombinant cells that can replicate HCV genomic RNA comprising an RNA sequence portion encoding the NS3, NS4, NS5A, and NS5B proteins of the JFHl strain is referred to as "HCV genomic RNA-replicating cells."

Autonomous replication of HCV genomic RNA can be be confirmed, for example, by transfecting Huh7 cells with RNA as a target, culturing the Huh7 cells, and subjecting RNA extracted from the cells in the obtained culture to Northern blot hybridization, using a probe capable of specifically detecting the introduced RNA, but such confirmation method is not limited thereto. 2, Production of HCV particles

The HCV genomic RNA-replicating cells produced as described above are able to generate HCV virus particles in vitro. That is to say, the HCV genomic RNA-replicating cells of some embodiments are cultured in a suitable medium, and the generated virus particles are then collected from a culture (preferably, a culture solution), thereby easily obtaining HCV particles.

The virus particle-generating ability of the HCV genomic RNA-replicating cells can be confirmed by any known virus detection method. For example, a culture solution containing cells that presumably generate virus particles is fractionated in a sucrose density gradient manner, and the density, HCV core protein concentration, and HCV genomic RNA amount of each fraction are then measured. When the peak of the HCV core protein corresponds to that of the HCV genomic RNA, and when the density of a fraction in which the peak is detected is lower than the density of the same fraction, which is fractionated after the culture supernatant has been treated with 0.25% NP40 (polyoxyethylene(9)octylphenyl ether) (for example, between 1,15 mg and 1.22 mg), it can be confirmed that the cells have virus particle-generating ability.

HCV virus particles released into the culture solution can also be detected using an antibody reacting with a core protein, an El protein, or an E2 protein. Moreover, it is also possible to indirectly detect the existence of HCV virus particles by amplifying HCV genomic RNA contained in HCV virus particles in the culture solution and then detecting the amplified product according to the RT-PCR method using specific primers. 3. Infection of other cells with the HCV particles

The HCV virus particles generated by the method of some embodiments have an ability to infect HCV-sensitive cells. Some embodiments also provide a method for producing a hepatitis C virus-infected cell, which comprises culturing HCV genomic RNA- replicating cells and then infecting HCV-sensitive cells with virus particles contained in the obtained culture (preferably, a culture solution). The term "HCV-sensitive cells" is used herein to mean cells having infectivity to HCV. Such HCV-sensitive cells are typically hepatic cells. Specific examples of such hepatic cells may include primary hepatic cells, Huh7 cells, and HepG2 cells.

When HCV-sensitive cells are infected with HCV particles generated in the HCV genomic RNA-replicating cells of some embodiments, HCV genomic RNA is replicated in the infected cells, and virus particles are then formed. Thereafter, by allowing cells to be infected with the virus particles generated in the HCV genomic RNA-replicating cells of some embodiments, HCV genomic RNA can be replicated in the cells, and virus particles can be further produced.

When animals that can be infected with the HCV virus, such as chimpanzees, are infected with the HCV virus particles generated in the HCV genomic RNA-replicating cells of some embodiments, the particles may cause hepatitis derived from HCV to the animals. 4. Purification of HCV particles

A solution containing HCV viruses used in purification of the HCV particles may be derived from one or more selected from the blood derived from a patient infected with HCV, HCV-infected cultured cells, a cell culture medium containing cells that generate HCV particles as a result of genetic recombination, and a solution obtained from a homogenate of the cells.

A solution containing HCV viruses is subjected to centrifugation and/or filtration through a filter, so as to eliminate cells and cell residues. The solution obtained by elimination of such residues can be concentrated at a magnification between 10 and 100 times, using an ultrafiltration membrane with a molecular weight cut-off between 100,000 and 500,000.

The solution containing HCV, from which residues have been eliminated, can be purified by either one of chromatography and density gradient centrifugation as described below, or by the combined use of chromatography with density gradient centrifugation in any order. Representative chromatography and density gradient centrifugation methods will be described below, but some embodiments are not limited thereto.

Gel filtration chromatography can be used to purify HCV particles, preferably using a chromatography carrier having, as a gel matrix, a cross-linked polymer consisting of allyl dextran and N,N'-methylenebisacrylamide, and more preferably using Sephacryl(R) S-300, S-400, or S-500.

Ion exchange chromatography can be used to purify HCV particles, preferably using Q-Sepharose(R) as an anion exchange resin, and preferably using SP Sepharose(R) as a cation exchange resin.

Affinity chromatography can be used to purify HCV particles, preferably using, as a carrier, a resin as a ligand to which a substrate selected from heparin, sulfated cellulofine, lectin, and various pigments is allowed to bind. Such affinity chromatography can be used to purify HCV particles, more preferably using HiTrap Heparin HP(R), HiTrap Blue HP(R), HiTrap Benzamidine FF(R), sulfated cellulofine, or carriers to which LCA, ConA, RCA-120, and WGA bind. Such affinity chromatography can be used to purify HCV particles, most preferably using sulfated cellulofine as a carrier. In purification by density gradient centrifugation, as a solute that forms a density gradient, cesium chloride, sucrose, Nycodenz(R), or a sugar polymer such as Ficoll(R) or Percoll(R), can preferably be used. More preferably, sucrose can be used. In addition, as a solvent used herein, water or a buffer solution such as a phosphate buffer, a Tris buffer, an acetate buffer, or glycine buffer, can preferably be used.

The temperature applied to purification is preferably between O⁰C and 40°C, more preferably between 0°C and 25⁰C, and most preferably between 0⁰C and 1O⁰C.

In a purification method involving density gradient centrifugation, the centrifugal force applied to the purification is preferably between 1 x 10⁴ and 1 x 10⁹ g, more preferably between 5 x 10⁴ and 1 x 10⁷ g, and most preferably between 5 x 10⁴ and 5 x 10⁵ g.

With regard to the combined use of purification methods, density gradient centrifugation and column chromatography may be combined in any order. Preferably, after HCV particles have been purified by multiple types of column chromatography, the sample is subjected to density gradient centrifugation. More preferably, anion exchange column chromatography, and then, affinity chromatography are performed, so as to obtain a fraction containing HCV particles, and thereafter, the obtained fraction is purified by density gradient centrifugation. Most preferably, a fraction containing HCV particles obtained by column chromatography using Q-Sepharose(R) is further purified using a column with sulfated cellulofine, and thereafter, the obtained fraction containing HCV particles are purified by density gradient centrifugation. Moreover, dialysis or ultrafiltration can be carried out between the process of column chromatography and the process of density gradient centrifugation, so as to conduct substitution of a solute in the solution containing HCV particles and/or concentration of the HCV particles. 5. Other embodiments

HCV genomic RNA is replicated at high efficiency in the HCV genomic RNA- replicating cells of some embodiments. Accordingly, using the HCV genomic RNA- replicating cells of some embodiments, HCV genomic RNA can be produced at high efficiency.

In some embodiments, HCV genomic RNA-replicating cells are cultured, and RNA is extracted from the culture (cultured cells and/or a culture medium). The extracted RNA is then electrophoresed, so as to isolate and purify the separated HCV genomic RNA, thereby producing HCV genomic RNA. The thus produced RNA comprises an HCV genomic sequence. By providing such a method for producing the RNA comprising the HCV genomic sequence, it becomes possible to analyze the HCV genome more in detail.

Moreover, the HCV genomic RNA-replicating cells of some embodiments can preferably be used to produce an HCV protein. Such an HCV protein may be produced by any known method. For example, HCV genomic RNA is introduced into cells, so as to produce recombinant cells. Thereafter, the recombinant cells are cultured, and a protein is recovered from the obtained culture (cultured cells and/or a culture medium) by common methods.

HCV virus particles may have hepatic cell directivity. Thus, a hepatic cell-directed virus vector can be produced using the HCV genomic RNA of some embodiments. This virus vector is preferably used for gene therapy. In some aspects, RNA encoding a foreign gene is incorporated into HCV genomic RNA, and the RNA is then introduced into cells, so as to introduce the above foreign gene into the cells. Thereafter, the foreign gene can be replicated and then expressed in the cells.

Furthermore, RNA is produced by exchanging the El protein coding sequence and/or E2 protein coding sequence in the HCV genomic RNA with the coat protein of a virus derived from other living species. The produced RNA is then introduced into cells, so as to produce virus particles. Thus, it becomes also possible to allow the cells of various living species to be infected with the RNA. In this case also, a foreign gene is further incorporated into the HCV genomic RNA, and the obtained RNA can be used as a cell-directed virus vector for allowing the foreign gene to be expressed in various types of cells, depending on the directivity of a recombinant virus coat protein.

Some aspects also relate to a method for producing a virus vector containing a foreign gene, which comprises inserting RNA encoding the foreign gene into HCV genomic RNA, introducing genomic RNA into cells, and culturing the cells, so as to allow the cells to generate virus particles.

Some embodiments also provide a method for producing a hepatitis C immunogenic substance and/or vaccine using the HCV particles as an antigen, or using particles produced by genetic recombination of the virus coat protein for alteration of cell directivity or a portion thereof as an antigen. Moreover, a neutralizing antibody to HCV infection can also be produced, using the HCV particles of some embodiments as an antigen, or using particles produced by genetic recombination of the virus coat protein for altering of cell directivity or a portion thereof as an antigen.

Vaccine or Immunogenic Compositions

1. Viral particles

Results from recent HCV vaccination studies are promising. Houghton, M. and Abrignani, S. 2005 Nature 436:961-96. Many of these studies use recombinant HCV envelope glycoproteins gpEl and gpE2 as immunogenic and/or vaccine antigens.

Some embodiments described herein provide a hepatitis C vaccine or immunogenic composition that comprises an HCV particle prepared in accordance with the teachings provided herein. Methods of making and using these compositions to induce an immune response specific for HCV are also embodied.

In particular, HCV particles prepared as described above may be used directly as a vaccine or immunogenic composition or may be used after attenuation or inactivation, as known in the art. For example, a HCV immunogen or vaccine stock solution can be obtained by purifying the HCV particles using column chromatography, filtration, centrifugation and the like. An attenuated live HCV vaccine/immunogen or an inactivated HCV vaccine/immunogen may be prepared from these stock solutions by reacting the compositions with an inactivation agent such as formalin, β-propiolactone, glutaraldehyde and the like.

For production of a HCV vaccine or immunogen, as described herein, an HCV RNA, wherein the pathogenicity is attenuated or lost by an introduced mutation can be employed.

A vaccine/immunogen as described herein may be obtained by growing a virus to high titer followed by inactivation of the virus. In other embodiments, an attenuated vaccine/immunogen can be prepared, which contains one or more of the mutations described herein.

The HCV particle vaccine and/or immunogenic compositions described herein can be used preventively against the possible new HCV infection by administering to healthy individuals to induce the immune response to HCV. The HCV particle vaccine and/or immunogenic compositions described herein can also be used as a therapeutic vaccine to eliminate or reduce the proliferation of HCV by administering to patients infected with HCV and inducing a strong immune response to HCV in the body. Some methods described herein include the steps of identifying a subject in need of an immune response directed to HCV, providing said identified subject one or more of the HCV nucleic acids and/or proteins described herein (e.g., SEQ. ID. No: 1 and SEQ ID NO: 2), and, optionally, measuring a reduction in viral titer or an inhibition of HCV proliferation. Subjects in need of an immune response to HCV can be identified using conventional diagnostic procedures or routine clinical evaluation. The reduction in viral titer or an inhibition of HCV proliferation can be accomplished using RT-PCR, immunology, and T cell analytical techniques. 2. Genetic Immunogens or Vaccines

DNA immunogens or vaccines provide several advantages over protein-based vaccines, including the ability to express diverse antigens, tolerability in various, hosts, and ease of delivery. DNA vaccination has also been shown to be safe and effective (Robinson HL and Torres CA 1997 Semin Immunol 9:271-283; Kodihalli S. et al. 2000 Vaccine 18:2592-2599; McCluskie MJ et al. 1999 MoI Med 5:287-300; Oshop GL et al. 2002 Vet Immunol Immunopathol 89:1-12; and Rao SS et al. 2006 Vaccine 24:367-373). DNA can be synthesized in a relatively short period of time, and the constructs can be rapidly modified to target mutations that are specific for particular viral genotypes. In this manner, a focused and enhanced immune response can be obtained (Gurunathan S et al. 2000 Annu Rev Immunol 18:927-974; Fomsgaard A 1999 Immunol Lett 65:127-131 ; Wan H and Perez DR 2007 J Virol 81:5181-5191).

Some embodiments described herein concern DNA immunogens and vaccines that contain nucleic acids encoding for Hepatitis C Virus proteins. HCV sequences containing the adaptive mutations disclosed herein are used in the preparation of these compositions. Codon optimization of the HCV genes for the particular recipient of the immunogen and/or vaccine is also desired for some embodiments as it may allow for better expression of the construct in the subject. Studies have confirmed the minimal chance of host integration and toxicity with codon-optimized constructs (Sheets RL et al. 2006 Toxicol Sci 91:610-619; Epstein JE et al. 2004 Vaccine 22:1592-1603; and Wang Z et al. 2004 Gene Ther 11:711- 721).

In other embodiments, DNA immunogens and vaccines that contain nucleic acids encoding for HCV proteins are codon-optimized for human expression. These human codon- optimized constructs can be administered to other animals (e.g., mammals). As human codon-optimized constructs are capable of inducing an immune response in non-human animals, use of the human codon-optimized immunogens and/or vaccines provides the ability to monitor the safety and efficacy of the immunogens and/or vaccines in animals. Additionally, the human codon-optimized constructs are available for administration to humans without undue modification to allow for more efficient expression.

In some embodiments, an HCV immunogen and/or vaccine that comprises, consists of, or consists essentially of a nucleic acid that encodes an HCV as described herein induces a protective immune response in the host. In desired embodiments, an HCV immunogen and/or vaccine that comprises, consists of, or consists essentially of a nucleic acid that encodes an HCV induces a protective immune response in the host against a matching live virus challenge and may elicit a robust protective immune response against other homologous and heterologous HCV genotypes.

In some embodiments, nucleic acids encoding an HCV are inserted into expression vectors capable of expression in the intended host.

An effective amount of the DNA immunogen and/or vaccine can be incorporated into a pharmaceutical composition with or without a carrier. Routes of administration of the immunogen and/or vaccine include, but are not limited to, topical, intranasal, intramuscular, transdermal, intradermal, parenteral, gastrointestinal and transbronchial. The embodiments, as described herein can be delivered by any modality of DNA vaccination, such as topical, intranasal, transdermal, intradermal, intramuscular and parenteral.

In some embodiments, subjects are provided one or more of the constructs described herein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times so as to elicit an immune response. In a preferred embodiment, the constructs are provided a total of 3 times. In an especially preferred embodiment, the constructs are provided to the subject twice. The nucleic acid embodiments can also be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same HCV genotype 2a amino acid sequence as depicted in SEQ ID NO: 2 can be used in some embodiments. These include, but are not limited to, nucleic acid sequences comprising all or portions of the nonstructural and structural proteins.

The variant nucleic acids used in some of the embodiments described herein also include nucleic acids encoding HCV genotype 2a polypeptides or peptides having a non- conservative change that affects the functionality of the molecule. Additional mutants include nucleic acids encoding molecules, wherein the N-terminal region or the C-terminal region is deleted. Further, some mutant nucleic acids encode one or more protein domains combined in a novel fashion so as to create a "chimeric" molecule, also referred to as a "hybrid". Several assays can be employed to evaluate these molecules for their ability to induce an immune response. Hybrids that are identified for their ability to induce an immune response can be used in biotechnological assays and can be formulated in immunogenic and/or vaccine compositions, as described herein.

The nucleotide sequences encoding the full-length HCV genotype 2a proteins, or fragments thereof as described herein, can be modified to generate sequences optimized for expression in human, avian or other animal cells without altering the encoded polypeptide sequences. Computer algorithms are available for codon optimization. For example, web- based algorithms (e.g., Sharp et al. 1988 Nucleic Acids Res 16:8207-11, hereby incorporated by reference) can be used to generate a nucleotide sequence with optimized expression in a suitable host (e.g., human, horse, dog, cat, pig, or rodent).

Compositions comprising a nucleic acid encoding at least one HCV genotype 2a protein or fragment thereof and an adjuvant enhance and/or facilitate an animal's immune response to the antigen. Adjuvant activity is manifested by a significant increase in immune- mediated protection against the antigen, an increase in the titer of antibody raised to the antigen, and an increase in proliferative T cell responses.

Methods of enhancing or promoting an immune response in a human or other animal to an antigen prepared as described herein are also provided. Such methods can be practiced, for example, by identifying a subject in need of an immune response to HCV and providing said subject a composition comprising one or more of the nucleic acids, as described herein, and, optimally, an amount of adjuvant that is effective to enhance or facilitate an immune response to the antigen/epitope. In some embodiments, the antigen and the adjuvant are administered separately, instead of in a single mixture. Preferably, in this instance, the adjuvant is administered a short time before or a short time after administering the antigen. Preferred methods involve providing the subject in need with a nucleic acid encoding an HCV genotype 2a with or without an adjuvant or a codon-optimized nucleic acid encoding an HCV genotype 2a thereof with or without an adjuvant,

The constructs and methods disclosed herein provide a model for the production of immunogens and/or vaccines against other genotypes of HCV including genotypes listed in Table 1.

Some embodiments include a method of making an immunogenic composition comprising identifying a virus that infects both humans and an animal host, including horses, cats, dogs, and rodents. The animal host is inoculated with an immunogenic composition that comprises a nucleic acid comprising a genomic or subgenomic region of an HCV as disclosed herein. The sera of the animal is analyzed for an immune response against the corresponding virus. When an immune response is detected, the nucleic acid encoding the genomic or subgenomic region of an HCV as disclosed herein is formulated for introduction into a human.

Screening Methods

The HCV RNA-replicating cell or HCV genomic RNA-replicating cell or the hepatitis C virus -infected cell, which is infected with virus particles generated in these cells, can be used as a test system for screening a substance (anti-hepatitis C virus substance), which promotes or inhibits, for example, the replication of hepatitis C virus, re-construction of virus particles and release of virus particles. In particular, for example, the substance that promotes or inhibits the growth of hepatitis C virus can be screened by determining whether the test substance promotes or inhibits the replication of the HCV RNA or the HCV genomic RNA, or formation or release of the virus particles, culturing these cells in the presence of the test substance and detecting the HCV RNA or the HCV genomic RNA, or the virus particles in the obtained culture. In this case, the detection of the HCV RNA or the HCV genomic RNA in the culture may be carried out by determining the amount, the ratio or the presence of the HCV RNA or the HCV genomic RNA in the RNA preparation extracted from cells described above, The detection of the virus particles in the culture (mainly culture supernatant) may be carried out by measuring the amount, the ratio or the presence of HCV protein in the culture supernatant.

Various screening assays may be used to identify compounds that alter virus replication. One embodiment is a method of screening for an anti-hepatitis C virus substance comprising the steps of: a) mixing a hepatitis C virus particle that comprises a viral RNA having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V with a substance; b) exposing a hepatitis C virus-permissive cell to the virus/substance mixture of step (a); and c) measuring viral replication in the cells; wherein a reduction or increase in the replication of virus that was pre-exposed to the substance relative to the replication of control virus that were not pre-exposed to the substance demonstrates that the substance alters viral replication.

Another embodiment is a method of screening for an anti-hepatitis C virus substance comprising the steps of: a) treating hepatitis C virus-permissive cells with a substance; b) exposing the treated cells to a hepatitis C virus particle that comprises a viral RNA having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375 G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and c) measuring viral replication in said treated cells; wherein a reduction or increase in the replication of the virus in the treated cells relative to the replication of virus in control cells that were not treated with the substance demonstrates that the substance alters viral replication.

Another embodiment is method of screening for an anti-hepatitis C virus substance comprising the steps of: a) infecting a hepatitis C virus-permissive cell with a hepatitis C virus particle that comprises a viral RNA having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation ri618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q 1012R₁ and L2175V; b) treating the infected cells with a substance; and c) measuring viral replication in said treated cells, wherein a reduction or increase in the replication of the virus in the treated cells relative to the replication of the virus in control cells that were not treated with said substance demonstrates that the substance alters viral replication.

The HCV particles, generated in the HCV RNA-replicating cell or the HCV genomic RNA-replicating cell described herein, and HCV permissive cell can be used as a test system for screening a substance which may stimulate or inhibit the binding of HCV to cells. In particular, for example, substances, which may promote or inhibit the growth of hepatitis C virus, can be screened by culturing the HCV particles generated in the HCV RNA-replicating cell of some embodiments together with a HCV permissive cell in the presence of a test substance, detecting the HCV RNA or virus particles in the culture obtained and determining whether the test substance promotes or inhibits the replication of the viral RNA or formation of virus particles

Such detections of HCV RNA or HCV genomic RNA, or virus particles can be carried out according to the techniques described above. The screening protocols described above can be used for the production and evaluation of the preventive, therapeutic or diagnostic agents of hepatitis C virus infection.

In particular, examples of the usage of the test system of some embodiments described above include the following: (1) Screening for a substance which inhibits growth and infection of HCV. The substances which inhibit growth and infection of HCV include, for example, organic compounds which affect the growth and infection of HCV directly or indirectly, anti-sense oligonucleotides or the like which affect the growth of HCV or translation of HCV protein directly or indirectly by hybridizing with the target sequence in the HCV genome or its complementary strand. (2) Evaluation of various substances which have antivirus activity in cell culture. The aforementioned various substances include substances obtained by rational drug design or high-throughput screening (for example, purified and isolated enzyme). (3) Identification of a new target for the treatment of patients infected with HCV. For example, the HCV RNA-replicating cell or the HCV genomic RNA- replicating cells of some embodiments can be used for identifying host cellular proteins which may play an important role for the growth of HCV. (4) Evaluation of the ability of HCV for acquiring resistance to drugs and the like, and identification of the mutation related to the resistance. (5) Production of a virus protein as an antigen usable for development, production and evaluation of diagnostic and therapeutic agents for hepatitis C virus infection. (6) Production of a virus protein as an antigen usable for development, production and evaluation of the immunogen and/or vaccine for hepatitis C virus infection and production of attenuated HCV. (7) Production of monoclonal or polyclonal antibodies for diagnosis and treatment of hepatitis C virus infection.

Furthermore, it can be investigated whether immunoglobulin purified from the serum of an HCV infected patient can prevent infection with HCV particles of some embodiments. In this test, sera from mice, rats, rabbits and the like, which have been immunized with the HCV virus particles of some embodiments, can be used. Immunization by a HCV peptide, the HCV gene and the like may be utilized. This test may be performed on the other infection preventive substances in a similar manner.

The antibodies, which are generated against the HCV virus particles described herein include polyclonal antibodies and monoclonal antibodies. When the polyclonal antibody is preferred, selected mammals (e.g., mouse, rabbit, goat, sheep, horse and the like) are immunized with the HCV particles described herein, as the first step. Sera are collected from immunized animals and processed to isolate the antibodies (e.g., precipitation or affinity procedures). If the sera containing polyclonal antibodies to HCV epitopes contain antibodies to other antigens, these sera may be purified by immunoaffinity chromatography. The methods for generating polyclonal antisera and the methods for treatment of it are known in the art. Polyclonal antibodies may be isolated from mammals already infected with HCV.

Monoclonal antibodies to HCV epitopes can be produced easily by persons skilled in the art. The common method for producing hybridomas which generate monoclonal antibodies is known. For example, the methods described in Current Protocols in Immunology (John Wiley & Sons, Inc.) can be used.

The monoclonal antibody-generating cell lines may be produced by cell fusion, or by other method such as direct transformation of B lymphocytes with tumor gene DNA or transduction with Epstein-Barr virus.

Monoclonal antibodies and polyclonal antibodies obtained by these methods are useful for diagnosis, treatment and prevention of HCV.

The antibodies produced by using the HCV particles of some embodiments are administered with pharmaceutically acceptable solubilizer, additive, stabilizer, buffer and the like. Any administration route can be chosen but subcutaneous, intradermal and intramuscular administrations are preferred and intravenous administration is more preferred.

Industrial Applicability

By using a viral RNA described herein, RNA containing the HCV genomic RNA can be produced efficiently in a cell culture system. Furthermore, by using the cells, in which the HCV RNA or the HCV genomic RNA is introduced, the HCV RNA or the HCV genomic RNA can be replicated, and the HCV virus particles can be produced continuously in the cell culture system. The cells, in which the HCV RNA or the HCV genomic RNA is introduced, can also be used as a test system for screening various substances which influence the process of HCV replication, virus particle formation and extracellular release of virus particles. The HCV RNA and HCV genomic RNA, and virus particles described herein are also useful as a viral vector for a foreign gene. The virus particles can be included in a vaccine or immunogenic composition to elicit an immune response against hepatitis C virus. Further, the system, in which the virus particles described herein and other cells are cultured together, can be utilized as a test system for screening various substances which have an influence on the infection of cells with virus particles. The HCV RNA or the HCV genomic RNA are useful as a template which enables simple reproduction of the HCV genome sequence.

The HCV genomic RNA-replicating cells of some embodiments, or HCV-infected cells that are infected with virus particles generated in the HCV genomic RNA-replicating cells can be used, for example, for replication of HCV or reconstruction of the virus particles, or as a test system for screening for a substance that promotes or inhibits the release of the virus particles (an anti-hepatitis C virus substance). Specifically, for example, such cells are cultured in the presence of a test substance, and HCV genomic RNA or virus particles contained in the obtained culture is detected. Thereafter, it is determined whether or not the above test substance promotes or inhibits the replication of viral RNA or HCV genomic RNA, the formation of such virus particles, or the release thereof, thereby screening for a substance that promotes or inhibits the growth of hepatitis C viruses. In this case, HCV genomic RNA contained in the culture may be detected by measuring the amount of the HCV genomic RNA in the RNA extracted from the aforementioned cells, the ratio thereof, or the presence or absence thereof. Virus particles contained in the culture (mainly, a culture solution) may be detected by measuring the amount of an HCV protein contained in the culture solution, the ratio thereof, or the presence or absence thereof.

HCV particles generated in the HCV genomic RNA-replicating cells of some embodiments and HCV-sensitive cells can be used as test systems for screening for a substance that promotes or inhibits the binding of HCV to cells. Specifically, for example, HCV-sensitive cells may be cultured together with HCV particles generated in the HCV genomic RNA-replicating cells in the presence of a test substance. Thereafter, HCV genomic RNA or virus particles is detected in the obtained culture. It is determined whether or not the above test substance promotes or inhibits the replication of the HCV genomic RNA or the formation of the virus particles, thereby screening for a substance that promotes or inhibits the growth of hepatitis C viruses.

Such HCV genomic RNA or virus particles can be detected in accordance with the aforementioned means. The above-described test system can be used for production or evaluation of a preventive agent, a therapeutic agent, or a diagnostic agent for hepatitis C virus infection. Specific examples of the use of the aforementioned test system of some embodiments are given here: (1) Screening for a substance that inhibits the growth of HCV and the infection therewith. Examples of a substance that inhibits the growth of HCV and the infection therewith may include: an organic compound that directly or indirectly affects the growth of HCV and the infection therewith; and an antisense oligonucleotide that hybridizes with the target sequence of HCV genome or a complementary strand thereof, so as to directly or indirectly affect the growth of HCV or the translation of an HCV protein. (2) Evaluation of various substances having antiviral activity in cell culture. An example of the aforementioned various substances may be a substance obtained using rational drug design or high throughput screening (for example, isolated and purified enzyme). (3) Identification of a novel target to be used for treatment of patients infected with HCV. In order to identify a host cell protein playing an important role in replication of an HCV virus, the HCV genomic RNA-replicating cells of some embodiments can be used, for example. (4) Evaluation of ability of HCV virus to acquire resistance to agents or the like, and identification of a mutation associated with such resistance. (5) Production of virus proteins used as antigens that can be applied for development, production, and evaluation of diagnostic agents or therapeutic agents for hepatitis C virus infection. (6) Production of virus proteins and attenuated HCV used as antigens that can be applied for development, production, and evaluation of a vaccine against hepatitis C virus infection

Example 1 Single-Cycle Production Assay to Study Cell Culture- Adaptive Mutations of Hepatitis C

Virus Selection of mutations during serial passage

Huh-7.5 cells were transfected with in vitro transcribed genomes of JFHl and cells, and later media, were passaged sequentially. Consensus sequencing of the entire coding region at various time-points demonstrated the acquisition and fixation of 5 mutations between day 20 post-transfection and round 2 of virus passage (Table 2). The mutations included one synonymous mutation at nt 1681 of E2 and one non-synonymous mutation in each of E2, p7, NS2, and NS5A (Table 2). Since all but the p7 mutation were unique to this virus, the question was asked, which, if any, increased the efficiency of virus production and at which step?

Mutated virus produces more progeny

Since mutations in E2, p7, and NS2 often have been associated with adaptation in other studies (Zhong, J. et al. 2006 J Virol 80:11082-11093; Delgrange, D. et al. 2007 J Gen Virol 88:2495-2503; Kaul, A. et al. 2007 J Virol 81:13168-13179; Gottwein, J.M. et al. 2007 Gastroenterology 133:1614-1626; Yi₅ M. et al, 2007 J Virol 81:629-638), the 4 mutations in these genes were cloned into the wild-type JFHl genome in the absence (JFH-AMl) or presence (JFH-AM2) of the NS 5 A mutation and the replication capacity of these recombinant viruses was compared to that of wild-type. Following transfection, at day 5 both viruses had produced 100- to 1000-fold more infectious virus than had wild-type. Kinetics of virus production were determined by inoculating equal amounts of the infectious virus produced by transfection onto naϊve cells at an m.o.i. of 0.0001 and monitoring viral RNA and infectious virus accumulation in the medium. Target cell cultures grew at similar rates until just before cytopathic effects (CPE) similar to those previously reported (Zhong, J. et al. 2006 J Virol 80: 11082-11093) were observed at peak virus titer, i.e. slower cell growth and the appearance of dead cells floating in the culture fluids. At m.o.i.s ranging from 0.001-1.00, the only differences noted were that CPE and peak titers occurred at earlier time-points. In general, RNA levels and infectious titer decreased immediately after severe CPE was observed. Culture fluids from cells transfected with a JFHl subgenomic replicon (SGR-JFHl), which does not make infectious virus particles, were used as a mock infection control. The RNA patterns (Fig. 4A) and production of infectious virions (Fig. 4B) were virtually identical for JFH-AMl and -AM2, and both released over 3 logs more virus than wild-type at the time of peak production. Therefore, adaptive mutations had been selected. Since these data suggested that the NS 5 A mutation was not important for increased virus yield, it was omitted from subsequent experiments. Effect of individual mutations during prolonged culture

Recombinant genomes containing only one of the four selected mutations were constructed to test individual effects. At day 5 post-transfection, virus production by wild- type and the E2 mutant with the synonymous mutation were roughly equivalent, whereas that of each of the other three mutants was at least 1 log higher. For precise comparison, equal amounts of infectious virus harvested at day 4 post-transfection were inoculated onto naive Huh-7.5 cells (m.o.i. = 0.0005) and virus release was monitored. RNA analyses of culture fluids indicated that input RNA (SGR negative control) was no longer detected by day 4 (Fig. 5A). Wild-type JFHl RNA levels remained constant until day 14 then increased. In contrast, JFH- AM2 RNA levels immediately and rapidly increased until peaking at ~10⁷ copies/ml by day 8. Although all of the viruses with a single coding mutation lagged behind JFH- AM2, they all produced RNA levels significantly higher than that of JFHl, suggesting that each coding mutation could increase virus production: both the E2 and p7 mutants produced the same peak titer of RNA as the JFH-AM2 mutant, but it took much longer.

The wild-type and E2 synonymous mutant again produced only low levels of infectious progeny (Fig. 5B). As with the RNA levels, the E2 non-synonymous mutant produced the same high levels of infectious virus as JFH- AM2, but again with delayed kinetics: however, the p7 mutant was less effective in producing infectious virus, although it and the NS2 mutant each produced over 2 logs more infectious virus than JFHl, confirming that these two mutations were also adaptive. Similar patterns of virus replication kinetics were consistently observed in at least two independent experiments. These results seemed to demonstrate that the E2 mutation was the most critical for efficient virus production and that the NS2 and p7 mutations were roughly equivalent. However, the relative differences in level of virus production changed with time suggesting that virus spread and/or newly- acquired mutations were a confounding factor. Indeed, we found that the virus at day 16 in the culture fluids of the E2 and p7 mutants retained the original mutations, but also had acquired additional consensus sequence amino acid changes (P251L in El and T2438S in NS5A for the E2 mutant; V1132A in NS3 and Y2158H in NS5A for the p7 mutant). It is possible one or more of these affected the replication kinetics with the degree of impact coupled to the time of acquisition and, therefore, unpredictable (Fig. 5A). Regardless, these results indicated that a mutant virus with improved, but suboptimal, replication capacity may continue to adapt in culture, and very quickly, making it difficult to ascertain the contribution of a specific adaptive mutation if multiple infectious cycles are permitted. Development of a single cycle assay In order to determine whether a mutation promoted virion entry or release, it was useful to separate these steps. Therefore, a cell line was sought that was resistant to infection but released infectious HCV following transfection. A panel of Huh-7 subclones that had been randomly generated by limiting dilution cloning for use in other studies was screened for these characteristics and the S29 cells were found to meet these criteria. These cells were at least 1000-fold less susceptible than Huh-7.5 cells to infection by exogenous JFH- AM2 virus. Only rarely was an infected cell detected. Since CD81 is the major receptor protein for HCV (Drummer, H.E. et al. 2002 J Virol 76:11143-11147; Drummer, H.E. et al. 2005 Biochem Biophys Res Commun 328:251-257; Flint, M. et al. 1999 J Virol 73:6235-6244; Higginbottom, A. et al. 2000 J Virol 74:3642-3649; Pileri, P. et al. 1998 Science 282:938- 941), we determined if it was present on S29 cells. Immunofluorescent (IF) staining for CD81 did not detect CD81 on S29 cells whereas it was easily detected on Huh-7.5 cells.

The resistance of the majority of S29 cells to infection was confirmed by co- cultivation of JFH-AM2-infected Huh-7.5 cells with either uninfected Huh-7.5 or uninfected S29 cells at a ratio of 1 infected per 100 uninfected cells. IF staining for HCV core antigen demonstrated that although virus rapidly spread through the entire culture of Huh-7.5 cells, it was unable to infect the S29 cells (Fig. 6A). In a repeat experiment, co-cultured cells were dually stained for HCV antigens and CD81; after 3 days of co-culture the largest focus in the S29 target cell population contained only 4 HCV-positive cells, which probably reflected division of a single pre-infected Huh-7.5 cell since all also were CD81 -positive. In contrast, the Huh-7.5 target cell population displayed multiple foci containing as many as 50 infected cells each. In both cultures, virus was found only in cells that stained positive for CD81. This result suggested that infectability of S29 cells might be restored if CD81 were provided. Therefore, S 29 cells transfected with a CD 81 expression vector or an irrelevant vector were inoculated 1 day post-transfection with JFH-AM2 virus at an m.o.i. of 2.5 and doubly stained 2 days later for CD81 and HCV core protein. CD81 staining of control S29 cells was not observed. However, in the culture transfected with the CD 81 vector, every cell that was successfully transfected with CD81 was also stained for HCV antigen (Fig. 6B). Therefore, the CD 81 deficiency alone was responsible for the resistance to infection and all other factors, including receptor co-factors, required for progression through the entire HCV replication cycle were present. Additionally, these data suggested that HCV could not pass directly from one cell to another by a receptor-independent mechanism. Single cycle virus production

S29 cells were transfected with the panel of HCV genomes and the yield of infectious virus was determined by assays of focus-forming units in Huh-7.5 cells. Transfection efficiencies were comparable since real-time RT-PCR assays on O.όng of total cellular RNA indicated that intracellular viral RNA levels at 6 hrs post-transfection differed no more than 3 -fold and transfected cultures contained similar numbers of core-positive cells at day 6 (Fig. 7A). The results were strikingly different from those obtained by transfecting Huh-7.5 cells with the same plasmid preparations. Measurement on day 2 of total intracellular and extracellular infectious virus (Fig. 7B) indicated that the E2 coding mutant no longer produced the greatest amounts of virus, but rather was similar to the wild-type and E2 synonymous viruses. In agreement with the Huh-7.5 results, both the p7 and NS2 mutants were virtually identical and produced approximately one log more infectious virus than wild- type in each compartment (Fig. 7C). The transfection was repeated and this time virus at day 3 was quantified: again, both p7 and NS2 mutants released more infectious virus into the medium than did the E2 mutants.

Since the E2 adaptive mutation did not increase virus production from S29 cells, the effect of this mutation on virus entry was tested in a pseudoparticle system (HCVpp) that incorporated JFHl E1E2. Unexpectedly, the JFHl HCVpp containing the E2 adaptive mutation were approximately 90% less infectious than HCVpp containing wild-type E1E2. This result could reflect differences between the structure and/or conformation of the glycoproteins found on 293T-derived HCVpp versus Huh-7.5 -derived HCVcc, but that remains to be determined.

Finally, S29 cells were transfected with the various viruses and the levels of infectious virus produced per day were monitored (Fig. 8). One day post-transfection wild- type, the E2 silent mutant, and the E2 mutant each either produced no or very little detectable infectious virus. In contrast, the p7 and NS2 mutants both produced significant amounts during the first day (almost 3 logs). Interestingly, aside from JFH-AM2, all viruses reached a similar maximal level of daily virus production: JFH-AM2, containing all 5 mutations, plateaued at approximately one log higher than all other mutants.

Cell culture-derived adaptive mutations can greatly improve the in vitro replication capacity of the JFHl strain of HCV (Zhong, J. et al. 2006 J Virol 80:11082-11093, Delgrange, D, et al. 2007 J Gen Virol 88:2495-2503; Kaul, A. et al. 2007 J Virol 81:13168- 13179; Gottwein, J.M. et al. 2007 Gastroenterology 133:1614-1626; Yi, M. et al. 2007 J Virol 81:629-638; Yi, M. et al. 2006 Proc Natl Acad Sci USA 103:2310-2315). However, the mechanism of these adaptive mutations and the steps at which they exert their effects are difficult to ascertain with highly permissive cell lines such as Huh-7.5 cells that support multiple rounds of the complete viral life cycle. Not only can different rates of entry, replication, or release affect the kinetics of virus production in available in vitro systems, but multiple infection cycles or generation of a virus stock with sufficient titer to permit a high m.o.i. infection can select mutations in addition to those being studied. For example, the majority of the p7 and E2 mutant viruses shown in Fig. 5 had already acquired two new consensus mutations each after only 20 days (4 post-transfection and 16 post-infection days) in culture. Therefore, we utilized a cell line (S29) that produced infectious HCVcc in the absence of cell-to-cell spread, due to an almost complete absence of CD81, thereby providing a single-cycle virus production assay. By comparing results obtained using this subclone of Huh-7 cells with those obtained with permissive subclones such as 7.5, it is possible to separate entry-enhancing mutations from production-enhancing mutations. This system represents the first specific test for distinguishing the effects of such mutations. In agreement with previous studies (Zhong, J. et al. 2006 J Virol 80:11082-11093, Delgrange, D. et al. 2007 J Gen Virol 88:2495-2503; Kaul, A. et al. 2007 J Virol 81:13168-13179; Gottwein, J.M. et al. 2007 Gastroenterology 133:1614-1626; Yi, M. et al. 2007 J Virol 81:629-638; Yi, M. et al. 2006 Proc Natl Acad Sci USA 103:2310-2315), this report describes a set of amino acid changes within E2, p7, and NS2 that individually enhanced virus production from JFHl- transfected and -infected cells. In Huh-7.5 cells, the E2 adaptive mutation by itself increased infectious titers to a range of 10⁵-10⁶ ffu/ml. However, in the S29-based single-cycle virus production assay, adaptive mutations in p7 and NS2 significantly increased the rate of virion production, whereas the E2 mutation had a minimal effect. The most logical explanation for the different effects in the two cell lines is that the E2 mutation enhanced virus entry thus leading to rapid amplification via efficient spreading in a fully permissive culture, whereas the p7 and NS 2 mutations promote assembly and/or egress, thus resulting in a more rapid accumulation of extracellular virus. The fact that at similar RNA concentrations the E2 mutant produced more foci than the p7 mutant (Fig. 5) supports the contention that the E2 mutant was able to infect cells more efficiently. And, the fact that the p7 and NS2 mutants produced more intracellular infectious virus (as well as extracellular) than did wild-type (Fig. 7) is consistent with these mutations playing a role in steps leading to or involved in virion assembly. However, the "all or none kinetics of virus release observed upon daily sampling (Fig. 8) were unexpected and are not possible to explain at this time.

At present we can only speculate on how the N to S E2 mutation might exert such a positive effect. The mutated Asn at position 417 is highly conserved, with 98% (198/202) of the E2 sequences listed in the Los Alamos HCV Sequence Database containing N, and none containing S. This Asn residue is a known glycosylation site, and mutation at this position in a genotype Ia pseudoparticle system resulted in >50% lower HCVpp infectivity (Falkowska, E. et al. 2007 J Virol 81:8072-8079; Goffard, A. et al. 2005 J Virol 79:8400-8409), which is consistent with the 90% decrease we observed in the HCVpp system. Until more is known about glycoprotein assembly in the context of authentic HC Vcc it will be difficult to explain exactly how these mutations affect virus production or entry, but two recent reports confirmed that mutation of N417 in the context of HCVpp caused impairment in entry (Falkowska, E. et al. 2007 J Virol 81:8072-8079, Owsianka, A.M. et al. 2006 J Virol 80:8695-8704). In contrast, the observation with HCVcc that preventing addition of glycan at this position increased infectivity (Fig. 5) indicates that there may be discrepancies with respect to the structure of the glycoproteins contained on pseudotyped viruses versus authentic HCV particles. In this regard, the loss of a glycan causing a more favorable interaction with an HCV receptor has recently been proposed to explain the N543K JFHl adaptive mutation in E2 (Delgrange, D. et al. 2007 J Gen Virol 88:2495-2503).

The p7 protein contains only 63 amino acids, and mutations can have profound effects on the virus. It is essential for infectivity in chimpanzees: two basic residues in the cytoplasmic loop are necessary for infectivity in vivo (Sakai, A. et al. 2003 Proc Natl Acad

-Al- Sci USA 100:1 1646-11651). Basic residues in this loop are also critical for virus production in vitro (Jones, CT. et al. 2007 J Virol 81:8374-8383). It is proposed to oligomerize to form ion channels (Griffin, S. et al. 2005 J Virol 79:15525-15536; Griffin, S.D. et al. 2003 FEBS Lett 535:34-38; Pavlovic, D. et al. 2003 Proc Natl Acad Sci USA 100:6104-6108), but its exact function is unknown. The mutated Asn at residue 765 is unique to JFHl and the Asp that it mutated to was found in only one other sequence (genotype Ib virus). All other genotype 2 sequences have a Ser at this position. Yet, the Asn to Asp mutation clearly increased the levels of infectious virus in culture fluids of both Huh-7.5 and S29 cells (Figs. 5 and 7). This same mutation was recently isolated in another laboratory suggesting that it plays a precise and critical role in adaptation (Kaul, A. et al. 2007 J FrVo/ 81:13168-13179).

Most studies have concluded that p7 functions at the stage of virion assembly. Yi et al. propose that it protects the infectivity of newly assembled virions during release (Yi, M. et al. 2007 J Virol 81:629-638), whereas Jones et al. concluded that p7 functions at an early stage of virion morphogenesis (Jones, CT. et al. 2007 J Virol 81:8374-8383). A recent mutatgenesis study demonstrated that p7 is essential for assembly and release of HCVcc (Steinmann, E. et al. 2007 PLoS Pathog 3:el03). Our data provide further support for the prevalent conclusion that p7 plays a central role in virus assembly and/or release. Based on the observation that the levels of intracellular, in addition to extracellular, infectious virus increased over 10-fold in the presence of the p7 mutation, we propose that the mutation did not simply accelerate virus release, which would deplete the intracellular pool, but rather increased the absolute number of assembled virions within the cell.

The NS2 mutant behaved much like the p7 mutant, even though as a membrane- associated cysteine protease, it appears to be a very different protein. Its exact function is as unclear as that of p7 (Grakoui, A. et al. 1993 Proc Natl Acad Sci USA 90:10583-10587; Lorenz, I. C et al. 2006 Nature 442:831-835). However, like p7 it is thought to play a role in production of infectious virus (Yi, M. et al. 2007 J Virol 81:629-638; Pietschmann, T. et al. 2006 Proc Natl Acad Sci USA 103:7408-7413) and to be important at an early stage of virion morphogenesis (Jones, CT. et al. 2007 J Virol 81:8374-8383). Again, our data, especially those demonstrating the increased level of intracellular infectious virus support these conclusions. At the next level, it should be very informative to test different combinations of mutations in the single-cycle system to determine if there is a synergistic or additive effect, or inhibition.

The fact that single site mutants reached a similar plateau of virus production and that the best combination was only 10-fold higher suggests there are real constraints on virus replication and assembly in vitro. From the practical standpoint, it will be interesting to see if virus production in vitro can be pushed to levels high enough to make vaccine candidates feasible. From the biological standpoint, it will be very interesting to determine the factors which restrict virus multiplication in vitro since it can replicate so efficiently in vivo. Currently, we and others have identified a number of adaptive mutations in JFHl and related inter-genotypic chimeras (Zhong, J. et al. 2006 J Virol 80:11082-11093, Delgrange, D. et al. 2007 J Gen Virol 88:2495-2503; Kaul, A. et al. 2007 J Virol 81:13168-13179; Gottwein, J.M. et al. 2007 Gastroenterology 133:1614-1626; Yi, M. et al. 2007 J Virol 81:629-638; Yi, M. et al. 2006 Proc Natl Acad Sci USA 103:2310-2315). Such viruses with the ability to replicate to high levels will be useful for both basic virological and immunological studies, and can be used to test putative HCV antiviral agents. The establishment of the S29-based single cycle virus production assay provides a further advance toward determination of the degree and stage of the viral life cycle these mutations affecting. Further elucidation of the mechanisms employed by these mutations will undoubtedly provide a better understanding of the various complex interactions required for the HCV life cycle. For example, biologically relevant adaptive mutations have now been found in all of the HCV structural genes, and it will be interesting to determine how these modified proteins work together to assemble an infectious virus particle. In this regard, the S29-based single cycle virus production assay provides an additional tool for the study of HCV particle assembly and release in general, as well as the involvement of cellular factors in these processes. For example, a paper was recently published suggesting HCV can spread from cell-to-cell by a receptor-independent mechanism (Timpe, J.M. et al. 2008 Hepatology 47: 17-24): The results from experiments performed in the CD81 -deficient S29 cells (Fig. 6) are in conflict with this conclusion since they suggested that HCV absolutely requires CD81 for cell-to-cell spread. Additional experiments will be needed to resolve this question further. Example 2

Greater Detail on the Reagents and Methods Disclosed Herein Cells

HCVcc transfections and infections were performed in the Huh-7.5 human hepatoma cell line (Blight, KJ. et al. 2002 J Virol 76:13001-13014) (gift from C. Rice) or S29 cells, a subclone of Huh-7 cells (Nakabayashi, H, et al. 1982 Cancer Res 42:3858-3863) that was generated in-house by limiting dilution cloning. All cells were grown as monolayers in complete medium consisting of Dulbecco's modified Eagle's medium with L-glutamine (DMEM; Invitrogen) supplemented with penicillin/streptomycin (Sigma) and 9% fetal bovine serum (BioWhittaker) at 37°C in the presence of 5% CO₂. Plasmid Constructs

The JFHl and SGR-JFHl plasmids were gifts from Takaji Wakita and the human CD81 expression vector was a gift from T. Jake Liang. Custom plasmids incorporating cell culture-selected mutations were commercially synthesized (GeneScript Corp and Gene Oracle) and used to generate JFH-AMl and JFH- AM2, as well as constructs containing single selected mutations. A custom plasmid incorporating cell culture-selected mutations (JFHRRl) was commercially synthesized (GeneScript Corp) encoding nucleotides (nt) 1331- 2972 of JFHl containing the BsiWI and Notl restriction sites, and the Al 590G and A2633G nt changes generating N417S in E2, and N765D in p7, respectively, as well as the Tl 681C synonymous mutation (italicized to indicate a nucleotide change only). A second plasmid (JFHRR2) encoding nt2923-3881 of JFHl, containing the Notl and Avrll restriction sites and including the A3375G nt change generating Q1012R in NS2, was also custom synthesized (Gene Oracle). The BsiWI/Notl fragment was isolated from JFHRRl along with a Notl/Avrll fragment from JFHRR2 and substituted for the BsiWI/Avrll region of JFHl by a triple ligation to create JFH-AMl (AM for adaptive mutations) containing the N417S, N765D, and Q1012R amino acid changes, as well as the T1681C synonymous mutation. A third plasmid (JFHRR3) encoding nt6812-7475 of JFHl, including the SanDI and RsrII restriction sites as well as the T6863G nt change coding for L2175V in NS5A was custom synthesized (Gene Oracle). The SanDI/RsrII fragment containing L2175V was isolated from JFHRR3 and substituted into the JFH-AMl construct to create JFH-AM2 containing all five mutations. JFHl plasmids containing single cell culture-selected mutations were cloned from the above plasmids. All HCV wild-type and mutant sequences were confirmed by double- stranded DNA sequencing. RNA Transfection

JFHl plasmids were linearized with Xbal and purified by phenol/chloroform/isoamylalcohol extraction, followed by ethanol precipitation. One microgram of linearized DNA from comparable plasmid preparations was transcribed in vitro with the T7-MEGAscript kit (Ambion) as per supplied protocol. One million Huh-7.5 cells were seeded in 100mm culture dishes and allowed to adhere overnight. The following day, cells were transfected with DMRIE-C (Invitrogen). Briefly, 4μl of the 20μl transcription reaction was diluted in 500μl of serum-free (SF) DMEM containing 50μl of DMRIE-C reagent. The transfection mix was added to cells already containing 2ml of SF-DMEM and incubated at 37^°C for 5hrs, then washed once with complete medium and cultured at 37°C. For S29 cell transfections, in wYra-transcription reactions were treated with 2 units of DNaseI for 30min before being added to the cells since RT-PCR for HCV was to be performed on total cellular RNA. Virus stocks were harvested at indicated times post-transfection, passed through a 0.45μm filter, and stored at -80^°C for subsequent titrations and infections. HCV- positive cells were visualized by immunofluorescence (IF) microscopy against HCV core antigen. TaqMan Real Time RT-PCR Quantitative Assay

Quantitative RT-PCR was performed as previously described (Engle, R.E. et al. 2008 J Med Virol 80:72-79). Briefly, infection culture media were passed through a 0.45μm filter and viral RNA was extracted from 140μl of filtered infection culture media with the QIAamp Viral RNA Mini Kit (Qiagen) to yield 60 μl of RNA. Primers and probe were selected from a highly conserved region of the 5' UTR (Bukh, J. et al. 1992 Proc Natl Acad Sci USA 89:187- 191). RT-PCR reactions were performed with the TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems) and each 50μl reaction volume included lOμl of RNA. Total cellular RNA was extracted from transfected cells and treated for 15min at 37⁰C with DNaseI to remove residual DNA from in vitro transcription/transfection. Cellular RNA samples were normalized for concentration and 0.6ng of each was amplified by quantitative RT-PCR. Duplicate samples were tested in every run, along with negative, positive, and no-template controls. Data analyses were carried out using ABI's SDS version 2.2 and numbers generated were converted to copies/ml. The cutoff of the assay was 3.36 logio copies/ml. Virus Sequencing

Viral RNA was extracted from lOOμl of filtered transfection and infection culture media with TRIzol (Invitrogen) and long RT-PCR was performed as previously described (Tellier, R. et al. 1996 J Clin Microbiol 34:3085-3091; Tellier, R. et al. 2003 Methods MoI Biol 226:173-178; Yanagi, M. et al. 1997 Proc NatlAcadSci USA 94:8738-8743; Yanagi, M. et al. 1999 Virology 262:250-263). Briefly, isolated RNA was heat-denatured for 2min at 65 ^"C in the presence of DTT (Promega) and RNasin (Promega), and reverse transcribed with Superscript™ II RNase H- Reverse Transcriptase (Invitrogen) at 42° C for 60min using the reverse primer 9470R(24) JFHl in the presence of RNasin. All primer sequences are listed below. cDNA preparations were RNase H and Tl treated at 37°C for 20min and 2.5μl of the cDNA reaction mix was subjected to long PCR using Advantage KlenTaq Polymerase (BD, Clontech) using the primer pair -285 SjHCV and 947OR(24)_JFH1. Long PCR cycling steps included 35 cycles of denaturation at 99°C for 35 sec, primer annealing at 67°C for 30 sec, and extension at 68⁰C for 10 min (5 cycles), 11 min (10 cycles), 12 min (10 cycles) and 13 min (10 cycles). Second round PCR was performed with modifications that allow more efficient amplification. A 2.5μl volume of the 50μl reaction mix was subjected to nested PCR (primers and sequences listed below) with PrimeSTAR HS DNA Polymerase (Takara) for 30 cycles including denaturation at 98°C for 10 sec, primer annealing at 60°C for 5 sec, and extension at 72^°C for 70 sec. Resulting PCR amplicons were sequenced by double- stranded DNA sequencing. Primers cDNA synthesis: Long PCR:

947OR(24)_JFH1 -285S_HCV and 947OR(24)_JFH1

Nested PCR:

1: -84SJiCV & 1109RJ6 7: 4528S_J6 & 5446RjFHl

2: 946S_J6 & 2111R_JFH1 8: 5272SjFHl & 6460RJ6

3: 1849S J6 & 2763R J6 9: 6186S JFHl & 7234R JFHl 4: 2546SJFH1 & 3329RJFH1 10: 6862SJFH1 & 7848RJFH1 5: 3O81S_JFH1 & 4118R_JFH1 11 : 7741 S_J6 & 8703RJFH1 6: 3880S J6 & 4796R JFHl 12: 8137SJFH1 & 9464R(24)_JFH1

Primer Sequences:

947OR(24)JFH1 : S'-CTATGGAGTGTACCTAGTGTGTGC-S' (SEQ ID NO: 3) -285S_HCV: S'-ACTGTCTTCACGCAGAAAGCGTCTAGCCAT-S' (SEQ ID NO: 4)

-84S_HCV: S'-GTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGAT-S' (SEQ ID NO: 5)

1109RJ6: 5'-TTTGCCCACGCTCCCTGCATAGAGAA-S' (SEQ ID NO: 6)

946SJ6: 5'-CACCGCATGGCGTGGGACATGATG-S' (SEQ ID NO: 7)

211 IRJFHl : 5'-TGTACGTCCACGATGTTCTGGTG-S' (SEQ ID NO: 8)

1849SJ6: S'-TACAGGCTCTGGCATTACCCCTGCAC-S' (SEQ ID NO: 9)

2763RJ6: S'-AGCGTGAGCCCTGACGAAGTACGG-S' (SEQ ID NO: 10)

2546SJFH1 : 5'-GGTTGTGCTATCTCCTGACCCTGG-S' (SEQ ID NO: 11)

3329RJFH1 : 5'-CCCTCAGCACTCAAGTACATCTG-S' (SEQ ID NO: 12)

3O81S_JFH1: S'-GAAGCTCCTTGCTCCCATCACTGC-S' (SEQ ID NO: 13)

41 18RJFH1 : 5^I-CGCCCGAGGCCTACCTCTTCTATATC-3^I (SEQ ID NO: 14)

3880S_J6: 5'-CCCATCACGTACTCCACATATGGC-S' (SEQ ID NO: 15)

4796R_JFH1 : 5'-GCGCACACCGTAGCTTGGTAGG-S' (SEQ ID NO: 16)

4528SJ6: S'-GAGCGAGCCTCAGGAATGTTTGACA-S' (SEQ ID NO: 17)

5446RJFH1: S'-TGATGTTGAGAAGGATGGTGGTAC-S' (SEQ ID NO: 18)

5272SJFH1: S'-TGGCCCAAAGTGGAACAATTTTGG-S' (SEQ ID NO: 19)

6460RJ6: S'-CAACGCAGAACGAGACCTCATCCC-S' (SEQ ID NO: 20)

6186S_JFH1: S'-GACCTTTCCTATCAATTGCTACAC-S' (SEQ ID NO: 21)

7234RJFH1 : 5'-GAAGCTCTACCTGATCAGACTCCA-S' (SEQ ID NO: 22)

6862SJFH1 : 5'-TGGGCACGGCCTGACTACAA-S' (SEQ ID NO: 23)

7848RJFH1 : 5'-GGCCATTTTCTCGCAGACCCGGAC-S' (SEQ ID NO: 24)

7741 S 36: 5^r-ATGGCCAAAAATGAGGTGTTCTGC-3¹ (SEQ ID NO: 25) 8703RJFH1 : S'-AAGGTCCAAAGGATTCACGGAGTA-S' (SEQ ID NO: 26)

8137SJFH1 : S'-GGTCAAACCTGCGGTTACAGACGTTG-S' (SEQ ID NO: 27)

9464R(24)_JFH 1 : S'-GTGTACCTAGTGTGTGCCGCTCTA-S' (SEQ ID NO: 28) Immunofluorescence Microscopy

Culture medium was removed from chambered culture slides, and cells were washed in phosphate-buffered saline (PBS) and fixed in acetone for Imin. Slides were incubated at room temperature for 20min with monoclonal antibody recognizing core protein (Anogen) diluted 1:200 in PBS containing 5% bovine serum albumin. Slides were washed in PBS and incubated for 20min with Alexa Fluor® 488 anti-mouse secondary antibody (Invitrogen) diluted 1 :100 in PBS. Slides were washed in PBS, covered with mounting medium containing DAPI for nuclear staining, and visualized by fluorescence microscopy (Axioskop 2 plus; Zeiss). Human CD81 was visualized directly using a FITC-conjugated mouse monoclonal Ab against human CD81 (BD Pharmingen). For CD81 transfection experiments (Fig. 6), JFHl -infected cells were stained with immune serum from an HCV-infected chimpanzee, followed by anti-human AlexaFluor® 568. Virus Titration

Virus titers were determined by endpoint dilution assays of focus-forming units as previously described (Zhong, J. et al. 2005 Proc Natl Acad Sci USA 102:9294-9299). Briefly, Huh-7.5 cells were seeded at 4x10⁴ cells/well in 8-chamber culture slides and allowed to adhere overnight. Next day, culture fluids from transfected or infected cells were serially diluted 10-fold and lOOμl was inoculated into each well. After 5hrs, virus inoculum was replaced by 400μl of complete medium. Cells were cultured for 3 days and then analyzed by IF against core. Virus titer was determined by counting the number of foci observed in the highest positive dilution and expressed as ffu/ml. The cutoff of the assay was lOffu/ml. Infection by JFHl Viruses

One million Huh-7.5 cells were seeded in 100mm dishes and cultured overnight. Next day, culture fluids were removed and HCVcc-containing filtered supernatants were diluted to 3ml in complete medium to give desired m.o.i., treated with RNase A at 40μg/ml for lhr at 37°C (Fig. 5), and incubated with cells for 5hrs. Virus inoculum was replaced by 7.5ml of complete medium and cells were incubated at 37°C. For all mutant and wild-type virus cultures, cells were split at confluency every 3-4 days with all media replaced at each passage. Culture fluids were collected for virus quantification every 2 to 3 days, on the same day for each culture in a given experiment. Titration of Intracellular Virus

Levels of intracellular infectious virus were measured as previously described (Gastaminza, P. et al. 2006 J Virol 80:11074-11081). At day 2 post-transfection, virus- containing culture fluids were removed, cells were washed once, and then detached by treatment with trypsin-versene for 7min at 37°C. Cells were collected and the dish was washed with 6ml of complete medium which was combined with the cells. Cells were centrifuged at 250 x g for 5min at room temperature then resuspended in 1.2ml of complete medium, transferred to an Eppendorf tube, and frozen at -8O⁰C for subsequent processing. Cells were later thawed completely at 37°C for 5min, pulse-vortexed twice to mix, and frozen for 3min in a dry ice/methanol bath. Tubes were placed at 37°C for 3min to thaw, and this freeze/thaw cycle was repeated 3 more times. Lysates were centrifuged at 1500 x g for 5min at room temperature and infectious virus in the supernatant was quantified by the assay for focus-forming units described above. CD81 Transfection

Two hundred thousand S29 cells were seeded in 2-chamber culture slides and allowed to adhere overnight. Cells were transfected with 0.8μg of either human CD81 expression vector, or an irrelevant vector as a negative control, using Lipofectamine Plus reagents (Invitrogen). Plasmid DNA was diluted in 50μl of SF-DMEM and l.όμl of Plus reagent was added, The DNA solution was incubated at room temperature for 15min and then added to 50μl of SF-DMEM containing 2.4μl of Lipofectamine reagent. After 15min, cells were washed twice and covered with 400μl of SF-DMEM. The DNA/Lipofectamine mixture was added to the cells and placed at 37°C. Three hours later, plates were washed once and cultured in 2ml of complete medium for 24 hours before virus inoculation. Table 1. Confirmed HCV Genotypes/Subtypes

*Consensus proposed genotype/subtype names. For instances in which multiple sequences of a HCV genotype are available, two sequences have been listed, prioritized by (1) publication date, or (2) submission date when unpublished. f Locus (or isolate name, if locus is the same as the accession number). % Sequence obtained from acute phase plasma of a chimpanzee experimentally infected with (human derived) isolate SAl 3.

Table 2. Amino acid and nucleotide mutations selected during JFHl culture.

Virus* Position Changed

E2 (£2)t P7 NS2 NS5A aa 417 nt 1590 nt (1681) aa 765 nt 2633 aa 1012 nt 3375 aa 2175 nt 6863

Wild Type N A (T) N A Q A L T

D12, D16 PT N A (T) N A Q A L T

D20 PT, D4 PI N/S A/G (T/C) N/D A/G Q/R AJG L/V T

RND2,3,4 VP S G (Q D G R G V T/G

* D, day; PT, post-transfection; PI, post-infection; Rnd VP, round of virus passage f The synonymous change in E2 is shown in parentheses aa: amino acid; nt: nucleotide. Numbering based on isolate JFH-I, Genbank Accession No. AB047639.

***

While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid comprising a genomic or subgenomic region of hepatitis C virus of genotype 2a, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation H618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, wherein the hepatitis C virus of genotype 2a is of JFHl strain.

2. A viral RNA, comprising a nucleotide sequence comprising a 5' untranslated region, a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein encoding sequence, an NS2 protein coding sequence, an NS3 protein coding sequence, an NS4A protein coding sequence, an NS4B protein coding sequence, an NS5A protein coding sequence, an NS5B protein coding sequence, and a 3' untranslated region of genomic RNA of hepatitis C virus of genotype 2a, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375 G, T6863G, and synonymous mutation Tl 618 C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, wherein the genomic RNA of hepatitis C virus of genotype 2a is an RNA of JFHl strain.

3. The viral RNA according to claim 2, wherein the genomic RNA of hepatitis C virus of genotype 2a is an RNA comprising a nucleotide sequence shown in SEQ ID NO: 1.

4. The viral RNA according to claims 2 or 3, wherein the 5¹ untranslated region comprises a nucleotide sequence shown from nucleotide 1 to nucleotide 340 in SEQ ID NO: 1 , the core protein coding sequence comprises a nucleotide sequence shown from nucleotide 341 to nucleotide 913 in SEQ ID NO: 1, the El protein coding sequence comprises a nucleotide sequence shown from nucleotide 914 to nucleotide 1489 in SEQ ID NO: 1, the E2 protein coding sequence comprises a nucleotide sequence shown from nucleotide 1490 to nucleotide 2590 in SEQ ID NO: 1, the p7 protein coding sequence comprises a nucleotide sequence shown from nucleotide 2591 to nucleotide 2779 in SEQ ID NO: 1, the NS2 protein coding sequence comprises a nucleotide sequence shown from nucleotide 2780 to nucleotide 3430 in SEQ ID NO: 1, the NS3 protein coding sequence comprises a nucleotide sequence shown from nucleotide 3431 to nucleotide 5323 in SEQ ID NO: 1, the NS4A protein coding sequence comprises a nucleotide sequence shown from nucleotide 5324 to nucleotide 5485 in SEQ ID NO: 1, the NS4B protein coding sequence comprises a nucleotide sequence shown from nucleotide 5486 to nucleotide 6268 in SEQ ID NO: 1, the NS5A protein coding sequence comprises a nucleotide sequence shown from nucleotide 6269 to nucleotide 7666 in SEQ ID NO: 1, the NS5B protein coding sequence comprises a nucleotide sequence shown from nucleotide 7667 to nucleotide 9439 in SEQ ID NO: 1, and the 3' untranslated region comprises a nucleotide sequence shown from nucleotide 9440 to nucleotide 9678 in SEQ ID NO: 1.

5. A viral RNA, comprising the following RNA (a) or (b):

(a) an RNA comprising a nucleotide sequence shown in SEQ ID NO: I₃ having at least one nucleotide mutation selected from the group consisting of A1590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V; or

(b) an RNA comprising a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 1 by deletion, substitution or addition of 1 to 100 nucleotides, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and having autonomous replication ability and virus particle production ability.

6. A method for producing a cell which replicates a hepatitis C virus RNA and produces a virus particle, comprising introducing the viral RNA of any one of claims 2 to 5 into a cell.

7. The method according to claim 6, wherein the cell is a proliferative cell.

8. The method according to claim 7, wherein the proliferative cell is a human liver-derived cell.

9. The method according to claim 8, wherein the human liver-derived cell is a Huh7 cell or a HepG2 cell.

10. A cell culture cell obtainable by the method according to any one of claims 6 to 9, which replicates the viral RNA and produces the virus particle.

11. A method for producing a hepatitis C virus particle, comprising culturing the cell according to claim 10 to allow the cell to produce the virus particle.

12. A hepatitis C virus particle obtainable by the method according to claim 11.

13. A method for producing a hepatitis C virus infected cell, comprising culturing the cell according to claim 10 and infecting other cells with the virus particle in the culture.

14. A hepatitis C virus infected cell obtainable by the method according to claim 13.

15. A method of screening for an anti-hepatitis C virus substance, comprising culturing, in the presence of a test substance, at least one selected from the group consisting of the following (a), (b) and (c):

(a) the cell according to claim 10, (b) the hepatitis C virus infected cell according to claim 14, and (c) the hepatitis C virus particle according to claim 12 and a hepatitis C virus permissive cell; and detecting the viral RNA or the virus particles in the resulting culture.

16. A hepatitis C vaccine or immunogenic composition, comprising the hepatitis C virus particle according to claim 12.

17. A method for producing a hepatitis C vaccine or immunogenic composition by using the hepatitis C virus particle according to claim 12 as an antigen.

18. A method for producing a hepatotropic virus vector for gene delivery by using the viral RNA according to any one of claims 2 to 5.

19. A hepatotropic virus vector obtainable by the method according to claim 18.

20. A hepatitis C vaccine or immunogenic composition comprising a nucleic acid molecule according to claim 1.

21. A method of inducing an immune response to a hepatitis C virus in a subject in need thereof, comprising: identifying a subject in need of an immune response against hepatitis C virus; and administering a nucleic acid according to claim 1 to said subject.

22. A method for replicating and/or expressing a foreign gene in a cell, comprising inserting an RNA encoding the foreign gene into the viral RNA according to any one of claims 2 to 5 and introducing it into said cell.

23. A method for producing a cell which replicates an RNA and produces a virus particle, comprising introducing into the cell the RNA comprising a nucleotide sequence shown in SEQ ID NO. 1, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation Ω618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V.

24. A method for producing a hepatitis C virus particle, comprising introducing into a cell the RNA comprising a nucleotide sequence shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of A 1590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and culturing the cell to allow the cell to produce a virus particle.

25. The method according to claim 23 or 24, wherein the cell is a proliferative cell.

26. A method for producing a virus vector comprising a foreign gene, comprising inserting an RNA encoding a foreign gene into an RNA comprising the nucleotide sequence shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of A1590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C₅ or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q 1012R₅ and L2175V, introducing it into a cell, and culturing the cell to allow the cell to produce a virus particle.

27. An antibody against the hepatitis C virus particle according to claim 13.

28. A modified hepatitis C virus genomic RNA comprising genomic RNA portions of two or more types of hepatitis C viruses, which comprises a 5' untranslated region, a core protein coding sequence, an El protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, a partial RNA sequence encoding NS3, NS4A, NS4B, NS5A, and NS5B proteins of a JFHl strain shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of A1590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and a 3' untranslated region, and which can be autonomously replicated.

29. A modified hepatitis C virus genomic RNA, which is produced by substituting a hepatitis C virus genomic RNA portion ranging from an NS3 protein coding sequence to an NS5B protein coding sequence with a partial RNA sequence encoding the NS3, NS4, NS5A, and NS5B proteins of a JFHl strain shown in SEQ ID NO: 1, having at least one nucleotide mutation selected from the group consisting of Al 590G, A2633G, A3375G, T6863G, and synonymous mutation 71618C, or encoding an amino acid mutation selected from the group consisting of N417S, N765D, Q1012R, and L2175V, and which can be autonomously replicated.

30. The modified hepatitis C virus genomic RNA according to claim 28 or 29, wherein the hepatitis C virus genotype is selected from the group consisting of 1, 2, 3, 4, 5 and 6.

31. The modified hepatitis C virus genomic RNA according to claim 30, wherein the hepatitis C virus strain is a strain of genotype Ib or genotype 2a.

32. A cell culture cell into which the modified hepatitis C virus genomic RNA according to any one of claims 28 to 31 is introduced, and which replicates the hepatitis C virus genomic RNA and can generate virus particles.

33. A method for producing hepatitis C virus particles, the method comprising culturing the cell according to claim 32 and recovering virus particles from the culture.

34. The method of claim 33, further comprising applying the recovered virus particles to column chromatography and/or density gradient centrifugation.

35. A method for screening an anti-hepatitis C virus substance, the method comprising culturing the cell according to claim 32 in the presence of a test substance and detecting hepatitis C virus RNA or virus particles in the culture, thereby evaluating the anti- hepatitis C virus effects of the test substance.

36. A hepatitis C vaccine or immunogenic composition comprising the modified hepatitis C virus genomic RNA according to claim 28 or 29.

37. A method of screening for an anti-hepatitis C virus substance comprising the steps of: a) mixing the hepatitis C virus particle of claim 12 with a substance; b) exposing a hepatitis C virus-permissive cell to the virus/substance mixture of step (a); and c) measuring viral replication in the cells; wherein a reduction or increase in the replication of virus that was pre-exposed to the substance relative to the replication of control virus that were not pre-exposed to the substance demonstrates that the substance alters viral replication.

38. A method of screening for an anti-hepatitis C virus substance comprising the steps of: a) treating hepatitis C virus-permissive cells with a substance; b) exposing the treated cells to a hepatitis C virus particle according to claim 12; and c) measuring viral replication in said treated cells; wherein a reduction or increase in the replication of the virus in the treated cells relative to the replication of virus in control cells that were not treated with the substance demonstrates that the substance alters viral replication.

39. A method of screening for an anti-hepatitis C virus substance comprising the steps of: a) infecting a hepatitis C virus-permissive cell with the hepatitis C virus particle of claim 12, b) treating the infected cells with a substance; and c) measuring viral replication in said treated cells, wherein a reduction or increase in the replication of the virus in the treated cells relative to the replication of the virus in control cells that were not treated with said substance demonstrates that the substance alters viral replication.