Near Full-genome Assay Of Hcv Drug Resistance

Abstract

Assays for characterization of genotypic mutations of Hepatitis C Virus (HCV) showing a resistance to anti-HCV drugs.

Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to assays that detect and characterize single or linked mutations in a genome of a Hepatitis C Virus (HCV) that are associated with resistance of a subject to an anti-HCV drug. The assays can also be used for predicting resistance to an anti-HCV drug of a subject infected with HCV prior to or early during antiviral therapy or for selecting an alternative therapy for an HCV-infected subject that has developed resistance to a particular therapeutic drug or drug combination. The invention also relates to nucleotide primer pairs and kits for carrying out these assays.

[0003] 2. Description of Related Art

[0004] HCV was cloned and characterized about 15 years ago by Choo and colleagues. Choo et al. (1989) Science 244, 359-362. HCV belongs to the family of Flaviviridae and comprises an enveloped nucleocapsid and a single-stranded RNA genome of positive polarity. (Bartenschlager et al. (2003) Antiviral Res 60, 91-102.) The HCV genome consists of 5' and 3' noncoding (UTR or NCR) regions that flank a single long open reading frame (ORF). This ORF encodes three structural proteins at the amino-terminal end and six nonstructural (NS) proteins at the carboxy-terminal end. The structural proteins are the nucleocapsid core protein (C) and the two glycoproteins envelope 1 (E1) and envelope 2 (E2). The non-structural proteins are named NS2, NS3, NS4a, NS4b, NS5a, NS5b. The 5'NCR is the most highly conserved region of the HCV genome, whereas the sequences of the two envelope proteins (E1 and E2) are highly variable among different HCV isolates. The highest degree of variation has been observed in a region within E2, now commonly termed hypervariable region 1.

[0005] Since the initial identification of HCV, at least 7 different major viral types have been identified and designated genotype 1 through 7. Within these genotypes are numerous subtypes (e.g. HCV1a, 1b, 1c). Genotype and subtype of a virus with which a subject is infected may affect clinical prognosis as well as responsiveness to various drug treatments. (Simmonds et al. (1995) Hepatology 21, 570-582; Bukh et al. (1995) Semin Liver Dis 15, 41-63; Chevaliez and Pawlotsky (2007) World J Gastroenterol 13, 2461-2466).

[0006] HCV infection remains a serious medical problem to this date. There are currently about 170 million people infected with HCV. HCV is transmitted primarily by blood and blood products as well as by vertical transmission during pregnancy. The initial course of infection is typically mild. However, the immune system is often incapable of clearing the virus, and subjects with persistent infection are at a high risk for liver cirrhosis and hepatocellular carcinoma. (Poynard et al. (1997) Lancet 349, 825-832).

[0007] Current standard treatment for chronic HCV infection is based on a combination of pegylated interferon alpha and ribavirin. This therapy produces a sustained anti-viral response in 85-90% of subjects infected with genotypes 2 and 3, but, unfortunately, only in about 45% of subjects infected with the prevalent genotype 1. (Stribling et al. (2006) Gastroenterol Clin North Am vol, 463-486.) Additional therapies using other drugs and drug combinations that are endowed with higher antiviral activity and superior safety profiles are clearly required, in particular for the prevention of HCV recurrence.

[0008] Introduction of diagnostic tests for screening blood products has significantly reduced the rate of new infection. Availability of in vitro models, i.e., HCV subgenomic replicon models and an infectious cell culture model, and improvements in molecular research techniques such as the Polymerase Chain Reaction (PCR) have facilitated development of additional potent inhibitors of HCV replication targeting directly a viral protein or acting indirectly through host proteins involved in viral infection. (Bartenschlager (2002) Nat Rev Drug Discov 1, 911-916 ; Wakita et al. (2005) Nat Med 11, 791-796.) Several of these new compounds have entered clinical trials or are already on the market (http://www.hcvadvocate.org/hepatitis/hepC/HCVDrugs.sub.--2007.pdf).

[0009] Assays have been developed that are aimed at providing prognostic information about the likelihood of responsiveness to an anti-HCV therapy.(Gretch et al. (1997) Hepatology 26, 43s-47s; Podzorski (2002) Arch Pathol Lab Med 126, 285-290). These assays include serological tests and qualitative or quantitative molecular tests. Examples of PCR-based assays of HCV viral load are Cobas Amplicor.RTM. (Roche) and m2000 Real-Time PCR Diagnostics System.RTM. (Abott). Other PCR-based assays that include, e.g., Versant.RTM. HCV Genotyping Assay (Bayer Diagnostics), INNO-LiPA HCV II.RTM. (Innogenetics), GEN-ETI-K DEIA kit (Sorin, Saluggia, Italy) and TRUGENE HCV 5'NC genotyping kit (Visible Genetics Europe, Evry, France) identify HCV genotype and subtype. Systematic assessment of HCV genotype prior to therapy has been advocated recently because HCV genotype will determine choice and dose regimen of the most effective anti-HCV drug, e.g. ribavirin or interferon, as well as duration of treatment. Current genotype identification relies primarily on sequencing of a small subregion of an HCV genome, e.g., the 5'UTR, but not of a full or nearly full HCV genome.

SUMMARY OF THE INVENTION

[0010] In its most general embodiment, the present invention relates to an assay for identifying a mutation in the genome of an HCV present in a sample. The assay comprises the following steps that are carried out in sequence: [0011] a) extraction of viral RNA from the sample containing the HCV; [0012] b) determination of genotype and subtype of the HCV; [0013] c) synthesis of partial cDNAs of the genome of the HCV in three separate reverse transcription reactions, the first reverse transcription reaction initiated from a first outer antisense primer selected to specifically hybridize to a sequence in the 3'UTR of a prototype HCV genome of the same genotype and subtype, the second reverse transcription reaction initiated from a second outer antisense primer selected to specifically hybridize to a sequence in the NS4B-NS5A region of the genome of the prototype HCV and the third reverse transcription reaction initiated from a third outer antisense primer selected to specifically hybridize to a sequence in the NS2 region of the genome of the prototype HCV; [0014] d) second strand synthesis and amplification of the partial cDNAs of step c) in three separate PCR reactions, the first PCR reaction comprising an aliquot of the first reverse transcription reaction, the first outer antisense primer and a first outer sense primer selected to specifically hybridize to a complementary sequence in the NS4B-NS5A region of the genome of the prototype HCV, the second PCR reaction comprising an aliquot of the second reverse transcription reaction, the second outer antisense primer and a second outer sense primer selected to specifically hybridize to a complementary sequence in the NS2 region of the genome of the prototype HCV, and the third PCR reaction comprising an aliquot of the third reverse transcription reaction, the third outer antisense primer and a third outer sense primer selected to specifically hybridize to a complementary sequence in the 5'UTR region of the genome of the prototype HCV, wherein the second outer antisense primer hybridizes to a sequence in the NS4B-NS5A region of the genome of the prototype HCV that is located 3' to the region that is complementary to the first outer sense primer and wherein the third outer antisense primer hybridizes to a sequence in the NS2 region of the genome of the prototype HCV that is located 3' to the region that is complementary to the second outer sense primer; [0015] e) further amplification of the partial cDNAs of step d) in three separate nested PCR reactions, the first nested PCR reaction comprising an aliquot of the first PCR reaction and first inner antisense and sense primers, the second nested PCR reaction comprising an aliquot of the second PCR reaction and second inner antisense and sense primers, and the third nested PGR reaction comprising an aliquot of the third PCR reaction and third inner antisense and sense primers, wherein the inner primers do not overlap the outer primers, the second inner antisense primer hybridizes to a sequence in the NS4B-NS5A region of the genome of the prototype HCV that is located 3' to the region that is complementary to the first inner sense primer and the third inner antisense primer hybridizes to a sequence in the NS2 region of the genome of the prototype HCV that is located 3' to the region that is complementary to the second inner sense primer; [0016] f) sequence analysis of the further amplified cDNAs of step e); and [0017] g) comparison of the sequences obtained from step f) with that of the prototype HCV.

[0018] In an another embodiment, an assay of the invention is used to identify and characterize individual and linked mutations associated with resistance of an HCV-infected subject to particular anti-HCV drugs or drug combinations and to generate or expand a data bank of HCV mutations associated with anti-HCV drug resistance. The assay entails the following steps that are carried out in sequence: [0019] a) extraction of viral RNA from a sample taken from a subject carrying an HCV that is resistant to an anti-HCV drug or drug combination with which the subject has been treated as indicated by treatment failure; [0020] b) determination of genotype and subtype of the HCV; [0021] c) synthesis of partial cDNAs of the genome of the HCV in three separate reverse transcription reactions, the first reverse transcription reaction initiated from a first outer antisense primer selected to specifically hybridize to a sequence in the 3'UTR of a prototype HCV genome of the same genotype and subtype, the second reverse transcription reaction initiated from a second outer antisense primer selected to specifically hybridize to a sequence in the NS4B-NS5A region of the genome of the prototype HCV and the third reverse transcription reaction initiated from a third outer antisense primer selected to specifically hybridize to a sequence in the NS2 region of the genome of the prototype HCV; [0022] d) second strand synthesis and amplification of the partial cDNAs of step c) in three separate PCR reactions, the first PCR reaction comprising an aliquot of the first reverse transcription reaction, the first outer antisense primer and a first outer sense primer selected to specifically hybridize to a complementary sequence in the NS4B-NS5A region of the genome of the prototype HCV, the second PCR reaction comprising an aliquot of the second reverse transcription reaction, the second outer antisense primer and a second outer sense primer selected to specifically hybridize to a complementary sequence in the NS2 region of the genome of the prototype HCV, and the third PCR reaction comprising an aliquot of the third reverse transcription reaction, the third outer antisense primer and a third outer sense primer selected to specifically hybridize to a complementary sequence in the 5'UTR region of the genome of the prototype HCV, wherein the second outer antisense primer hybridizes to a sequence in the NS4B-NS5A region of the genome of the prototype HCV that is located 3' to the region that is complementary to the first outer sense primer and wherein the third outer antisense primer hybridizes to a sequence in the NS2 region of the genome of the prototype HCV that is located 3' to the region that is complementary to the second outer sense primer; [0023] e) further amplification of the partial cDNAs of step d) in three separate nested PCR reactions, the first nested PCR reaction comprising an aliquot of the first PCR reaction and first inner antisense and sense primers, the second nested PCR reaction comprising an aliquot of the second PCR reaction and second inner antisense and sense primers, and the third nested PCR reaction comprising an aliquot of the third PCR reaction and third inner antisense and sense primers, wherein the inner primers do not overlap the outer primers, the second inner antisense primer hybridizes to a sequence in the NS4B-NS5A region of the genome of the prototype HCV that is located 3' to the region that is complementary to the first inner sense primer and the third inner antisense primer hybridizes to a sequence in the NS2 region of the genome of the prototype HCV that is located 3' to the region that is complementary to the second inner sense primer; [0024] f) sequence analysis of the further amplified cDNAs of step e); [0025] g) comparison of the sequences obtained from step f) with that of the prototype HCV and identification of mutations; and [0026] h) entry of the mutations identified in a data bank of HCV mutations associated with anti-HCV drug resistance.

[0027] In a related embodiment, an assay of the invention is employed to identify and characterize individual and linked mutations associated with resistance of an HCV-infected subject to the treatment administered and, making use of a data bank of HCV mutations associated with anti-HCV drug resistance to select an alternative anti-HCV drug or drug combination to which the virus variant of the subject is not expected to be resistant for further therapy of the subject. The assay comprises the same steps as that of the preceding embodiment, except for step h) that is replaced by a step entailing a search of a data bank of HCV mutations associated with anti-HCV drug resistance for the mutations identified and selection for subsequent treatment of the subject of an anti-HCV drug or drug combination to which the HCV is not expected to be resistant.

[0028] In another embodiment, the assay of the preceding embodiment is employed for analysing a sample taken from a subject infected with HCV prior to commencement of any pharmacological therapy of the subject. Information obtained from this analysis will permit a treating physician to select an anti-HCV drug or drug combination for treatment of the subject, to which anti-HCV drug or drug combination the HCV variant or variants present in the subject are not expected to be resistant.

[0029] A more specific embodiment of the invention relates to an assay for identifying a mutation in the genome of an HCV1b present in a sample. The assay comprises the following steps that are carried out in sequence: [0030] a) extraction of viral RNA from the sample containing the HCV; [0031] b) determination of genotype and subtype of the HCV; [0032] c) provided that step b) indicated that the HCV is of type 1b, synthesis of partial cDNAs of the genome of the HCV in three separate reverse transcription reactions, the first reverse transcription reaction initiated from primer poly-A, the second reverse transcription reaction initiated from primer HCV1bOR6312 and the third reverse transcription reaction initiated from primer HCV1bOR3306; [0033] d) second strand synthesis and amplification of the partial cDNAs of step c) in three separate PCR reactions, the first PCR reaction comprising an aliquot of the first reverse transcription reaction and primer pair poly-A/HCV1bOF6074, the second PCR reaction comprising an aliquot of the second reverse transcription reaction and primer pair HCV1bOR6312/HCV1bOF1977 and the third PCR reaction comprising an aliquot of the third reverse transcription reaction and primer pair HCV1bOR3306/HCVOF129; [0034] e) further amplification of the partial cDNAs of step d) in three separate nested PCR reactions, the first nested PCR reaction comprising an aliquot of the first PCR reaction and primer pair HCV1bIR9339/HCV1bIF6126, the second nested PCR reaction comprising an aliquot of the second PCR reaction and primer pair HCV1bIR6282/HCV1bIF2523, and the third nested PCR reaction comprising an aliquot of the third PCR reaction and primer pair HCV1bIR2770/HCVIF278; [0035] f) sequence analysis of the further amplified cDNAs of step e); and [0036] g) comparison of the sequences obtained from step f) with that of a prototype HCV1b.

[0037] In another embodiment, an assay of the invention is used to identify and characterize individual and linked mutations associated with resistance of a subject infected with an HCV1b to a particular anti-HCV drug or drug combination and to generate or expand a data bank of HCV mutations associated with anti-HCV drug resistance. The assay entails the following steps that are carried out in sequence: [0038] a) extraction of viral RNA from a sample taken from a subject harboring an HCV that is resistant to an anti-HCV drug or drug combination with which the subject has been treated; [0039] b) determination of genotype and subtype of the HCV; [0040] c) provided that step b) indicated that the HCV is of type 1b, synthesis of partial cDNAs of the genome of the HCV in three separate reverse transcription reactions, the first reverse transcription reaction initiated from primer poly-A, the second reverse transcription reaction initiated from primer HCV1bOR6312 and the third reverse transcription reaction initiated from primer HCV1bOR3306; [0041] d) second strand synthesis and amplification of the partial cDNAs of step c) in three separate PCR reactions, the first PCR reaction comprising an aliquot of the first reverse transcription reaction and primer pair poly-A/HCV1bOF6074, the second PCR reaction comprising an aliquot of the second reverse transcription reaction and primer pair HCV1bOR6312/HCV1bOF1977 and the third PCR reaction comprising an aliquot of the third reverse transcription reaction and primer pair HCV1bOR3306/HCVOF129; [0042] e) further amplification of the partial cDNAs of step d) in three separate nested PCR reactions, the first nested PCR reaction comprising an aliquot of the first PCR reaction and primer pair HCV1bIR9339/HCV1bIF6126, the second nested PCR reaction comprising an aliquot of the second PCR reaction and primer pair HCV1bIR6282/HCV1bIF2523, and the third nested PCR reaction comprising an aliquot of the third PCR reaction and primer pair HCV1bIR2770/HCVIF278; [0043] f) sequence analysis of the further amplified cDNAs of step e); [0044] g) comparison of the sequences obtained from step f) with that of a prototype HCV1b and identification of mutations; and [0045] h) entry of the mutations identified in a data bank of HCV mutations associated with anti-HCV drug resistance.

[0046] Once a useful data bank has been assembled, a similar assay can be utilized to analyze samples from treatment-naive HCV1b-infected subjects or from subjects infected with HCV1b that have been treated and developed resistance to the treatment regimen to select an appropriate anti-HCV drug or drug combination for treatment or further treatment, respectively. In such embodiments, step h) of the assay described immediately above is replaced by a step entailing a search of a data bank of HCV mutations associated with anti-HCV drug resistance for the mutations identified and selection for subsequent treatment of the subject of an anti-HCV drug or drug combination to which the HCV is not expected to be resistant.

[0047] The invention also relates to primer pairs consisting of poly-A and HCV1bOF6074, HCV1bOR6312 and HCV1bOF1977, HCV1bOR3306 and HCVOF129, HCV1bIR9339 and HCV1bIF6126, HCV1bIR6282 and HCV1bIF2523, and HCV1bIR2770 and HCVIF278. Kits for detecting mutations in an HCV genome are also an object of the invention. These kits comprise at least one or all of the aforementioned primer pairs and can include additional reagents such as e.g., polymerase, buffers, and nucleoside triphosphates.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 schematically represents the partial cDNA synthesis and amplifcation steps of assays of the invention on the example of an HCV type 1b.

[0049] FIG. 2 compares the sensitivity of the partial cDNA amplification method of the invention with that of amplifcation of full-length viral genomes.

[0050] FIGS. 3a and b compare translated HCV amino acid sequences from two infected subjects obtained by an assay of the invention with the sequence of a prototype HCV genome.

DETAILED DESCRIPTION OF THE INVENTION

[0051] To aid in understanding the invention, several terms are defined below.

[0052] The terms "nucleic acid" and "oligonucleotide" refer to primers and oligomer fragments to be amplified or detected, and shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), also named DNA, to polyribonucleotides (containing D-ribose), also named RNA, and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base. There is no intended distinction in length between the terms "nucleic acid" and "oligonucleotide", and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.

[0053] The term "cDNA" refers to complementary DNA which is DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase or reverse transcriptase or DNA polymerase. These terms include double- and single-stranded complementary DNA.

[0054] Oligonucleotides can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (1979) Meth Enzymol 68, 90-99; the phosphodiester method of Brown et al. (1979) Meth Enzymol 68, 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetrahedron Lett 22, 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.

[0055] The terms "hybridization" and "hybridize" refer to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully (exactly) complementary nucleic acid strands or between "substantially complementary" nucleic acid strands that contain minor regions of mismatch. Conditions under which only fully complementary nucleic acid strands will hybridize are referred to as "stringent hybridization conditions". Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically following the guidance provided by the art (see, e.g., Sambrook et al. (1985) Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Generally, stringent conditions are selected to be about 5o C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the base pairs have dissociated. Relaxing the stringency of the hybridization conditions will allow sequence mismatches to be tolerated; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Hybridization of both exactly complementary and substantially complementary nucleic acid strands is referred to herein as "specific".

[0056] The term "primer" refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 15 to about 35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis. The region of the primer which is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.

[0057] As used herein, a "sense" or "upstream" primer refers to a primer whose extension product is a subsequence of the coding strand; an "antisense" or "downstream" primer refers to a primer whose extension product is a subsequence of the complementary non-coding strand.

[0058] The terms "outer primer" or "outer primers" refer to the first primer or pair of primers that are used to reverse-transcribe or initially amplify a stretch of a nucleic acid. The terms "inner primer" or "inner primers" refer to a primer or to a pair of primers that are used to further amplify the initial amplification product. Inner primers do not share significant sequence homology with corresponding outer primers, and amplification by inner primers typically produces a secondary amplification product that is slightly shorter than the initial amplification product.

[0059] As used herein, an oligonucleotide primer is "specific" for a target sequence if the number of mismatches present between the oligonucleotide and the target sequence is less than the number of mismatches present between the oligonucleotide and non-target sequences. Hybridization conditions can be chosen under which stable duplexes are formed only if the number of mismatches present is no more than the number of mismatches present between the oligonucleotide and the target sequence. Under such conditions, the target-specific oligonucleotide can form a stable duplex only with a target sequence. The use of target-specific primers under suitably stringent amplification conditions enables the specific amplification of those target sequences that contain the target primer binding sites.

[0060] The terms "target region" and "target nucleic acid" refers to a region of a nucleic acid that is to be amplified or otherwise analyzed. The sequence to which a primer hybridizes can be referred to as a "target".

[0061] The terms "variant" or "HCV variant" refers to an HCV having a genome that differs from that of the corresponding prototype virus by the presence of at least one mutation that causes at least a one amino acid change in a viral protein product. Accordingly, the term "mutation" refers to such changes in the genome of a variant that result in a viral protein product that differs from that of the prototype virus by at least one amino acid.

[0062] The term "prototype virus" or "prototype HCV" means a reference HCV virus that provides a reference genome with which the viral sequences produced in the assays of the invention are compared and which serve as a basis for designing the cDNA synthesis, amplification and sequencing primers used in the assays.

[0063] Prototype virus may refer to a specific virus isolate. Alternatively, it may refer to a hypothetical HCV virus that contains a consensus genome derived from comparison of genomic sequences of multiple virus isolates. One such prototype virus, HCV strain H77 (Genbank Accession Number AF011751), was used in example 1 for designing HCV 1b primers. HCV cont (Genbank Accession Number AJ238799) was used as prototype HCV 1b genomic sequence in the assays of example 3. A person skilled in the art will know how to select prototype HCV sequences for other genotypes and subtypes. For example, HCV strains HC-J6 (Genbank Accession Number D00944) or JFH1 (Genbank Accession Number AB047639) may be considered as prototype HCV 2a.

[0064] The term "quasispecies" relates to a group of highly related HCV of identical genotype and subtype frequently present in an infected subject.

[0065] The terms "anti-HCV drug or drug combination" refer to any drug or drug combination, respectively, that is capable of decreasing HCV viral load or viral titer in at least a subset of HCV-infected subjects. In particular, these terms also refer to any drug or drug combination, respectively, that is capable of substantially or significantly decreasing HCV viral load or viral titer in at least a subset of HCV-infected subjects. Anti-HCV drugs include but are not limited to HCV replication inhibitors such as polymerase inhibitors, protease inhibitors and cyclophilin inhibitors, immune or host response modulators, virus entry inhibitors and host factor inhibitors.

[0066] The term "data bank of HCV mutations associated with anti-HCV drug resistance" refers to a compilation, in any form, of mutations that are associated with resistance to any anti-HCV drug or drug combination, resistance being indicated by a failure of a course of treatment with an anti-HCV drug or drug combination to substantially reduce viral load (also referred to as treatment failure). Such a data bank may be assembled using information available in the art or becoming available in the art as well as information obtained from analyses of samples from drug-resistant, infected subjects conducted employing the assays of the present invention.

[0067] Presently available assays do not indicate whether a member of a population of HCV variants present in a subject (quasispecies) or even whether a predominant variant will be resistant to a treatment with a particular anti-HCV drug or a combination of anti-HCV drugs. Moreover, these tests do not determine which individual mutations, linked mutations or fingerprint within a viral genome are associated with viral resistance to a particular anti-HCV therapy. Most therapies against HCV are very expensive and lengthy. Treating an HCV-infected subject without knowing whether it is a priori resistant to the particular anti-HCV drug or drug combination utilized may result in adverse effects in the subject due to the consequences of the continued presence of virus at high levels as well as to secondary effects (toxicity) of the anti-HCV treatment.

[0068] A diagnostic assay for characterizing mutations in a complete or nearly complete HCV genome of a particular genotype and/or subtype that are associated with resistance to a particular anti-HCV drug or drug combination is a long felt need for the person skilled in the art, i.e. a physician, a clinician or a nurse at a hospital or medical care facility. The present invention provides such an assay. The assay relies on amplification by reverse transcription--polymerase chain reaction (RT-PCR) of an HCV genome as three overlapping DNA fragments of similar lengths. The inventors found that amplification of the genome as three fragments represents a best compromise between the need for sensitivity of the assay and avoidance of selection of viral variant sequences. Within limits, sensitivity of the assay would increase with the number of discrete fragments amplified, whereas minimization of selection would require a decrease in the number of discrete fragments amplified. The assays of the invention are highly sensitive and, in contrast to assays based on reverse transcription and amplification of full-length viral genomes, permit detection and analysis of HCV genomes from samples taken from subjects with very low viral loads.

[0069] The assays of the invention will enable the systematic assembly of a data bank of mutations that are associated with resistance to particular anti-HCV drugs and, once a useful data set has been assembled, can be employed as prognostic assays for determining the presence in an infected subject of an HCV variant that is resistant to treatment with a particular anti-HCV drug(s). Based on such information, it will be possible to select an appropriate anti-HCV drug or drug combination for a therapy of the infected subject that will not be hampered by drug resistance of a detectable HCV variant already present in the subject prior to therapy. Moreover, once a particular therapeutic regimen is selected and treatment of the subject has been initiated, virus isolated from the subject could be introduced into an infectious cell culture model and allowed to undergo a few rounds of replication under the selective pressure of the drug or drug combination used on the subject. An assay of the invention could then be performed on the selected virus population to determine whether amplification had occurred of a minor drug-resistant variant that could not be detected prior to such amplification. Results obtained could be utilized to rapidly adapt or change the drug regimen administered to the subject.

[0070] In addition to measurements of viral load, an assay of the invention could be employed for monitoring development of drug resistance in a subject treated with an anti-HCV drug or drug combination. Virus would be isolated at the end of a course of therapy or at various times during treatment and analysed using an assay of the invention. Detection of a major variant containing a known resistance mutation or set of linked mutations would provide an indication, which is independent from determinations of viral load, that the therapeutic regimen needs be adapted or replaced by another regimen. The above-mentioned data bank of mutations would assist the treating physician in the choice of an adapted or alternative regimen.

[0071] The assays of the present invention are highly sensitive and enable detection, by a single procedure, of individual mutations or linked mutations occurring essentially anywhere in an HCV genome that are associated with resistance to a treatment with any combination of one or more anti-HCV drugs.

[0072] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, polypeptide and nucleic acid synthesis, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook et al., (1989) Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; DNA Cloning--a Practical Approach, volumes 1 and 2 (D. M. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984); Transcription and Translation (B. D. Hames & S. J. Higgins, eds., 1984); Animal Cell Culture--a Practical Approach (R. I. Freshney, ed., 1986); Immobilized Cells and Enzymes--A Practical Approach (J. Woodward, ed., 1985); B. Perbal (1984) A Practical Guide to Molecular Cloning, John Wiley & Sons, New York, N.Y.; the series Methods in Enzymology, Academic Press, Inc., including volume 154 (R. Wu & L. Grossman, eds., 1987) and volume 155 (R. Wu, ed., 1987); Gene Transfer Vectors for Mammalian Cells (J. H. Miller & M. P. Calos, eds., 1987); Immunochemical Methods in Cell and Molecular Biology (R. J. Mayer & J. H. Walker, eds., 1987), Protein Purification--Principles and Practice (R. K. Scopes, 1987); and Handbook of Experimental immunology, volumes 1-4 (D. M. Weir & C. C. Blackwell, eds., 1986).

[0073] In the most general embodiment, the present invention relates to an assay that is capable of identifying and characterizing single and linked mutations in a genome of an HCV variant present in a sample, the assay comprising the following steps that are carried out sequentially: [0074] Step 1: extraction of viral RNA from a sample containing an HCV; [0075] Step 2: determination of genotype and subtype of the HCV; [0076] Step 3: synthesis of partial cDNAs of the genome of the HCV in three separate reverse transcription reactions, the first reverse transcription reaction initiated from a first outer antisense primer selected to specifically hybridize to a sequence in the 3'UTR of a prototype HCV of the same genotype and subtype, the second reverse transcription reaction initiated from a second outer antisense primer selected to specifically hybridize to a sequence in the NS4B-NS5A region of the genome of the prototype HCV and the third reverse transcription reaction initiated from a third outer antisense primer selected to specifically hybridize to a sequence in the NS2 region of the genome of the prototype HCV; [0077] Step 4: second strand synthesis and amplification of the partial cDNAs of the genome of the HCV in three separate PCR reactions, the first PCR reaction comprising an aliquot of the first reverse transcription reaction, the first outer antisense primer and a first outer sense primer selected to specifically hybridize to a complement of a sequence in the NS4B-NS5A region of the genome of the prototype HCV, the second PCR reaction comprising an aliquot of the second reverse transcription reaction, the second outer antisense primer and a second outer sense primer selected to specifically hybridize to a complement of a sequence in the NS2 region of the genome of the prototype HCV, and the third PCR reaction comprising an aliquot of the third reverse transcription reaction, the third outer antisense primer and a third outer sense primer selected to specifically hybridize to a complement of a sequence in the 5'UTR region of the genome of the prototype HCV, wherein the second outer antisense primer hybridizes to a sequence in the NS4B-NS5A region of the genome of the prototype HCV that is located 3' to the region that is complementary to the first outer sense primer and wherein the third outer antisense primer hybridizes to a sequence in the NS2 region of the genome of the prototype HCV that is located 3' to the region that is complementary to the second outer sense primer; [0078] Step 5: further amplification of the partial cDNAs of step 4 in three separate nested PCR reactions, the first nested PCR reaction comprising an aliquot of the first PCR reaction and first inner antisense and sense primers, the second nested PCR reaction comprising an aliquot of the second PCR reaction and second inner antisense and sense primers, and the third nested PCR reaction comprising an aliquot of the third PCR reaction and third inner antisense and sense primers, wherein the inner primers do not overlap the outer primers, the second inner antisense primer hybridizes to a sequence in the NS4B-NS5A region of the genome of the prototype HCV that is located 3' to the region that is complementary to the first inner sense primer and the third inner antisense primer hybridizes to a sequence in the NS2 region of the genome of the prototype HCV that is located 3' to the region that is complementary to the second inner sense primer; [0079] Step 6: sequence analysis of the further amplified cDNAs of step 5; and [0080] Step 7: comparison of the sequences obtained with that of the prototype HCV.

[0081] In a more specific embodiment, the present invention relates to an assay that is capable of identifying and characterizing single and linked mutations in a genome of an HCV variant resistant to an anti-HCV drug or drug combination in a sample taken from a subject that has been undergoing treatment with the anti-HCV drug or drug combination. The assay comprises the following steps that are carried out sequentially: [0082] Step 1: extraction of viral RNA from a sample taken from a subject harboring an HCV that is resistant to an anti-HCV drug or drug combination with which the subject has been treated;

[0083] Steps 2-6 as in the assay described above; [0084] Step 7: comparison of the sequences obtained in step 6 with that of the genome of the prototype HCV and identification of mutations; and [0085] Step 8: entry of the mutations identified in a data bank of mutations associated with anti-HCV drug resistance.

[0086] Once a useful data bank of mutations associated with drug resistance has been established, the present invention will also relate to an assay that is identical to the assay described immediately before, except that step 8 will consist of a search of the data bank for the mutations identified and selection of an alternative anti-HCV drug or drug combination to which the HCV is not expected to be resistant for subsequent treatment of the subject.

[0087] The latter assay of the invention may also be carried out on a sample taken from a subject infected with HCV prior to commencement of any pharmacological therapy of the subject. Results from the assay will allow the physician to select an anti-HCV drug or drug combination to which the HCV the subject is infected with is not resistant. It is realized that a subject frequently is infected with a quasispecies of HCV comprising a dominant variant and multiple, minor variants. The assay primarily will uncover mutations present in the dominant variant. Whether mutations present in minor variants will also be discoverable will depend on multiple factors, in particular on the relative abundance of minor variants and the sequencing technology employed. Mutations in a minor variant should be identifiable by routine capillary sequencing, i.e., standard automated sequencing, if the relative abundance of the minor variant is at least about 20%. If representation of a variant is lower, an advanced sequencing methodology such as high-throughput sequencing-by-synthesis technologies will need to be utilized to obtain mutational information. Such sequencing technologies were developed, e.g., by Illumina Inc., San Diego, Calif., 454 Life Sciences, Branford, Conn., and Applied Biosciences, Foster City, Calif. Sequencing equipment and services are commercially available.

[0088] Specific embodiments of assays for use with samples containing an HCV 1b variant are presented below and are further illustrated in the Example section. One such assay comprises the following steps that are carried out in sequence: [0089] Step 1: extraction of viral RNA from a sample containing an HCV; [0090] Step 2: determination of genotype and subtype of the HCV; [0091] Step 3: provided that step 2 indicated that the HCV is of type 1b, synthesis of partial cDNAs of the genome of the HCV in three separate reverse transcription reactions, the first reverse transcription reaction initiated from primer poly-A, the second reverse transcription reaction initiated from primer HCV1bOR6312 and the third reverse transcription reaction initiated from primer HCV1bOR3306; [0092] Step 4: second strand synthesis and amplification of the partial cDNAs of the genome of the HCV in three separate PCR reactions, the first PCR reaction comprising an aliquot of the first reverse transcription reaction and primer pair poly-A/HCV1bOF6074, the second PCR reaction comprising an aliquot of the second reverse transcription reaction and primer pair HCV1bOR6312/HCV1bOF1977 and the third PCR reaction comprising an aliquot of the third reverse transcription reaction and primer pair HCV1bOR3306/HCVOF129; [0093] Step 5: further amplification of the partial cDNAs of step 4 in three separate nested PCR reactions, the first nested PCR reaction comprising an aliquot of the first PCR reaction and primer pair HCV1bIR9339/HCV1bIF6126, the second nested PCR reaction comprising an aliquot of the second PCR reaction and primer pair HCV1bIR6282/HCV1bIF2523, and the third nested PCR reaction comprising an aliquot of the third PCR reaction and primer pair HCV1bIR2770/HCVIF278; [0094] Step 6: sequence analysis of the further amplified cDNAs of step 5; and [0095] Step 7: comparison of the sequences obtained with that of a prototype HCV1b.

[0096] Another such assay for use with samples obtained from a subject harboring an HCV1b that is resistant to the therapy the subject has received comprises the following steps that are carried out in sequence: [0097] Step 1: extraction of viral RNA from a sample taken from a subject infected with an HCV that is resistant to an anti-HCV drug or drug combination with which the subject has been treated; [0098] Steps 2-6 as in the preceding assay; [0099] Step 7: comparison of the sequences obtained in step 6 with that of the genome of the prototype HCV and identification of mutations; and [0100] Step 8: entry of the mutations identified in a data bank of mutations associated with anti-HCV drug resistance.

[0101] A similar assay that can be carried out once a useful data bank of HCV1b mutations associated with drug resistance has been generated is identical to the assay described immediately before, except that step 8 will consist of a search of the latter data bank for the mutations identified and selection of an alternative anti-HCV drug or drug combination to which the HCV is not expected to be resistant for subsequent treatment of the subject.

[0102] The above assay may also be carried may also be carried out on a sample taken from a subject infected with HCV prior to commencement of any pharmacological therapy of the subject. Provided that the HCV of the subject is of type 1b, results from the assay would allow the physician to select an anti-HCV drug or drug combination to which the HCV the subject is infected with is not resistant.

[0103] Key aspects of the above HCV1b-specific assays are also illustrated in FIG. 1 and in the Example section. The nucleotide sequences of cDNA synthesis and amplification primers as well as a set of proposed sequencing primers are provided in Table 1.

TABLE-US-00001 TABLE 1 HCV 1b primers Location in the genome (H77 as SEQ ID Primer name Purpose Direction Primer sequence reference) NO: HCV1bOR3306 cDNA/outer PCR antisense GATGATGTCCCCAC 3332-3306 3 5'UTR-NS2 region ACGCCGCGGTGTC HCVOF129 outer PCR 5'UTR- sense CCGGGAGAGCCAT 129-157 6 NS2 region AGTGGTCTGCGGA ACC HCVIF278 inner PCR 5'UTR- sense GCCTTGTGGTACTG 278-307 12 NS2 region CCTGATAGGGTGCT TG HCV1bIR2770 inner PCR 5'UTR- antisense TCCGCACGATGCA 2798-2770 9 NS2 region GCCATCTCCCGGTC CA HCV1bOR6312 cDNA/outer PCR antisense GGCAGGAGCTTGG 6343-6312 2 NS2-NS5A region ACTGGAGCCAGGT CTTGAA HCV1bOF1977 outer PCR NS2- sense CAAGGCAACTGGTT 1977-2008 5 NS5A region CGGCTGTACATGGA TGAA HCV1bIF2523 inner PCR NS2- sense GACGCGCGCGTCT 2523-2551 11 NS5A region GYGCCTGCTTRTGG AT HCV1bIR6282 inner PCR NS2- antisense TCAGTCAACACCGT 6310-6282 8 NS5A region GCATATCCAGTCCC A poly-A cDNA/outer PCR antisense 30-mer 9447-9418 1 NS4B-3'UTR region HCVOF6074 outer PCR NS4B- sense GGCTGTGCAGTGG 6074-6103 4 3'UTR region ATGAACCGGCTGAT AGC HCV1bIF6126 inner PCR NS4B- sense GTCTCCCCCACGCA 6126-6151 10 3'UTR region CTATGTGCCTGA HCV1bIR9339 inner PCR NS4B- antisense GGGAGCAGGTAGA 9367-9342 7 3'UTR region TGCCTACCCCTAC HCV1bSF310 sequencing 5'UTR- sense GAGTGCCCCGGGA 309-332 13 NS2 region GGTCTTCGTAGA HCV1bSF807 sequencing 5'UTR- sense CCGGGTTCTGGAG 806-829 14 NS2 region GACGGCGTGAA HCV1bSR850 sequencing 5'UTR- antisense AGGAAGATAGAGAA 874-849 15 NS2 region AGAGCAACCGGG HCV1bSF1202 sequencing 5'UTR- sense TCTCCCAGCTGTTC 1201-1222 16 NS2 region ACCTTCTC HCV1bSR1302 sequencing 5'UTR- antisense GACCAGTTCATCAT 1321-1301 17 NS2 region CATATCC HCV1bSF1597 sequencing 5'UTR- sense GGCAGCTGGCACA 1593-1615 18 NS2 region TCAACAGGAC HCV1bSR1653 sequencing 5'UTR- antisense GTAGAACAGCGCG 1674-1653 19 NS2 region GCAAGGAAC HCV1bSF1854 sequencing 5'UTR- sense TGGTCCAGTGTATT 1850-1872 20 NS2 region GYTTCACCC HCV1bSR1990 sequencing 5'UTR- antisense TTCATCCATGTACA 2008-1986 21 NS2 region GCCGAACCA HCV1bSF2242 sequencing 5'UTR- sense GTGGGGGGCGTGG 2238-2259 22 NS2 region AGCACAGGC HCV1bSR2433 sequencing 5'UTR- antisense CGTACAGGTATTGC 2451-2429 23 NS2 region ACGTCCACG HCV1bSF2640 sequencing NS2- sense TCTCTCCTTCCTTG 2636-2658 24 NS5A region TGTTCTTCT HCV1bSF2860 sequencing NS2- antisense AACCACCATATGAG 2882-2860 25 NS5A region CCTGGCGAG HCV1bSF3085 sequencing NS2- sense CGCGCTCAAGGGC 3081-3102 26 NS5A region TCATYCGTG HCV1bSR3280 sequencing NS2- antisense CAGGTGATGATCTT 3298-3276 27 NS5A region GGTCTCCAT HCV1bSF3541 sequencing NS2- sense ACRCAATCTTTCCT 3537-3559 28 NS5A region GGCGACCTG HCV1bSR3643 sequencing NS2- antisense GTCCTGGTCTACAT 3662-3639 29 NS5A region TGGTGTACAT HCV1bSF4004 sequencing NS2- sense CGCAGACATTCCAA 4000-4021 30 NS5A region GTGGCCCA HCV1bSR4237 sequencing NS2- antisense CAACCACCGTCGG 4255-4233 31 NS5A region CAAGGAACTT HCV1bSF4516 sequencing NS2- sense CTCATTTTCTGCCA 4512-4534 32 NS5A region TTCCAAGAA HCVlbSR4666 sequencing NS2- antisense TCAAAGTCGCCGGT 4687-4662 33 NS5A region AWAGCCCGTCAT HCV1bSF5048 sequencing NS2- sense TAGATGCCCACTTC 5044-5064 34 NS5A region TTGTCCC HCV1bSR5164 sequencing NS2- antisense AGCCGTATGAGACA 5182-5160 35 NS5A region CTTCCACAT HCV1bSF5524 sequencing NS2- sense CAATTCAAGCAGAA 5520-5542 36 NS5A region GGCGCTCGG HCV1bSR5639 sequencing NS2- antisense TGTATCCCGCTGAT 5659-5635 37 NS5A region GAARTTCCACA HCV1bSF5986 sequencing NS2- sense ACACGCTGTGATAA 5986-6008 38 NS5A region ATGTCTCCCCCGC HCV1bSR6092 sequencing NS2- antisense GAAGCGAACGCTAT 6112-6088 39 NS5A region CAGCCGGTTCA HCV1bSF6186 sequencing NS2- sense CCTCTCCAGCCTTA 6182-6205 40 NS5A region CCATCACTCA HCV1bSR6256 sequencing NS2- antisense TCCCTIAGCCACGA 6281-6256 41 NS5A region GCCGGAGCATGG HCV1bSF6416 sequencing NS4B- sense TCATGCAIACCACCT 6416-6437 42 3'UTR region GCCCATG HCV1bSR6616 sequencing NS4B- antisense TCCCCCACCCGCG 6639-6616 43 3'UTR region TGACCTCCAC HCV1bSF6853 sequencing NS4B- sense GTGCTCACTTCCAT 6849-6871 44 3'UTR region GCTCACCGA HCV1bSR6964 sequencing NS4B- antisense TCAAGGAAGGCGC 6975-6954 45 3'UTR region AGACAACTG HCV1bSF7066 sequencing NS4B- sense GGGAACATCACCC 7056-7078 46 3'UTR region GCGTGGAGTC HCV1bSR7273 sequencing NS4B- antisense GGGCACCCGTGTA 7285-7263 47 3'UTR region CCACCGGAGG HCV1bSF7504 sequencing NS4B- sense TACTCCTCCATGCC 7494-7516 48 3'UTR region CCCCCTTGA HCV1bSR7558 sequencing NS4B- antisense CTCGCTCACRGTAG 7570-7548 49 3'UTR region ACCAAGACCC HCV1bSF7968 sequencing NS4B- sense CTCCGTGTGGAAG 7961-7984 50 3'UTR region GACTTGCTGGA HCV1bSR8077 sequencing NS4B- antisense TCTGGGAATACGAT 8092-8070 51 3'UTR region AAGGCGAGC HCV1bSF8528 sequencing NS4B- sense AGCTCCAGGACTG 8521-8542 52 3'UTR region CACGATGCT HCV1bSR8622 sequencing NS4B- antisense TACCTAGTCATAGC 8638-8615 53 3'UTR region CTCCGTGAAG HCV1bSF9010 sequencing NS4B- sense ACACGCTGTGATAA 9010-9032 54 3'UTR region ATGTCTCCCCCGC HCV1bSR9094 sequencing NS4B- antisense GATGTCTCCAGACT 9108-9087 55 3'UTR region CGCAAGGG

[0104] Other specific embodiments of the invention relate to analogous assays for use with samples obtained from subjects infected with HCV of other genotypes and subtypes, in particular HCV 1a, HCV 2 and HCV 3. A person skilled in the art will know how to design appropriate cDNA synthesis and amplification primers according to the method of the invention as well as sequencing primers by reference to prototype viral genome sequences.

[0105] Samples may consist of but are not limited to blood samples of subjects infected by HCV or co-infected by HIV and HCV, which samples may be taken from the subjects prior to, during or subsequent to a course of treatment with an anti-HCV drug or drug combination. The assays of the invention may be used for other applications. For example, HCV variants present in a sample from a subject may be cloned into an infectious HCV vector and transduced into mammalian cells. (Kato et as. (2007) J Virol 81, 4405-4411). Infectious virus containing HCV variant sequences may thereafter be used to infect mammalian cultures, and samples obtained after one or several cycles of infection in the presence or absence presence of an anti-HCV drug or drug combination may be analysed by the assays of the invention.

[0106] HCV RNA extraction from samples may be performed by various methods including the use of commercially available kits, e.g., QIAamp.RTM. Viral RNA mini kit, QIAamp.RTM. Utralsens.TM. Virus Kit, Trizol reagens (Invitrogen) or Vivaspin concentration.

[0107] The reverse transcription and PCR reactions that are part of the assays are performed using standard reaction mixtures and conditions such as those described in the examples and the references cited herein. Typically, nucleic acids extracted from samples are subjected to 20-40 amplification cycles in the PCR reactions of steps 4 and 2-10 cycles in the nested PCR reaction of steps 5.

[0108] All publications and patents cited herein shall be considered as incorporated by reference in their entirety.

[0109] The invention is further elaborated by the following examples. The examples are provided for purpose of illustration to a person skilled in the art, and are not intended to be limiting the scope of the invention as described in the claims. Thus, the invention should not be construed as being limited to the examples provided, but should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

EXAMPLES

Example 1

Detailed Methods used in the Assays of the Invention

[0110] Samples: Plasma samples from therapy-naive patients were obtained from two different hospitals. Viral load was determined using the HCV Viral Load COBAS AMPLICOR system from Roche Molecular Diagnostics (Basel, Switzerland).

[0111] Samples were selected based on the presence of HCV of type 1b as determined by InnoLipa test (Innogenetics, Zwijnaarde, Belgium). To confirm genotype, after cDNA synthesis and PCR amplification with one set of outer primer pairs, sequencing was performed with one of the latter primers. The resulting sequence was genotyped and subtyped using the Oxford Automated HCV Subtyping tool. (De Oliveira et al. (2005) Bioinformatics 21, 3797-3800.)

[0112] Primer selection and synthesis: Primers for reverse transcription, PCR amplification and sequencing were developed using the OLIGO software (Medprobe, Oslo, Norway). Aligned near-full length genome sequences were downloaded from the Los Alamos HCV database (http://hcv.lanl.gov/content/sequence/HCV/ToolsOutline.html). The primer sequences for HCV1b are given in Table 1. The primers were synthesised by Applera Europe (Lennik, Belgium).

[0113] RNA extraction: RNA extraction was performed using the QIAamp Viral RNA mini kit from Qiagen (Westburg, Leusden, The Netherlands) according to the manufacturer's protocol.

[0114] cDNA synthesis and PCR amplification: RNA was reverse-transcribed in three separate reactions (primed by the three outer antisense primers) with Transcriptor RT from Roche (Roche Diagnostics, Mannheim, Germany). First, 2.5 .mu.M outer antisense primer and 10 .mu.l RNA were denatured in a microtube for 5 min at 65.degree. C. and snap-cooled. Next, a reverse transcription mixture was assembled in a final volume of 20 .mu.l, the mixture including the following additional components: 1.times. Transcriptor RT-buffer, 1 mM dNTP's, 20U of Protector RNase Inhibitor, 10U of Transcriptor Reverse Transcriptase and MilliQ water. cDNA synthesis was performed at 50.degree. C. for 90 minutes, and reactions were then cooled down to 4.degree. C. PCR amplification was done in two steps using the Expand Long Template PCR System from Roche (Roche Diagnostics, Mannheim, Germany). A first set of PCR was performed under the following conditions: lx Expand Long Template Buffer 1, 0.350 mM dNTP's, 0.3 .mu.M outer sense primer, 0.3 .mu.M outer antisense primer, 3.75U Expand Long Template DNA Polymerase, 5 pl template cDNA and MilliQ water in a final volume of 50 .mu.l. Subsequent to a denaturation step at 94.degree. C. for 2 min, 10 cycles of 10 sec at 94.degree. C., 30 sec at 57.degree. C., and 4 min at 68.degree. C. were performed. This amplification was followed by 25 cycles of 15 sec at 94.degree. C., 30 sec at 57.degree. C., and 4 min at 68.degree. C., with a time increment of 20 sec/cycle and a final elongation step of 7 min at 68.degree. C. The reactions were then cooled to 4.degree. C. A second set of PCR was performed under the following conditions: 1.times. Expand Long Template Buffer 1, 0.350 mM dNTP's, 0.3 .mu.M inner sense primer, 0.3 .mu.M inner antisense primer, 3.75U Expand Long Template DNA Polymerase, 2 .mu.l amplification product from the first set of PCR and MilliQ water in a final volume of 50 .mu.l. Cycling conditions were identical to those of the first set of PCR except that only three cycles were performed.

[0115] Gel electrophoresis: PCR products were analyzed by agarose gel electrophoresis. 7.5 .mu.l of PCR product were loaded on a 1.5% agarose gel, and electrophoresis was performed at 100V. Gels were stained for 10 min with ethidium bromide for visualising DNA fragments.

[0116] PCR product purification: PCR products were purified using the Qiaquick

[0117] PCR purification kit from Qiagen (Westburg, Leiden, The Netherlands) according to the manufacturer's protocol. Concentration of purified PCR products was determined spectrophotometrically. Sequencing reactions required about 2ng/100 by of DNA.

[0118] Nucleotide sequencing: Sequencing reactions were performed at Fasteris SA (Geneva, Switzerland) using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems Inc., Foster City, USA). Contigs were assembled and the data were analysed in Seqscape (Applied Biosystems Inc., Foster City, USA).

Example 2

Validation of Assays of the Invention: PCR Success Rate, and Reproducibility and Sensitivity of an Assay of the Invention

[0119] The rate of success in obtaining visualizable and sequencable quantities of three overlapping PCR products representing nearly the entire viral genome from HCV 1b-containing samples was determined using 60 samples from infected subjects that were originally genotyped as HCV1b by Inno-LiPA HCV I. Genotypes/subtypes were re-analyzed by sequencing and phylogenetic analysis for samples with equivocal results.

[0120] Reproducibility of the assay was assessed for each partial genome PCR by triplicate testing of samples of confirmed genotype 1b. At least 2 of the 3 repeat testings were performed starting from plasma sample. Exceptionally, the 3rd repeat testing was started from the extracted RNA for samples with insufficient plasma volumes.

[0121] Sensitivity of the assay was validated on 10 samples.

[0122] Specificity of the assay was estimated by partial sequencing of PCR products produced from a subset of 8 samples. All sequences were either HCV1b or 1a. Results of these analyses are summarized in Table 2 below. Example results of the sensitivity determinations are presented in FIG. 2.

[0123] The data presented in Table 2 demonstrate that PCR success rate approached 100% after subtype correction. The assay was HCV genotype 1-specific. Good reproducibility was observed. Sensitivity was excellent for the 5'UTR-NS2 and NS2-NS5A regions, and acceptable for the NS4B-NS5B region.

TABLE-US-00002 TABLE 2 PCR success rate, and reproducibility and sensitivity of an assay of the invention PCR PCR success success rate rate HCV InnoLipa- sequence- Sensitivity genome typed as typed as (copies/ Pairs of primers region HCV1b HCV1b Reproducibility reaction) HCV1bOR3306/ 5'UTR-NS2 46/60 45/47 27/45 (3 of 3 tests) 1-10 HCVOF129 (82%) (96%) 16/45 (2 of 3 tests) 2/45 (1 of 3 tests) HCV1bOR6312/ NS2-NS5A 55/60 44/47 17/44 (3 of 3 tests) 10-100 HCV1bOF1977 (92%) (94%) 22/44 (2 of 3 tests) 5/44 (1 of 3 tests) poly-A/ NS4B- 54/60 (45/47) 19/45 (3 of 3 tests) 100-1000 HCV1bOF6074 NS5B (90%) (96%) 18/45 (2 of 3 tests) 8/45 (1 of 3 tests)

Example 3

Analysis of Samples from Treatment-Naive, HCV-Infected Subjects by Means of an Assay of the Invention

[0124] Aliquots of plasma samples of treatment-naive, HCV-infected subjects were analysed by an assay of the invention (the assay of claim 5). Nucleic acid sequences obtained after sequencing were automatically translated into amino acids and compared/aligned with clustalw2.0 program (at EBisite) to an HCV 1b consensus sequence, HCV coni (Genbank Accession Number AF011751). Sequences 5306 and 5415 are from two therapy-naive, infected subjects. Translated amino acid sequences derived from HCV present in the two subjects are compared to the coni sequence in FIGS. 3a and 3b. Sequence identity with the consensus sequence is indicated by dash in the sequences from the infected subjects. The beginning of the coding sequence for each viral protein is indicated (CORE, E1, E2, P7, NS2,NS3, NS4A, NS4B, NS5A, NS5B).

[0125] The amino acid sequences derived from HCV present in the naive, infected subjects reveal the presence of mutations L91 M in the core protein associated with resistance to IFN/Ribavirin therapy and V499A in NS5B associated with resistance to non-nucleoside inhibitors or benzimidazole compounds. See Table 3 for relevant published information.

TABLE-US-00003 TABLE 3 Summary of some amino acid mutations found in the HCV1b genome of subjects or HCV replicons that are associated with drug resistance Protein region in HCV1b Mutations Drug Resistance Publication reference Core L91M IFN/Ribavirin therapy Akuta et al, Virology, 2005 NS5B V499A Non-nucleoside Kukolj et al., JBC, inhibitor, 2005; benzimidazole Hwu et al., Antivir. compounds Res., 2008 NS5A T245A IFN/Ribavirin therapy Nousbaum et al., J. in HCV 1a-infected Virology, 2000 subjects NS3 C16S/C ACH806 Yang et al., Antimicrob. Agent Chem., 2008

Sequence CWU 1

1

58130DNAArtificial SequencePrimer HCVORpolyA 1aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 30232DNAArtificial SequencePrimer HCV1bOR6312 2ggcaggagct tggactggag ccaggtcttg aa 32327DNAArtificial SequencePrimer HCV1bOR3306 3gatgatgtcc ccacacgccg cggtgtc 27430DNAArtificial SequencePrimer HCV1bOF6974 4ggctgtgcag tggatgaacc ggctgatagc 30532DNAArtificial SequencePrimer HCV1bOF1977 5caaggcaact ggttcggctg tacatggatg aa 32629DNAArtificial SequencePrimer HCVOF129 6ccgggagagc catagtggtc tgcggaacc 29726DNAArtificial SequencePrimer HCV1bIR9339 7gggagcaggt agatgcctac ccctac 26829DNAArtificial SequencePrimer HCV1bIR6282 8tcagtcaaca ccgtgcatat ccagtccca 29929DNAArtificial SequencePrimer HCV1bIR2770 9tccgcacgat gcagccatct cccggtcca 291026DNAArtificial SequencePrimer HCV1bIF6126 10gtctccccca cgcactatgt gcctga 261129DNAArtificial SequencePrimer HCV1bIF2523 11gacgcgcgcg tctgygcctg cttrtggat 291230DNAArtificial SequencePrimer HCVIF278 12gccttgtggt actgcctgat agggtgcttg 301325DNAArtificial SequencePrimer 1bSF310 13gagtgccccg ggaggtcttc gtaga 251424DNAArtificial SequencePrimer HCV1bSF807 14ccgggttctg gaggacggcg tgaa 241526DNAArtificial SequencePrimer HCV1bSR850 15aggaagatag agaaagagca accggg 261622DNAArtificial SequencePrimer HCV1bSF1202 16tctcccagct gttcaccttc tc 221721DNAArtificial SequencePrimer HCV1bSR1302 17gaccagttca tcatcatatc c 211823DNAArtificial SequencePrimer HCV1bSF1597 18ggcagctggc acatcaacag gac 231922DNAArtificial SequencePrimer HCV1bSR1653 19gtagaacagc gcggcaagga ac 222023DNAArtificial SequencePrimer HV1bSF1854 20tggtccagtg tattgyttca ccc 232123DNAArtificial SequencePrimer HCV1bSR1990 21ttcatccatg tacagccgaa cca 232222DNAArtificial SequencePrimer HCV1bSF2242 22gtggggggcg tggagcacag gc 222323DNAArtificial SequencePrimer HCV1bSR2433 23cgtacaggta ttgcacgtcc acg 232423DNAArtificial SequencePrimer HCV1bSF2640 24tctctccttc cttgtgttct tct 232523DNAArtificial SequencePrimer HCV1bSF2860 25aaccaccata tgagcctggc gag 232622DNAArtificial SequencePrimer HCV1bSF3085 26cgcgctcaag ggctcatycg tg 222723DNAArtificial SequencePrimer HCV1bSR3280 27caggtgatga tcttggtctc cat 232823DNAArtificial SequencePrimer HCV1bSF3541 28acrcaatctt tcctggcgac ctg 232924DNAArtificial SequencePrimer HCV1bSR3643 29gtcctggtct acattggtgt acat 243022DNAArtificial SequencePrimer HCV1bSF4004 30cgcagacatt ccaagtggcc ca 223123DNAArtificial SequencePrimer HCV1bSR4237 31caaccaccgt cggcaaggaa ctt 233223DNAArtificial SequencePrimer HCV1bSF4516 32ctcattttct gccattccaa gaa 233326DNAArtificial SequencePrimer HCV1bSR4666 33tcaaagtcgc cggtawagcc cgtcat 263421DNAArtificial SequencePrimer HCV1bSF5048 34tagatgccca cttcttgtcc c 213523DNAArtificial SequencePrimer HCV1bSR5164 35agccgtatga gacacttcca cat 233623DNAArtificial SequencePrimer HCV1bSF5524 36caattcaagc agaaggcgct cgg 233725DNAArtificial SequencePrimer HCV1bSR5639 37tgtatcccgc tgatgaartt ccaca 253827DNAArtificial SequencePrimer HCV1bSF5986 38acacgctgtg ataaatgtct cccccgc 273925DNAArtificial SequencePrimer HCV1bSR6092 39gaagcgaacg ctatcagccg gttca 254024DNAArtificial SequencePrimer HCV1bSF6186 40cctctccagc cttaccatca ctca 244126DNAArtificial SequencePrimer HCV1bSR6256 41tccctnagcc acgagccgga gcatgg 264222DNAArtificial SequencePrimer HCV1bSF6416 42tcatgcanac cacctgccca tg 224323DNAArtificial SequencePrimer HCV1bSR6616 43tcccccaccc gcgtgacctc cac 234423DNAArtificial SequencePrimer HCV1bSF6853 44gtgctcactt ccatgctcac cga 234522DNAArtificial SequencePrimer HCV1bSR6964 45tcaaggaagg cgcagacaac tg 224623DNAArtificial SequencePrimer HCV1bSF7066 46gggaacatca cccgcgtgga gtc 234723DNAArtificial SequencePrimer HCV1bSR7273 47gggcacccgt gtaccaccgg agg 234823DNAArtificial SequencePrimer HCV1bSF7504 48tactcctcca tgccccccct tga 234924DNAArtificial SequencePrimer HCV1bSR7558 49ctcgctcacr gtagaccaag accc 245024DNAArtificial SequencePrimer HCV1bSF7968 50ctccgtgtgg aaggacttgc tgga 245123DNAArtificial SequencePrimer HCV1bSR8077 51tctgggaata cgataaggcg agc 235222DNAArtificial SequencePrimer HCV1bSF8528 52agctccagga ctgcacgatg ct 225324DNAArtificial SequencePrimer HCV1bSR8622 53tacctagtca tagcctccgt gaag 245427DNAArtificial SequencePrimer HCV1bSF9010 54acacgctgtg ataaatgtct cccccgc 275522DNAArtificial SequencePrimer HCV1bSR9094 55gatgtctcca gactcgcaag gg 22563010PRTHepatitis C virus con1 56Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly 65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190 Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser 195 200 205 Asn Ala Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro 210 215 220 Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val 225 230 235 240 Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr 245 250 255 Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Leu Cys 260 265 270 Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ala 275 280 285 Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys 290 295 300 Asn Cys Ser Ile Tyr Pro Gly His Val Thr Gly His Arg Met Ala Trp 305 310 315 320 Asp Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ser Gln 325 330 335 Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His 340 345 350 Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp 355 360 365 Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp Gly Gly 370 375 380 Thr Tyr Val Thr Gly Gly Thr Met Ala Lys Asn Thr Leu Gly Ile Thr 385 390 395 400 Ser Leu Phe Ser Pro Gly Ser Ser Gln Lys Ile Gln Leu Val Asn Thr 405 410 415 Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430 Leu Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Val His Lys Phe Asn 435 440 445 Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Ser Pro Ile Asp Ala 450 455 460 Phe Ala Gln Gly Trp Gly Pro Ile Thr Tyr Asn Glu Ser His Ser Ser 465 470 475 480 Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile 485 490 495 Val Pro Ala Ala Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510 Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Ser 515 520 525 Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro 530 535 540 Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe 545 550 555 560 Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ile Gly Asn 565 570 575 Lys Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala 580 585 590 Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu 595 600 605 Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe 610 615 620 Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu 625 630 635 640 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu Asp 645 650 655 Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp 660 665 670 Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly 675 680 685 Leu Ile His Leu His Gln Asn Val Val Asp Val Gln Tyr Leu Tyr Gly 690 695 700 Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp Glu Tyr Val Leu 705 710 715 720 Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp 725 730 735 Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val 740 745 750 Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe 755 760 765 Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro 770 775 780 Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu 785 790 795 800 Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala 805 810 815 Ser Cys Gly Gly Ala Val Phe Val Gly Leu Ile Leu Leu Thr Leu Ser 820 825 830 Pro His Tyr Lys Leu Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr 835 840 845 Phe Ile Thr Arg Ala Glu Ala His Leu Gln Val Trp Ile Pro Pro Leu 850 855 860 Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu Leu Thr Cys Ala Ile 865 870 875 880 His Pro Glu Leu Ile Phe Thr Ile Thr Lys Ile Leu Leu Ala Ile Leu 885 890 895 Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Lys Val Pro Tyr Phe 900 905 910 Val Arg Ala His Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Val 915 920 925 Ala Gly Gly His Tyr Val Gln Met Ala Leu Met Lys Leu Ala Ala Leu 930 935 940 Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Arg Asp Trp Ala 945 950 955 960 His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe 965 970 975 Ser Asp Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala 980 985 990 Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Arg 995 1000 1005 Glu Ile His Leu Gly Pro Ala Asp Ser Leu Glu Gly Gln Gly Trp 1010 1015 1020 Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly 1025 1030 1035 Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Arg Asn 1040 1045 1050 Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser 1055 1060 1065 Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His 1070 1075 1080 Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr 1085 1090 1095 Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala 1100 1105 1110 Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser 1115 1120 1125 Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg 1130 1135 1140 Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val 1145 1150 1155 Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser 1160 1165 1170 Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly 1175 1180 1185 Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr 1190 1195 1200 Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala 1205 1210 1215 Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly 1220 1225 1230 Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly 1235 1240 1245 Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 1250 1255 1260 Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile 1265 1270 1275 Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr 1280 1285 1290 Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly 1295 1300 1305 Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser 1310 1315 1320 Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr 1325 1330 1335 Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly 1340 1345 1350 Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser 1355 1360 1365 Ser Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu 1370 1375 1380 Thr Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys 1385 1390 1395 Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Leu Asn 1400 1405 1410 Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr 1415 1420 1425 Ser Gly Asp Val Ile Val Val Ala Thr Asp Ala Leu Met Thr Gly 1430

1435 1440 Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 1445 1450 1455 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 1460 1465 1470 Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg 1475 1480 1485 Gly Arg Thr Gly Arg Gly Arg Met Gly Ile Tyr Arg Phe Val Thr 1490 1495 1500 Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys 1505 1510 1515 Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala 1520 1525 1530 Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu 1535 1540 1545 Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr 1550 1555 1560 Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln 1565 1570 1575 Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val 1580 1585 1590 Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp 1595 1600 1605 Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro 1610 1615 1620 Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Thr Thr 1625 1630 1635 His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu 1640 1645 1650 Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala 1655 1660 1665 Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val 1670 1675 1680 Gly Arg Ile Ile Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg 1685 1690 1695 Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ala Ser 1700 1705 1710 His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe 1715 1720 1725 Lys Gln Lys Ala Ile Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala 1730 1735 1740 Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Thr Leu Glu 1745 1750 1755 Ala Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln 1760 1765 1770 Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala 1775 1780 1785 Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr 1790 1795 1800 Gln His Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala 1805 1810 1815 Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly 1820 1825 1830 Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu 1835 1840 1845 Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu 1850 1855 1860 Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp 1865 1870 1875 Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val 1880 1885 1890 Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro 1895 1900 1905 Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala 1910 1915 1920 Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser 1925 1930 1935 Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Ser Leu Thr Ile 1940 1945 1950 Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp Cys 1955 1960 1965 Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp 1970 1975 1980 Ile Cys Thr Val Leu Thr Asp Phe Lys Thr Trp Leu Gln Ser Lys 1985 1990 1995 Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg 2000 2005 2010 Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Gln Thr Thr 2015 2020 2025 Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser 2030 2035 2040 Met Arg Ile Val Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly 2045 2050 2055 Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser 2060 2065 2070 Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala Ala Glu 2075 2080 2085 Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr 2090 2095 2100 Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val Pro Ala 2105 2110 2115 Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His Arg Tyr 2120 2125 2130 Ala Pro Ala Cys Lys Pro Leu Leu Arg Glu Glu Val Thr Phe Leu 2135 2140 2145 Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu 2150 2155 2160 Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro 2165 2170 2175 Ser His Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly 2180 2185 2190 Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala 2195 2200 2205 Pro Ser Leu Lys Ala Thr Cys Thr Thr Arg His Asp Ser Pro Asp 2210 2215 2220 Ala Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly 2225 2230 2235 Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu 2240 2245 2250 Asp Ser Phe Glu Pro Leu Gln Ala Glu Glu Asp Glu Arg Glu Val 2255 2260 2265 Ser Val Pro Ala Glu Ile Leu Arg Arg Ser Arg Lys Phe Pro Arg 2270 2275 2280 Ala Met Pro Ile Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu 2285 2290 2295 Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val His Gly 2300 2305 2310 Cys Pro Leu Pro Pro Ala Lys Ala Pro Pro Ile Pro Pro Pro Arg 2315 2320 2325 Arg Lys Arg Thr Val Val Leu Ser Glu Ser Thr Val Ser Ser Ala 2330 2335 2340 Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Glu Ser Ser 2345 2350 2355 Ala Val Asp Ser Gly Thr Ala Thr Ala Ser Pro Asp Gln Pro Ser 2360 2365 2370 Asp Asp Gly Asp Ala Gly Ser Asp Val Glu Ser Tyr Ser Ser Met 2375 2380 2385 Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly 2390 2395 2400 Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys 2405 2410 2415 Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys 2420 2425 2430 Ala Ala Glu Glu Thr Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser 2435 2440 2445 Leu Leu Arg His His Asn Leu Val Tyr Ala Thr Thr Ser Arg Ser 2450 2455 2460 Ala Ser Leu Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val 2465 2470 2475 Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys 2480 2485 2490 Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys 2495 2500 2505 Lys Leu Thr Pro Pro His Ser Ala Arg Ser Lys Phe Gly Tyr Gly 2510 2515 2520 Ala Lys Asp Val Arg Asn Leu Ser Ser Lys Ala Val Asn His Ile 2525 2530 2535 Arg Ser Val Trp Lys Asp Leu Leu Glu Asp Thr Glu Thr Pro Ile 2540 2545 2550 Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro 2555 2560 2565 Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp 2570 2575 2580 Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val 2585 2590 2595 Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly Phe Gln 2600 2605 2610 Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala Trp Lys 2615 2620 2625 Ala Lys Lys Cys Pro Met Gly Phe Ala Tyr Asp Thr Arg Cys Phe 2630 2635 2640 Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile 2645 2650 2655 Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Arg 2660 2665 2670 Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser 2675 2680 2685 Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val 2690 2695 2700 Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala 2705 2710 2715 Ala Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu 2720 2725 2730 Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr 2735 2740 2745 Gln Glu Asp Glu Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr 2750 2755 2760 Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Lys Pro Glu Tyr Asp 2765 2770 2775 Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His 2780 2785 2790 Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr 2795 2800 2805 Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro 2810 2815 2820 Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu 2825 2830 2835 Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu 2840 2845 2850 Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly 2855 2860 2865 Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln 2870 2875 2880 Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro 2885 2890 2895 Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val 2900 2905 2910 Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala 2915 2920 2925 Arg Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr 2930 2935 2940 Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile 2945 2950 2955 Pro Ala Ala Ser Gln Leu Asp Leu Ser Ser Trp Phe Val Ala Gly 2960 2965 2970 Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro 2975 2980 2985 Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly 2990 2995 3000 Ile Tyr Leu Leu Pro Asn Arg 3005 3010 573003PRTHepatitis C virus 57Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly 65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190 Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser 195 200 205 Asn Thr Ser Ile Val Tyr Glu Thr Ala Asp Met Ile Met His Thr Pro 210 215 220 Gly Cys Val Pro Cys Val Arg Glu Ala Asn Ser Ser Arg Cys Trp Ala 225 230 235 240 Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Thr Ser Ile Pro Thr Thr 245 250 255 Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Leu Cys 260 265 270 Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 275 280 285 Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys 290 295 300 Asn Cys Ser Leu Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp 305 310 315 320 Asp Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Ala Ser Gln 325 330 335 Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His 340 345 350 Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp 355 360 365 Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp Gly Ala 370 375 380 Pro Asn Ala Ile Ala Ser Ser Pro Ala Arg Gly Ala Arg Gly Ile Thr 385 390 395 400 Ser Leu Phe Thr Pro Gly Ala Ser Gln Asn Val Gln Leu Ile Asn Thr 405 410 415 Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430 Leu His Thr Gly Phe Leu Ala Ala Leu Phe Tyr Val Asn Lys Phe Asn 435 440 445 Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Asp Gln 450 455 460 Phe Ala Gln Gly Trp Gly Pro Ile Thr His Val Val Pro Lys Asp Leu 465 470 475 480 Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile 485 490 495 Val Arg Ala Ser Glu Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510 Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Asn 515 520 525 Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro 530 535 540 Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe 545 550 555 560 Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Leu Gly Asn 565 570 575 Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala 580 585 590 Thr Tyr Ile Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu 595 600 605 Val Asn Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe 610 615 620 Thr Ile Thr Gln Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu 625 630 635 640 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu Asp 645

650 655 Arg Asp Arg Ala Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp 660 665 670 Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly 675 680 685 Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly 690 695 700 Ile Gly Ser Ala Val Val Ser Tyr Val Ile Lys Trp Glu Tyr Val Leu 705 710 715 720 Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp 725 730 735 Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val 740 745 750 Val Leu Asn Ala Ala Ser Val Ala Gly Thr His Gly Ile Leu Ser Phe 755 760 765 Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro 770 775 780 Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu 785 790 795 800 Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala 805 810 815 Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser 820 825 830 Pro His Tyr Lys Pro Leu Leu Ala Lys Leu Ile Trp Trp Leu Gln Tyr 835 840 845 Leu Ile Thr Arg Ala Glu Ala His Leu Gln Val Trp Val Pro Pro Leu 850 855 860 Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Thr Cys Ala Leu 865 870 875 880 His Pro Glu Leu Thr Phe Asp Ile Thr Lys His Leu Leu Ala Ile Leu 885 890 895 Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Lys Val Pro Tyr Phe 900 905 910 Val Arg Ala Gln Gly Leu Ile Arg Val Cys Met Leu Val Arg Lys Val 915 920 925 Ala Gly Gly His Tyr Val Gln Met Ala Leu Met Lys Leu Ala Ala Leu 930 935 940 Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Arg Asp Trp Ala 945 950 955 960 His Gln Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe 965 970 975 Ser Asp Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala 980 985 990 Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Arg 995 1000 1005 Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Gln Gly Trp 1010 1015 1020 Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly 1025 1030 1035 Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn 1040 1045 1050 Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser 1055 1060 1065 Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Phe His 1070 1075 1080 Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr 1085 1090 1095 Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala 1100 1105 1110 Pro Ser Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser 1115 1120 1125 Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg 1130 1135 1140 Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val 1145 1150 1155 Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Leu 1160 1165 1170 Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly 1175 1180 1185 Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr 1190 1195 1200 Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala 1205 1210 1215 Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly 1220 1225 1230 Ser Gly Lys Ser Thr Arg Val Pro Ala Ala Tyr Ala Ala Gln Gly 1235 1240 1245 Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 1250 1255 1260 Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile 1265 1270 1275 Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr 1280 1285 1290 Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly 1295 1300 1305 Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser 1310 1315 1320 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr 1325 1330 1335 Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly 1340 1345 1350 Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser 1355 1360 1365 Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu 1370 1375 1380 Thr Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys 1385 1390 1395 Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Leu Asn 1400 1405 1410 Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr 1415 1420 1425 Ser Gly Asn Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly 1430 1435 1440 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 1445 1450 1455 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 1460 1465 1470 Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg 1475 1480 1485 Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr 1490 1495 1500 Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys 1505 1510 1515 Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala 1520 1525 1530 Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu 1535 1540 1545 Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr 1550 1555 1560 Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln 1565 1570 1575 Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val 1580 1585 1590 Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp 1595 1600 1605 Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro 1610 1615 1620 Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Val Leu Thr 1625 1630 1635 His Pro Val Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu 1640 1645 1650 Glu Ile Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala 1655 1660 1665 Ala Leu Ala Ala Tyr Cys Leu Thr Ala Gly Ser Val Val Ile Val 1670 1675 1680 Gly Arg Ile Ile Leu Ser Gly Lys Pro Ala Val Ile Pro Asp Arg 1685 1690 1695 Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser 1700 1705 1710 His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe 1715 1720 1725 Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala 1730 1735 1740 Glu Ala Val Ala Pro Val Val Glu Ser Lys Trp Gln Ala Leu Glu 1745 1750 1755 Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln 1760 1765 1770 Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala 1775 1780 1785 Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr 1790 1795 1800 Gln His Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala 1805 1810 1815 Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly 1820 1825 1830 Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu 1835 1840 1845 Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu 1850 1855 1860 Val Ala Phe Lys Ile Met Ser Gly Glu Met Pro Ser Thr Glu Asp 1865 1870 1875 Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val 1880 1885 1890 Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro 1895 1900 1905 Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala 1910 1915 1920 Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser 1925 1930 1935 Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Ser Leu Thr Ile 1940 1945 1950 Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp Cys 1955 1960 1965 Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp 1970 1975 1980 Ile Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser Lys 1985 1990 1995 Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Leu Ser Cys Gln Arg 2000 2005 2010 Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met His Thr Thr 2015 2020 2025 Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser 2030 2035 2040 Met Arg Ile Val Gly Pro Lys Thr Cys Ser Asn Thr Trp His Gly 2045 2050 2055 Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser 2060 2065 2070 Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala Ala Glu 2075 2080 2085 Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr 2090 2095 2100 Gly Ile Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val Pro Ala 2105 2110 2115 Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His Arg Tyr 2120 2125 2130 Ala Pro Ala Cys Lys Pro Leu Leu Arg Glu Glu Val Thr Phe Leu 2135 2140 2145 Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu 2150 2155 2160 Pro Glu Pro Asp Val Thr Val Leu Thr Ser Met Leu Thr Asp Pro 2165 2170 2175 Ser His Ile Thr Ala Glu Ala Ala Lys Arg Arg Leu Ala Arg Gly 2180 2185 2190 Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala 2195 2200 2205 Pro Ser Leu Lys Ala Thr Cys Thr Ala Cys His Asp Ser Pro Asp 2210 2215 2220 Ala Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly 2225 2230 2235 Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu 2240 2245 2250 Asp Ser Phe Asp Pro Leu His Ala Glu Glu Asp Glu Gly Glu Val 2255 2260 2265 Ser Ile Pro Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Arg 2270 2275 2280 Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu 2285 2290 2295 Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val His Gly 2300 2305 2310 Cys Pro Leu Pro Pro Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg 2315 2320 2325 Arg Lys Arg Thr Val Val Leu Thr Asp Ser Thr Val Ser Ser Ala 2330 2335 2340 Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Glu Ser Ser 2345 2350 2355 Ala Val Asp Ser Gly Thr Ala Thr Ala Pro Pro Asp Gln Pro Leu 2360 2365 2370 Asp Asp Gly Asn Thr Gly Ser Asp Ala Glu Ser Tyr Ser Ser Met 2375 2380 2385 Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly 2390 2395 2400 Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys 2405 2410 2415 Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys 2420 2425 2430 Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser 2435 2440 2445 Leu Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser 2450 2455 2460 Ala Ser Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val 2465 2470 2475 Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys 2480 2485 2490 Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Ile Glu Glu Ala Cys 2495 2500 2505 Lys Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly 2510 2515 2520 Ala Lys Asp Val Arg Asn Leu Ser Ser Lys Ala Val Asn His Ile 2525 2530 2535 Arg Ser Val Trp Gln Asp Leu Leu Glu Asp Ser Glu Thr Pro Ile 2540 2545 2550 Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro 2555 2560 2565 Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp 2570 2575 2580 Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val 2585 2590 2595 Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly Phe Gln 2600 2605 2610 Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala Trp Lys 2615 2620 2625 Ser Lys Lys Asn Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe 2630 2635 2640 Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Val Glu Glu Ser Ile 2645 2650 2655 Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Arg 2660 2665 2670 Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn Ser 2675 2680 2685 Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val 2690 2695 2700 Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala 2705 2710 2715 Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu 2720 2725 2730 Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr 2735 2740 2745 Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala Met Thr 2750 2755 2760 Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp 2765 2770 2775 Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His 2780 2785 2790 Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr 2795 2800 2805 Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro 2810 2815 2820 Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu 2825 2830 2835 Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu 2840 2845 2850 Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly 2855 2860

2865 Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln 2870 2875 2880 Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro 2885 2890 2895 Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val 2900 2905 2910 Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg Ala 2915 2920 2925 Lys Leu Leu Ser Leu Gly Gly Arg Ala Ala Thr Cys Gly Arg Tyr 2930 2935 2940 Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile 2945 2950 2955 Pro Ala Ala Ser Gln Leu Asp Leu Ser Gly Trp Phe Val Ala Gly 2960 2965 2970 Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro 2975 2980 2985 Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly 2990 2995 3000 583005PRTHepatitis C virus 58Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly 65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190 Glu Val Arg Asn Val Ser Gly Ala Tyr His Val Thr Asn Asp Cys Ser 195 200 205 Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile Met His Thr Pro 210 215 220 Gly Cys Val Pro Cys Val Arg Glu Asp Asn Thr Ser Arg Cys Trp Val 225 230 235 240 Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Met 245 250 255 Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Thr Ala Ala Phe Cys 260 265 270 Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 275 280 285 Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys 290 295 300 Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp 305 310 315 320 Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln 325 330 335 Leu Leu Arg Ile Pro Gln Val Val Met Asp Ile Val Thr Gly Ala His 340 345 350 Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Ala Gly Asn Trp 355 360 365 Ala Lys Val Leu Ile Val Leu Leu Leu Phe Ala Gly Val Asp Gly Arg 370 375 380 Thr Glu Thr Thr Gly Gly Val Ala Ala Arg Thr Thr His Gly Phe Thr 385 390 395 400 Ser Leu Phe Ser Val Gly Ser Lys Gln Thr Ile Gln Leu Ile Asn Thr 405 410 415 Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430 Leu Gln Thr Gly Phe Leu Ala Ala Leu Phe Tyr Val Asn Lys Phe Asn 435 440 445 Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Asp Lys 450 455 460 Phe Ala Gln Gly Trp Gly Pro Ile Ser Tyr Ala Lys Ala Ser Ser Ser 465 470 475 480 Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile 485 490 495 Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510 Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Val Pro Thr Tyr Asn 515 520 525 Trp Gly Glu Asn Glu Thr Asp Val Leu Ile Leu Asn Asn Thr Arg Pro 530 535 540 Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Tyr 545 550 555 560 Thr Lys Thr Cys Gly Gly Pro Pro Cys Asp Ile Gly Gly Val Gly Asn 565 570 575 Asp Ser Asn Arg Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 580 585 590 Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg 595 600 605 Cys Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val 610 615 620 Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His 625 630 635 640 Arg Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu 645 650 655 Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 660 665 670 Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser 675 680 685 Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu 690 695 700 Tyr Gly Val Gly Ser Ala Val Val Ser Leu Ala Ile Lys Trp Glu Tyr 705 710 715 720 Val Leu Leu Leu Phe Leu Phe Leu Ala Asp Ala Arg Val Cys Ala Cys 725 730 735 Val Trp Met Met Met Leu Ile Val Gln Ala Glu Ala Ala Leu Glu Asn 740 745 750 Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Glu His Gly Ile Leu 755 760 765 Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu 770 775 780 Val Pro Gly Ala Thr Tyr Ala Phe Tyr Ser Val Trp Pro Leu Leu Leu 785 790 795 800 Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Thr 805 810 815 Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Ala Leu Leu Thr 820 825 830 Leu Ser Pro His Tyr Lys Glu Leu Leu Ala Lys Leu Ile Trp Trp Leu 835 840 845 Gln Tyr Leu Ile Thr Arg Ala Glu Ala Gln Leu Gln Val Trp Val Pro 850 855 860 Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Thr Cys 865 870 875 880 Met Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Ile Leu Leu Ala 885 890 895 Ile Leu Gly Pro Leu Met Val Leu Gln Ala Ser Ile Thr Lys Met Pro 900 905 910 Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Ala Leu Val Arg 915 920 925 Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Met Lys Leu Ala 930 935 940 Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Arg Asp 945 950 955 960 Trp Ala His Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val 965 970 975 Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr 980 985 990 Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg 995 1000 1005 Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Glu Gly Gln 1010 1015 1020 Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr 1025 1030 1035 Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp 1040 1045 1050 Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr 1055 1060 1065 Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val 1070 1075 1080 Phe His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro 1085 1090 1095 Val Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp 1100 1105 1110 Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly 1115 1120 1125 Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro 1130 1135 1140 Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg 1145 1150 1155 Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys 1160 1165 1170 Ser Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr 1175 1180 1185 Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met 1190 1195 1200 Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro 1205 1210 1215 Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro 1220 1225 1230 Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala 1235 1240 1245 Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr 1250 1255 1260 Leu Ser Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro 1265 1270 1275 Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile 1280 1285 1290 Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser 1295 1300 1305 Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Ile 1310 1315 1320 Asp Ser Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala 1325 1330 1335 Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro 1340 1345 1350 Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala 1355 1360 1365 Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro 1370 1375 1380 Ile Glu Thr Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser 1385 1390 1395 Lys Lys Lys Cys Asp Glu Ile Ala Ala Lys Leu Ser Ser Leu Gly 1400 1405 1410 Leu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile 1415 1420 1425 Pro Thr Ser Gly Asn Val Val Val Val Ala Thr Asp Ala Leu Met 1430 1435 1440 Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr 1445 1450 1455 Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr 1460 1465 1470 Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln 1475 1480 1485 Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe 1490 1495 1500 Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val 1505 1510 1515 Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr 1520 1525 1530 Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro 1535 1540 1545 Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val 1550 1555 1560 Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr 1565 1570 1575 Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala 1580 1585 1590 Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln 1595 1600 1605 Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro 1610 1615 1620 Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Ile 1625 1630 1635 Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ala Ala 1640 1645 1650 Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val 1655 1660 1665 Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val 1670 1675 1680 Ile Val Gly Arg Ile Ile Leu Ser Gly Lys Pro Ala Val Ile Pro 1685 1690 1695 Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys 1700 1705 1710 Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu 1715 1720 1725 Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys 1730 1735 1740 Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala 1745 1750 1755 Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly 1760 1765 1770 Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala 1775 1780 1785 Ile Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu 1790 1795 1800 Thr Thr Gln His Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val 1805 1810 1815 Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly 1820 1825 1830 Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys 1835 1840 1845 Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly 1850 1855 1860 Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr 1865 1870 1875 Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala 1880 1885 1890 Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 1895 1900 1905 Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala 1910 1915 1920 Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro 1925 1930 1935 Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Ser Leu 1940 1945 1950 Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu 1955 1960 1965 Asp Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp 1970 1975 1980 Asp Trp Ile Cys Thr Val Leu Thr Asp Phe Lys Thr Trp Leu Gln 1985 1990 1995 Ser Lys Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys 2000 2005 2010 Gln Arg Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Gln 2015 2020 2025 Thr Thr Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn 2030 2035 2040 Gly Ser Met Arg Ile Ile Gly Pro Lys Thr Cys Ser Asn Thr Trp 2045 2050 2055 His Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr 2060 2065 2070 Pro Ser Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala 2075 2080 2085 Ala Glu Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr 2090 2095

2100 Val Thr Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Val 2105 2110 2115 Pro Ala Pro Glu Phe Tyr Thr Glu Val Asp Gly Val Arg Leu His 2120 2125 2130 Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Glu Glu Val Thr 2135 2140 2145 Phe Leu Val Gly Leu Asn Glu Tyr Pro Val Gly Ser Gln Leu Pro 2150 2155 2160 Cys Glu Pro Glu Pro Asp Val Thr Val Leu Ala Ser Met Leu Thr 2165 2170 2175 Asp Pro Ser His Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala 2180 2185 2190 Arg Gly Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu 2195 2200 2205 Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Thr Asn His Asp Ser 2210 2215 2220 Pro Asp Ala Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu 2225 2230 2235 Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val 2240 2245 2250 Ile Leu Asp Ser Phe Asp Pro Leu Arg Ala Glu Glu Asp Glu Arg 2255 2260 2265 Glu Val Ser Ile Pro Ala Glu Ile Leu Arg Lys Ser Lys Lys Phe 2270 2275 2280 Pro Ser Ala Met Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro 2285 2290 2295 Leu Leu Glu Ser Trp Lys Ser Pro Asp Tyr Val Pro Pro Val Val 2300 2305 2310 His Gly Cys Pro Leu Pro Pro Ala Lys Ala Pro Pro Val Pro Pro 2315 2320 2325 Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val Ser 2330 2335 2340 Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Ser Ser Ser Glu 2345 2350 2355 Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Ala Pro Pro Asp Gln 2360 2365 2370 Pro Leu Asp Asp Gly Asp Ala Gly Ser Asp Ala Gly Ser Tyr Ser 2375 2380 2385 Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser 2390 2395 2400 Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val 2405 2410 2415 Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr 2420 2425 2430 Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser 2435 2440 2445 Asn Ser Leu Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser 2450 2455 2460 Arg Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu 2465 2470 2475 Gln Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys 2480 2485 2490 Ala Lys Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu 2495 2500 2505 Ala Cys Lys Leu Thr Pro Pro His Ser Ala Arg Ser Lys Phe Gly 2510 2515 2520 Tyr Gly Ala Lys Asp Val Arg Asn Leu Ser Ser Lys Ala Val Asn 2525 2530 2535 His Ile His Ser Val Trp Lys Asp Leu Leu Glu Asp Ser Glu Thr 2540 2545 2550 Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val 2555 2560 2565 Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Tyr 2570 2575 2580 Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp 2585 2590 2595 Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly 2600 2605 2610 Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala 2615 2620 2625 Trp Lys Ser Lys Lys Cys Pro Met Gly Phe Ala Tyr Asp Thr Arg 2630 2635 2640 Cys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Val Glu Glu 2645 2650 2655 Ser Ile Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala 2660 2665 2670 Ile Arg Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr 2675 2680 2685 Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser 2690 2695 2700 Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu 2705 2710 2715 Lys Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr 2720 2725 2730 Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala 2735 2740 2745 Gly Thr Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala 2750 2755 2760 Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu 2765 2770 2775 Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val 2780 2785 2790 Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp 2795 2800 2805 Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Lys His 2810 2815 2820 Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro 2825 2830 2835 Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile 2840 2845 2850 Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile 2855 2860 2865 Tyr Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile 2870 2875 2880 Ile Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr 2885 2890 2895 Ser Pro Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu 2900 2905 2910 Gly Val Pro Pro Leu Arg Thr Trp Arg His Arg Ala Arg Ser Val 2915 2920 2925 Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala Ile Cys Gly 2930 2935 2940 Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr 2945 2950 2955 Pro Ile Pro Ala Ala Ser Arg Leu Asp Leu Ser Gly Trp Phe Val 2960 2965 2970 Ala Gly Tyr Gly Gly Gly Asp Ile Tyr His Ser Val Ser Arg Ala 2975 2980 2985 Arg Pro Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly 2990 2995 3000 Val Gly 3005

Claims

1-8. (canceled)

9. A pair of primers selected from the group consisting of: (a) poly-A and HCV1bO F6074; (b) HCV1bOR6312 and HCV1bOF1977; (c) HCV1bOR3306 and HCVOF129; (d) HCV1bIR9339 and HCV1bIF6126; (e) HCV1bIR6282 and HCV1bIF2523; (f) HCV1bIR2770 and HCVIF278; and combinations thereof.

10. The pair of primers of claim 9, wherein said pair of primers is HCV1bOR6312 and HCV1bOF1977.

11. The pair of primers of claim 9, wherein said pair of primers is HCV1bOR3306 and HCVOF129.

12. The pair of primers of claim 9, wherein said pair of primers is HCV1bIR9339 and HCV1bIF6126.

13. The pair of primers of claim 9, wherein said pair of primers is HCV1bIR6282 and HCV1bIF2523.

14. The pair of primers of claim 9, wherein said pair of primers is of HCV1bIR2770 and HCVIF278.

15. A kit for detecting mutations in an HCV genome, wherein said kit comprises the pair of primers of claim 9.

16. (canceled)

17. The pair of primers of claim 9, wherein said pair of primers consists of poly-A and HCV1bOF6074.