# Patent

## Title
Near full-genome assay of HCV drug resistance

## Abstract

The invention relates to assays for characterization of genotypic mutations of Hepatitis C Virus (HCV) showing a resistance to anti-HCV drugs.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Stage of International Application No. PCT/IB2008/002924, filed Oct. 31, 2008, which claims the benefit of International Application No. PCT/IB2007/003304, filed Oct. 31, 2007, the contents of which are incorporated by reference herein.
BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Description of Related Art
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.
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).
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).
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.
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—2007.pdf).
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® (Roche) and m2000 Real-Time PCR Diagnostics System® (Abott). Other PCR-based assays that include, e.g., Versant® HCV Genotyping Assay (Bayer Diagnostics), INNO-LiPA HCV II® (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
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:

    
    
        a) extraction of viral RNA from the sample containing the HCV;
        b) determination of genotype and subtype of the HCV;
        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;
        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;
        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;
        f) sequence analysis of the further amplified cDNAs of step e); and
        g) comparison of the sequences obtained from step f) with that of the prototype HCV.
    
    


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:

    
    
        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;
        b) determination of genotype and subtype of the HCV;
        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;
        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;
        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;
        f) sequence analysis of the further amplified cDNAs of step e);
        g) comparison of the sequences obtained from step f) with that of the prototype HCV and identification of mutations; and
        h) entry of the mutations identified in a data bank of HCV mutations associated with anti-HCV drug resistance.
    
    


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.
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.
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:

    
    
        a) extraction of viral RNA from the sample containing the HCV;
        b) determination of genotype and subtype of the HCV;
        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;
        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;
        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;
        f) sequence analysis of the further amplified cDNAs of step e); and
        g) comparison of the sequences obtained from step f) with that of a prototype HCV1b.
    
    


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 HCV 1b 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:

    
    
        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;
        b) determination of genotype and subtype of the HCV;
        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;
        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;
        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/HCV1F278;
        f) sequence analysis of the further amplified cDNAs of step e);
        g) comparison of the sequences obtained from step f) with that of a prototype HCV1b and identification of mutations; and
        h) entry of the mutations identified in a data bank of HCV mutations associated with anti-HCV drug resistance.
    
    


Once a useful data bank has been assembled, a similar assay can be utilized to analyze samples from treatment-naïve 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.
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
FIG. 1 schematically represents the partial cDNA synthesis and amplification steps of assays of the invention on the example of an HCV type 1b.
FIG. 2 compares the sensitivity of the partial cDNA amplification method of the invention with that of amplification of full-length viral genomes.
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
To aid in understanding the invention, several terms are defined below.
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.
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.
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.
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 5° 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”.
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.
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.
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.
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.
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”.
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.
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. 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 HCV1b primers. HCV con1 (Genbank Accession Number AJ238799) was used as prototype HCV1b 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 HCV2a.
The term “quasispecies” relates to a group of highly related HCV of identical genotype and subtype frequently present in an infected subject.
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.
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.
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.
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.
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.
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.
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.
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).
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:

    
    
        Step 1: extraction of viral RNA from a sample containing an HCV;
        Step 2: determination of genotype and subtype of the HCV;
        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;
        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;
        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;
        Step 6: sequence analysis of the further amplified cDNAs of step 5; and
        Step 7: comparison of the sequences obtained with that of the prototype HCV.
    
    


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:

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


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.
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.
Specific embodiments of assays for use with samples containing an HCV1b 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:

    
    
        Step 1: extraction of viral RNA from a sample containing an HCV;
        Step 2: determination of genotype and subtype of the HCV;
        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;
        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;
        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;
        Step 6: sequence analysis of the further amplified cDNAs of step 5; and
        Step 7: comparison of the sequences obtained with that of a prototype HCV1b.
    
    


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:

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


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.
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.
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 1







 HCV 1b primers
















Location in







the genome
SEQ






(H77 as
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
CCGGGAGAGCCATAG
129-157
 6



NS2 region

TGGTCTGCGGAACC




 


HCVIF278
inner PCR 5′UTR-
sense
GCCTTGTGGTACTGC
278-307
12



NS2 region

CTGATAGGGTGCTTG




 


HCV1bIR2770
inner PCR 5′UTR-
antisense
TCCGCACGATGCAGC
2798-2770
 9



NS2 region

CATCTCCCGGTCCA




 


HCV1bOR6312
cDNA/outer PCR
antisense
GGCAGGAGCTTGGAC
6343-6312
 2



NS2-NS5A region

TGGAGCCAGGTCTTG







AA




 


HCV1bOF1977
outer PCR NS2-
sense
CAAGGCAACTGGTTC
1977-2008
 5



NS5A region

GGCTGTACATGGATG







AA




 


HCV1bIF2523
inner PCR NS2-
sense
GACGCGCGCGTCTGY
2523-2551
11



NS5A region

GCCTGCTTRTGGAT




 


HCV1bIR6282
inner PCR NS2-
antisense
TCAGTCAACACCGTG
6310-6282
 8



NS5A region

CATATCCAGTCCCA




 


poly-A
cDNA/outer PCR
antisense
30-mer
9447-9418
 1



NS4B-3′UTR 







region






 


HCVOF6074
outer PCR NS4B-
sense
GGCTGTGCAGTGGAT
6074-6103
 4



3′UTR region

GAACCGGCTGATAGC




 


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 
sense
GAGTGCCCCGGGA
309-332
13



5′UTR-NS2 region

GGTCTTCGTAGA




 


HCV1bSF807
sequencing 
sense
CCGGGTTCTGGAG
806-829
14



5′UTR-NS2 region

GACGGCGTGAA




 


HCV1bSR850
sequencing 
antisense
AGGAAGATAGAGAA
874-849
15



5′UTR-NS2 region

AGAGCAACCGGG




 


HCV1bSF1202
sequencing 
sense
TCTCCCAGCTGTTC
1201-1222
16



5′UTR-NS2 region

ACCTTCTC




 


HCV1bSR1302
sequencing 
antisense
GACCAGTTCATCAT
1321-1301
17



5′UTR-NS2 region

CATATCC




 


HCV1bSF1597
sequencing 
sense
GGCAGCTGGCACA
1593-1615
18



5′UTR-NS2 region

TCAACAGGAC




 


HCV1bSR1653
sequencing 
antisense
GTAGAACAGCGCG
1674-1653
19



5′UTR-NS2 region

GCAAGGAAC




 


HCVlbSF1854
sequencing 
sense
TGGTCCAGTGTATT
1850-1872
20



5′UTR-NS2 region

GYTTCACCC




 


HCV1bSR1990
sequencing 
antisense
TTCATCCATGTACA
2008-1986
21



5′UTR-NS2 region

GCCGAACCA




 


HCV1bSF2242
sequencing 
sense
GTGGGGGGCGTGG
2238-2259
22



5′UTR-NS2 region

AGCACAGGC




 


HCV1bSR2433
sequencing 
antisense
CGTACAGGTATTGC
2451-2429
23



5′UTR-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









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 HCV1a, HCV2 and HCV3. 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.
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.
HCV RNA extraction from samples may be performed by various methods including the use of commercially available kits, e.g., QIAamp® Viral RNA mini kit, QIAamp® UtralsensTM Virus Kit, Trizol reagens (Invitrogen) or Vivaspin concentration.
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.
All publications and patents cited herein shall be considered as incorporated by reference in their entirety.
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
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).
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.)
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).
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.
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 μM outer antisense primer and 10 μl RNA were denatured in a microtube for 5 min at 65° C. and snap-cooled. Next, a reverse transcription mixture was assembled in a final volume of 20 μl, the mixture including the following additional components: 1× Transcriptor RT-buffer, 1 mM dNTP's, 20 U of Protector RNase Inhibitor, 10 U of Transcriptor Reverse Transcriptase and MilliQ water. cDNA synthesis was performed at 50° C. for 90 minutes, and reactions were then cooled down to 4° 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: 1× Expand Long Template Buffer 1, 0.350 mM dNTP's, 0.3 μM outer sense primer, 0.3 μM outer antisense primer, 3.75 U Expand Long Template DNA Polymerase, 5 μl template cDNA and MilliQ water in a final volume of 50 μl. Subsequent to a denaturation step at 94° C. for 2 min, 10 cycles of 10 sec at 94° C., 30 sec at 57° C., and 4 min at 68° C. were performed. This amplification was followed by 25 cycles of 15 sec at 94° C., 30 sec at 57° C., and 4 min at 68° C., with a time increment of 20 sec/cycle and a final elongation step of 7 min at 68° C. The reactions were then cooled to 4° C. A second set of PCR was performed under the following conditions: 1× Expand Long Template Buffer 1, 0.350 mM dNTP's, 0.3 μM inner sense primer, 0.3 μM inner antisense primer, 3.75 U Expand Long Template DNA Polymerase, 2 μl amplification product from the first set of PCR and MilliQ water in a final volume of 50 μl. Cycling conditions were identical to those of the first set of PCR except that only three cycles were performed.
Gel electrophoresis: PCR products were analyzed by agarose gel electrophoresis. 7.5 μl 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.
PCR product purification: PCR products were purified using the Qiaquick 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 2 ng/100 by of DNA.
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
The rate of success in obtaining visualizable and sequencable quantities of three overlapping PCR products representing nearly the entire viral genome from HCV1b-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.
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.
Sensitivity of the assay was validated on 10 samples.
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.
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 2







PCR success rate, and reproducibility and sensitivity of an assay of the invention













HCV
PCR success
PCR success

Sensitivity



genome
rate InnoLipa-
rate sequence-

(copies/


Pairs of primers
region
typed as HCV1b
typed as 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-Naïve, HCV-Infected Subjects by Means of an Assay of the Invention
Aliquots of plasma samples of treatment-naïve, 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 HCV1b consensus sequence, HCV con1 (Genbank Accession Number AF011751). Sequences 5306 and 5415 are from two therapy-naïve, infected subjects. Translated amino acid sequences derived from HCV present in the two subjects are compared to the con1 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).
The amino acid sequences derived from HCV present in the naïve, 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 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

## Claims

1. An assay for identifying a mutation in the genome of an HCV present in a sample, the assay comprising the sequential steps of:
a) extraction of viral RNA from the sample containing the HCV;
b) determination of genotype and subtype of the HCV;
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;
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;
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;
f) sequence analysis of the further amplified cDNAs of step e); and
g) comparison of the sequences obtained from step f) with that of the prototype HCV.

2. An assay for identifying and characterizing single and linked mutations in a genome of an HCV resistant to an anti-HCV drug, the assay comprising the sequential steps of:
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;
b) determination of genotype and subtype of the HCV;
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;
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;
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;
f) sequence analysis of the further amplified cDNAs of step e);
g) comparison of the sequences obtained from step f) with that of the prototype HCV and identification of mutations; and
h) entry of the mutations identified in a data bank of HCV mutations associated with anti-HCV drug resistance.

3. The assay of claim 2, wherein step h) consists of 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.

4. The assay of claim 3, wherein the sample is taken from a subject infected with HCV prior to commencement of any pharmacological therapy of the subject.

5. An assay for identifying a mutation in the genome of an HCV present in a sample, the assay comprising the sequential steps of:
a) extraction of viral RNA from the sample containing the HCV;
b) determination of genotype and subtype of the HCV;
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 (SEQ ID NO: 1), the second reverse transcription reaction initiated from primer HCV1bOR6312 (SEQ ID NO: 2) and the third reverse transcription reaction initiated from primer HCV1bOR3306 (SEQ ID NO: 3);
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 (SEQ ID NOs: 1/4), the second PCR reaction comprising an aliquot of the second reverse transcription reaction and primer pair HCV1bOR6312/HCV1bOF1977 (SEQ ID NOs: 2/5) and the third PCR reaction comprising an aliquot of the third reverse transcription reaction and primer pair HCV1 bOR3306/HCVOF129 (SEQ ID NOs: 3/6);
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 HCV1b1R9339/HCV1b1F6126 (SEQ ID NOs: 7/10), the second nested PCR reaction comprising an aliquot of the second PCR reaction and primer pair HCV1b1R6282/HCV1b1F2523 (SEQ ID NOs: 8/11), and the third nested PCR reaction comprising an aliquot of the third PCR reaction and primer pair HCV1 b1R2770/HCVIF278 (SEQ ID NOs: 9/12);
f) sequence analysis of the further amplified cDNAs of step e); and
g) comparison of the sequences obtained from step f) with that of a prototype HCV1b.

6. An assay for identifying and characterizing single and linked mutations in a genome of an HCV resistant to an anti-HCV drug, the assay comprising the sequential steps of:
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;
b) determination of genotype and subtype of the HCV;
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 (SEQ ID NO: 1), the second reverse transcription reaction initiated from primer HCV1bOR6312 (SEQ ID NO: 2) and the third reverse transcription reaction initiated from primer HCV1bOR3306 (SEQ ID NO: 3);
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 (SEQ ID NOs: 1/4), the second PCR reaction comprising an aliquot of the second reverse transcription reaction and primer pair HCV1bOR6312/HCV1bOF1977 (SEQ ID NOs: 2/5) and the third PCR reaction comprising an aliquot of the third reverse transcription reaction and primer pair HCV1bOR3306/HCVOF129 (SEQ ID NOs: 3/6);
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 HCV1b1R9339/HCV1b1F6126 (SEQ ID NOs: 7/10), the second nested PCR reaction comprising an aliquot of the second PCR reaction and primer pair HCV1b1R6282/HCV1b1F2523 (SEQ ID NOs: 8/11), and the third nested PCR reaction comprising an aliquot of the third PCR reaction and primer pair HCV1b1R2770/HCVIF278 (SEQ ID NOs: 9/12);
f) sequence analysis of the further amplified cDNAs of step e);
g) comparison of the sequences obtained from step f) with that of a prototype HCV 1b and identification of mutations; and
h) entry of the mutations identified in a data bank of HCV mutations associated with anti-HCV drug resistance.

7. The assay of claim 6, wherein step h) consists of 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.

8. The assay of claim 7, wherein the sample is taken from a subject infected with HCV prior to commencement of any pharmacological therapy of the subject.