RU2145635C1 - Oligomer (variants), method of detection of hcv sequence (variants), set for detection, method of blood preparing - Google Patents

Abstract

FIELD: genetic engineering, molecular biology. SUBSTANCE: invention relates to polynucleotide used for detection of HCV hepatitis virus and its fragments given in fig. 18. Primer for DNA synthesis, labeled probe carrying the label to be detected and capture probe are the polynucleotide fragment. Method involves incubation of polynucleotide fragment with a sample under conditions providing the formation of hybridized duplexes between polynucleotide reagent and HCV nucleic acid and formed duplexes are detected. Before incubation of polynucleotide reagent with a sample nucleic acid of the sample can be amplified additionally by the method of polymerase chain reaction using a primer. Nucleic acid is isolated from the blood sample where the presence of HCV nucleic acid is suspected. Nucleic acid is incubated with polynucleotide fragment as polynucleotide reagent under conditions providing the formation of hybridized duplexes between HCV nucleic acid and polynucleotide reagent indicated above. Invention ensures to detect HCV hepatitis virus in biological samples. EFFECT: improved method of detection. 45 cl, 8 dwg, 6 tbl, 1 ex

Description

Technical part
The invention relates to materials and methodologies that are guided in cases of infection with non-A, non-B hepatitis virus (NANBV). In more detail, it relates to the etiological agent of non-A, non-B hepatitis (NANBH), hepatitis C virus (HCV) and polynucleotides and their analogues, which are used to determine HCV in biological samples.

The grounds for the invention
Non-A, non-B hepatitis (NANBH) - a disease transmitted from one person to another or a family of diseases that is believed to be of viral origin and indistinguishable from other forms of virus-associated liver diseases, including those caused by known hepatitis viruses , i.e. hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis delta virus (HDV), as well as hepatitis caused by cytomegalovirus (CMV) or Epstein-Bar virus (EBV). NANBH was first detected in individuals who received a blood transfusion. A human chimpanzee transmission and serial passage in a chimpanzee revealed evidence that NANBH is due to a contagious infectious agent or agents.

 Epidemiological evidence suggests that three types of NANBH are possible: an epidemic type transmitted through water, through blood or needles, and a type that sporadically occurs (infection in other people's society). However, the number of agents that can cause NANBH is unknown.

 There are a number of candidates for NANBH. See, for example, the reviews of Prince (1983), Feinstone and Hoofnagle (1984), Overby (1985, 1986, 1987) and Iwarson (1987). However, there is no evidence that any of these candidates represents the etiological agent NANBH.

 The need for sensitive specific methods for screening and identification of carriers of NANBV and NANBV, infected blood or its components is very important. Post-transfusion hepatitis (PTH) occurs in approximately 10% of patients, and NANBH is responsible for a maximum of 90% of these cases. The main problem of this disease is the frequent progression to chronic liver damage (25-55%).

 Serving patients, as well as preventing infection of NANBH through blood and its components or through close personal contacts, requires reliable screening, diagnostic and prognostic tools to detect nucleic acids, antigens and antibodies related to NANBH.

 Methods for detecting specific polynucleotides by hybridization assays are known in the art. See, for example, Matthews and Kricka (1988), Analytical Biochemistry 169: 1; Landegren et al. (1988), Science 242: 229 and Mittlin (1989), Clinical chem 35: 1819, US patent N 4868105, issued Sept., 9, 1989, and in EPO publication N 225807 (published June 16, 1987) .

 The applicant has discovered a new virus, hepatitis C virus (HCV), which has been proven to be the main etiological agent of waterborne NANBH (BB-NANBH). The first work of the applicant, including the partial genomic sequence of the isolate of the prototype HCV, CDC / HCV1 /, also designated as HCV1, is described in EPO publication N 318216 PCT N WO 89/04669 (published May 31, 1989) and published June 1, 1989. These Patent applications, like any relevant national patent applications, are incorporated herein in footnotes. These statements teach recombinant DNA techniques — cloning of HCV sequences, diagnostic procedures for HCV breakdown, anti-HCV antibodies, and methods for isolating new sequences.

Disclosure of Invention
The present invention is based on the HCV sequences described by EPO publication N 318216 and PCT pub. N WO 89/04669, as well as on other HCV sequences, which are described here. Methods for the isolation and / or detection of specific polynucleotides by hybridization could not be used to screen for HCV until the applicant discovered HCV. Accordingly, one aspect of the invention is an oligomer capable of hybridizing with the HCV sequence in an analyte polynucleotide chain, where the oligomer is included in the HCV target sequence complementary to at least 4 adjacent nucleotides of the HCV cDNA shown in FIG. 18.

Another aspect of the invention is a process for detecting an HCV sequence in an analyte chain that is suspicious of containing an HCV polynucleotide, where the HCV polynucleotide includes a selective region of a target, said process comprising:
a) providing an oligomer capable of hybridizing with the HCV sequence in the analyte polynucleotide chain, where the oligomer comprises an HCV target sequence complementary to at least 4 adjacent HCV nucleotides of the cDNA shown in FIG. 18,
b) incubation of the analyte chain with the oligomer a), which allows the formation of specific hybrid duplexes between the target sequence and the test sequence and
c) detection of hybrids formed between the target region or any other and the oligomer.

Another aspect of the invention is a method for producing HCV-free blood, including:
a) providing analyte nucleic acids from blood samples suspicious of containing the HCV sequence of the target,
b) providing an oligomer capable of hybridizing with the HCV sequence in the analyte polynucleotide chain or any other, where the oligomer comprises an HCV sequence of a target complementary to the sequence of at least 8 nucleotides present in the conserved HCV nucleotide sequence in PHK,
c) reaction a) c b) under conditions that allow the formation of a polynucleotide duplex between the target sequence and the test sequence, if not any other,
d) detection of the formed duplex in c) or any other and
e) preservation of blood, in which the complexes in g) were not detected.

Brief Description of the Drawings
FIG. 1 shows the HCV cDNA sequence in clone 12f and the amino acid encoded by it.

 FIG. 2 shows the HCV cDNA sequence in clone K9-1 and the amino acids encoded by it.

 FIG. 3 shows the sequence of clone 15e and its encoded amino acids.

 FIG. 4 represents the nucleotide sequence of HCV cDNA in clone 13i, the amino acids encoded by it, and sequences that overlap with clone 12f.

 FIG. 5 shows the nucleotide sequence of HCV cDNA in clone 26j, the amino acids encoded by it, and sequences that span clone 13i.

 FIG. 6 shows the nucleotide sequence of HCV cDNA in clone CA59a, its encoded amino acids, and sequences that overlap with clones 26j and K9-1.

FIG. 7 to 17 show the nucleotide HCV cDNA, which overlaps, respectively:
in FIG. 7 in clone CA84a by clone CA59a;
in FIG. 8 in clone CA156e by clone CA84a;
in FIG. 9 in clone CA1676 by clone CA156e;
in FIG. 10 in clone CA216a by clone CA1676;
in FIG. 11 in clone CA290a by clone CA216a;
in FIG. 12 in clone ad30a by clone CA290a;
in FIG. 13 in clone CA205a by the HCV cDNA sequence in clone CA290a;
in FIG. 14 in clone 18e by the HCV cDNA sequence in clone ag30a;
in FIG. 15 in clone 16jh by the HCV cDNA sequence in clone 15e;
in FIG. 16 in clone 6k by the HCV cDNA sequence in clone 16jh;
in FIG. 17 in clone p131jh by the HCV cDNA sequence in clone 6k.

FIG. 18 is an HCV cDNA sequence composed of clones described herein and their HCV cDNA sequence sequence, presented in BRO Publication No. 318216. The clones from which these sequences originated are
5'-clone 32, b114a, 18g, ag30a, CA205a, CA290a, CA216a, pi14a, CA167b, CA156e, CA84a, CA59a, K9-1, 26j, 13i, 12f, 14i, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g, 39c, 35f, 19g, 26g, 15e, b5a, 16jh, 6k and p131jh. In the drawing, three horizontal dashes above the sequence indicate the position of the putative initiating methionine codon. The drawing also shows the amino acid sequence of the putative polyprotein encoded by HCV cDNA. The heterogeneity in the HCV1 cloned DNA is indicated by the amino acids given above of the putative large OPC coding sequence, parentheses indicate that heterogeneity is found at or near the 5 ′ or 3 ′ end of the HCV clone cDNA.

 FIG. 19 is a sequence of captured and labeled samples for detecting HCV PHK in biological samples.

 FIG. 20 shows a schematic view of the flavivirus polyprotein and the putative HCV polyprotein encoded in the large OPC of the HCV genome. The drawing also shows the possible functions of flavivirus polypeptides purified from flavivirus polyproteins. In addition, comparative locations of NANB 5-1-1 and C100 polypeptides are given relative to the putative HCV polyprotein.

 FIG. 21 shows the double stranded nucleotide sequence of the insertion of HCV cDNA in the clone and the putative amino acid sequence of the polypeptide encoded by it.

 FIG. 22 gives the HCV cDNA sequence in clone 36, a segment of which overlaps with the NANBV cDNA of clone 81, and the polypeptide sequence encoded by clone 36.

 FIG. 23 gives the HCV cDNA sequence in clone 37b, a segment of which overlaps with clone 35, and the polypeptide encoded by it.

 FIG. 24 gives autoradiography of HCV qPCR analysis for RNA obtained from chimpanzee liver samples with NANBH (FIG. 25A) and Italian patients with NANBH (FIG. 25B).

 FIG. 25A and 25B are graphs showing the temperature dependence between the degree of liver damage, the presence of HCV RNA, and the presence of anti-HCV antibodies for two chimpanzees with NANBH.

 FIG. 26 gives the nucleotide sequence of HCV cDNA in clone CAB4a, the amino acids encoded by it, and sequences overlapping with clone C459a.

 FIG. 27 gives the HCV cDNA sequence in clone 40b, a segment of which overlaps with clone 37b, and the polypeptide encoded by it.

 FIG. 28 is an autoradiography showing labeled amplified products of approximately 300, 30, and 3 CID HCV genomes.

 FIG. 29 gives the nucleotide sequence of HCV cDNA in clone 40a.

 FIG. 30 is an autoradiography showing amplified products obtained from primers originating from conserved regions of the HCV genome.

 FIG. 31 represents the HCV cDNA sequence in clone 35, the segment of which overlaps clone 36, and the polypeptide encoded by it.

 FIG. 32 is a diagram showing the relationship of samples and primers originating from the 5'-region of HCV RNA from which the HCV cDNA of clones ag30a and K9-1 are derived.

 FIG. 33 is an autoradiography of amplified products obtained from primer sets derived from ag30a and K9-1.

 FIG. 34 gives the line-by-line nucleotide sequences of human HCV isolates 23 and 27 1. Homologous sequences are indicated by the symbol (*). Non-homologous sequences in small print.

 FIG. 35 shows the line-by-line amino acid sequences of human isolates 23 and 27 of HCV 1. Homologous sequences are indicated by the symbol (*). Non-homologous sequences in small print.

 FIG. 36 shows a halftone image of a Northern RNA blot from a Northern RNA blot isolated from an oven infected with BB-NANBV chimpanzee hybridizing with BB-NANBV cDNA clone 81.

FIG. 37 gives a halftone image of a nucleic acid radiograph extracted from NANBV particles captured from infected plasma using anti-NANB 5-1-1 and hybridized with ³² P-labeled NANBV cDNA from clone 81.

FIG. 38A and B depict filter autographs of filters containing isolated NANBV nucleic acids hybridized with ³² P-labeled plus and minus DNA strands as samples that originated from NANBV cDNA in clone 81.

 FIG. 39 shows the nucleotide sequence of the consensus of human isolate 23, variant sequences are shown below the line of this sequence. The amino acids encoded by the consensus sequence are also shown.

 FIG. 40 is a graph showing the relationship of EnvR and EnvL primers with the Flavivirus polyprotein model and presumably HCV polyprotein.

 FIG. 41 gives a vertical comparison of the nucleotide sequences of the Thorn, ECI, HCT # 18 and HCV 1 isolates.

 FIG. 42 provides a comparison of the nucleotide sequences of EC10 and the composite HCV1 sequence, the EC10 sequence is located on the line above the pillboxes, and the sequence in the line below the pillboxes.

 FIG. 43 provides a comparison of amino acid sequences 117-308 (relative to HCV1) encoded in the "EnvL" regions of the consensus sequences of human isolates HCT # 18, JH23, JH27, Thorne, EC1 and HCV 1.

 FIG. 44 provides a comparison of amino acid sequences 330-360 (relative to HCV1) encoded in the "EnvR" regions of the consensus sequences of human isolates HCT # 18, JH23, JH27, Thorne, EC1 and HCV1.

 FIG. 45 gives the nucleotide sequences of the individual primers in the 5'-3 primer mixture.

MODES FOR CARRYING OUT THE INVENTION
The term "hepatitis C virus" (HCV) is pre-ordered by researchers in this area for the still unknown etiological agent NANBH. The HCV prototype isolate was identified by USSN 122, 714 (see also EPO publication N 318,216). The term HCV also includes new isolates of the same viral species. As an explanation of this terminology, an HCV disease, previously called water-borne NANB hepatitis (BB-NANBH), has been called hepatitis C. The terms NANBH and hepatitis C can be used interchangeably in this case.

HCV viral species through which pathogenic strains cause BB-NANBH. There may also be attenuated strains or defective interfering particles originating from them. As shown below, the HCV genome consists of RNA. RNA viruses are known to have relatively high rates of spontaneous mutations, i.e. clearly on the order of 10 ^-3 to 10 ^-4 per nucleotide that is involved (Eields and Knipe (1986)). Therefore, since the heterogeneity and “fluidity” of the genotype is inherited in RNA viruses, among their strains there are many strains (isolates) that can be virulent or avirulent. The formulations and methods described herein make it possible to distribute, identify, detect and isolate various HCV strains or isolates.

 Identified several different strains / isolates of HCV (see below). One such strain or isolate that is a prototype is designated CDC / HCV1 (also called HCV1). Information about one strain or isolate, such as a partial genomic sequence, is sufficient to allow those skilled in the art using standard techniques to isolate new strains / isolates and identify whether the new strains / isolates are also HCV. For example, several different strains / isolates are described below. These strains, which were obtained from a number of human sera (and from different geographical areas), were isolated using HCV1 genomic sequence information.

 Using the techniques described in EPO publication N 318,216 and below, the genomic structure and nucleotide sequence of HCV1 genomic RNA was determined. It turned out that the genome is a single-stranded RNA containing ≈ 10 thousand nucleotides. The genome is a positive chain and has a continuous translational open reading frame (OPC), which encodes a polyprotein of about 3,000 amino acids. Apparently, in OPC, structural proteins are encoded by approximately the first quarter of the N-terminal region, with most of the polyprotein responsible for non-structural proteins. When compared with all known viral sequences, small but important homologies with non-structural proteins of the flavivirus family and with pestiviruses (which are now also considered as part of the flavivirus family) are observed.

 A schematic alignment of possible regions of a flavivirus polyprotein (using yellow fever virus as an example) and presumably the polyprotein encoded in the main OPC of the HCV genome is shown in FIG. 20. The drawing shows the possible domains of the HCV polyprotein. The flavivirus polyprotein contains, from the amino end to the C-terminal end, nucleocapsid protein (C), matrix protein (M), coat protein (E), non-structural proteins (NS) 1, 2 (a + b), 3, 4 (a + b) and 5. Based on the putative amino acids encoded in the HCV1 nucleotide sequence, the small domain at the outer N-terminus of the HCV polyprotein appears to be similar in size and in high alkaline base content to the nucleocapsid protein (C) located at the N-terminus of flavivirus polyproteins. The non-structural proteins 2, 3, 4, and 5 (NS2-5) of HCV and yellow fever virus (YFV) appear to have opposite parts of similar size and hydrophilicity, although there is divergence in amino acid sequences. However, the HCV region, which could correspond to the YFV regions of the polyprotein, which contain M, E, and NS1 proteins, does not differ in sequence, but, apparently, are also completely different in size and hydrophilicity. Thus, while certain domains of the HCV genome can be named as, for example, NS1 or NS2, it should be borne in mind that these indications are speculative, there may be significant differences between the HCV family and flaviviruses, which still have to be evaluated.

 The different strains, isolates and subtypes of HCV are expected to contain differences in amino acids and nucleic acids compared to HCV1. Many isolates are expected to give greater homology (i.e., more than about 40%) of the overall amino acid sequence compared to HCV1.

 However, it may also turn out that there are other less homologous HCV isolates. They can be defined as HCV according to various criteria, such as, for example, an OPC with approximately 9,000 nucleotides to approximately 12,000 nucleotides encoding a polyprotein with similar HCV1 hydrophobic and / or antigenic properties, and the presence of line-by-line peptide sequences that are conserved with HCV 1. In addition, it is believed that the genome is a positive RNA strand.

 All HCV isolates encode at least one epiton that is immunologically identical (i.e., immunologically cross-reacts) with the epitope encoded in the HCV cDNA described herein. Preferably, this epiton is included in the amino acid sequence described herein and is unique to HCV when compared with previously known pathogens. The uniqueness of this epiton can be determined by its immunological reactivity and anti-HCV antibodies and the lack of immunological reactivity with antibodies to known pathogens.

HCV strains and isolates are evolutionarily related. Therefore, it is believed that the overall homology of the genomes at the nucleotide level is perhaps about 40% or higher, maybe about 50% or higher, maybe about 60% or higher, and even more likely that about 80% or higher, and in addition that be appropriate adjacent sequences of at least about 13 nucleotides. It should be noted, as shown below, that in the HCV genome there are variable and hypervariable regions, therefore, it is assumed that the homology of these regions is much smaller than the genome as a whole. The correspondence between the putative genomic sequence of the HCV chain and, for example, the cDNA sequence of CDC / HCV1 can be determined using methods known in science. For example, they can be determined by directly comparing the sequence information of the polynucleotide of the allegedly HCV and HCV cDNA sequences described herein. They can also determine the hybridization of the polynucleotides under conditions in which the formation of stable duplexes between homologous regions (for example, those that could be used before splitting S _1), followed by cleavage with a nuclease specific for single-stranded DNA, followed by determination of the digested fragments .

 Due to the evolutionary relationship of HCV strains and isolates, putative HCV strains or isolates are identical in their homology at the polypeptide level. It is generally assumed that HCV strains or isolates are at least 40% homologous, more than 50% homologous, possibly more than 70% homologous, and even more likely that approximately 90% homologous at the polypeptide level. Technologies for determining homology of amino acid sequence are known in science. For example, the amino acid sequence can be determined directly and compared with the sequences shown here. Similarly, it is possible to determine the nucleotide sequence of genomic material, presumably HCV (usually via the cDNA intermediate), the putative amino acid sequence encoded by it, and compare the corresponding regions.

As used herein, a polynucleotide “derived from” said sequence refers to a polynucleotide sequence that contains at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even preferably at least about 15-20 nucleotides corresponding to the region of said nucleotide sequence. “Corresponding” means homologous or complementary to the designated sequence. Preferably, the sequence of the region from which the polynucleotide originates is homologous or complementary to a sequence unique to the HCV genome. More preferably, the related sequence is homologous or complementary to a unique sequence in all or the vast majority of HCV isolates. The unique sequence in the HCV genome or not, can be determined by methods known to those skilled in the art. For example, this sequence can be compared with sequences in a data bank, for example in a gene bank, to determine if it is present in uninfected hosts or other organisms. The sequence can also be compared with known sequences of other viral agents, including those known to cause hepatitis, such as HAV, HBV and HDV, and are members of the Flaviviridae family. The correspondence or mismatch of the related sequence to other sequences can also be determined by hybridization under suitable stringency conditions. Hybridization methods for determining the complementarity of nucleic acid sequences in science are discussed here. See also, for example, Maniatis et al. (1982). In addition, unpaired nucleotides in a polynucleotide duplex formed by hybridization can be determined by known methods, including, for example, digestion with a nuclease, such as S ₁ , which specifically cleaves single stranded regions in a polynucleotide duplex. Areas from which typical DNA sequences may "originate" include, but are not limited to, for example, regions encoding specific epitons, as well as untranscribed and / or untranslated regions.

 A related polynucleotide does not necessarily physically come from the shown nucleotide sequence, but can be formed in any way, including, for example, chemical synthesis, or DNA replication, or reverse transcription, or simply transcription. In addition, combinations of regions corresponding to regions of a specified sequence can be modified using methods known in the art to harmonize them in light of their intended use.

 The term "recombinant polynucleotide", as used here, means a genomic polynucleotide, cDNA, polysynthetic, synthetic, which by the method of its origin or manipulation with it: 1) does not resemble the full or part of the polynucleotide with which it is associated in nature, 2) associated with a different polynucleotide than the one with which it is associated in nature, 3) does not occur in nature.

 The term "polynucleotide", as used here, refers to the polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes single and double stranded DNA and RNA. It also includes well-known types of modifications, for example, labeling known in science, methylation, "capping", substitution of one or more natural nucleotides for analogues, internucleotide modifications, such as, for example, modifications with unchanged bonds (for example, methylphosphanates, phosphotriethers, phosphoanhydrides, carbamates, etc.) and with altered bonds (for example, phosphorothiates, phosphorodithiates, etc.) containing parts "suspended" to them, for example, proteins (including, for example, nucleases, toxins, antibodies, signal peptides, poly-L lysine, etc.) with tercators (e.g. acridine, psoralen, etc.) containing chelates (e.g. metals, radioactive metals, boron, oxidized metals, etc.) containing alkylators with modified bonds (e.g. alpha anomeric nucleic acids, etc.) .d.), as well as unmodified forms of this polynucleotide.

 As used here, the "sense strand" of a nucleic acid contains a sequence that has a sequence homologous to mRNA. An “antisense chain” contains a sequence complementary to a “sense chain”.

 As used herein, a “positive chain genome” of a virus is a genome consisting of RNA or DNA, single-stranded, which encodes viral polypeptides. Examples of positive RNA viruses include togaviruses, coronaviruses, retroviruses, picornaviruses, and caliciviruses. Flaviviruses that were previously classified as togaviruses are also included. See Fields and Knipe (1986).

 The term “primer,” as used herein, refers to an oligomer that is capable of operating as a site for initiating synthesis of a polynucleotide chain when incubated under suitable conditions. The primer is complementary or nearly complementary to the region of the polynucleotide chain to be copied. Thus, under conditions leading to hybridization, the primer is annealed with a complementary region of the analyte chain. When suitable reagents (e.g., polymerase, triphosphate nucleotide and the like) are added, the primer grows with polymerizing agents, forming a copy of the analyte chain. The primer may be single-stranded or, alternatively, may be partially or fully double-stranded.

 The terms “analyte polynucleotide” and “analyte chain” refer to a single or double stranded nucleic acid molecule that is suspected to contain a target sequence and which may be present in a biological sample.

 As used here, the term "oligomer" refers to primers and samples. The term oligomer has no additional meaning for the length of the molecule. However, usually oligomers are no longer than 1000 nucleotides, more typically no longer than 500 nucleotides and even more typically no longer than 250 nucleotides, they can be no longer than 100 nucleotides, and no longer than 75 nucleotides, and also can be no more than 50 nucleotides in length.

 As used here, the term “probe” refers to a structure comprising a polynucleotide that forms a hybrid structure with a target sequence due to the complementarity of at least one sequence in a sample with a sequence in the region of the target. Polynucleotide regions of the samples may consist of DNA and / or RNA and / or synthetic nucleotide analogues. With the word “sample”, combinations of “captured samples” and “labeled samples” are used. Preferably, the sample does not contain a sequence complementary to the sequence (s) used as primers for the PCR reaction.

 As used here, the term “target region” refers to the sequence with which a sample or primer forms a stable hybrid under the required conditions.

 The term "capture sample", as used here, refers to a polynucleotide consisting of a single-stranded polynucleotide annealed with a binding partner. A single stranded polynucleotide comprises a polynucleotide sequence for a target that is complementary to the target sequence in the region of the target to be detected in the analyte polynucleotide. This complementary region is long enough and complementary to the target sequence to allow the formation of a sufficiently stable duplex to immobilize the analyte polynucleotide on the surface of the carrier (via binding partners). A binding partner may be bonded to the surface of a solid support, or may be indirectly bonded through other structures or binding partners to a solid support.

 The term “target polynucleotide sequence” as used herein refers to a polynucleotide sequence that includes nucleotides complementary to the target nucleotide sequence, this sequence of sufficient length and complementarity with the target sequence to form a duplex of sufficient stability for the intended purpose.

 The term “binding partner”, as used here, refers to a molecule capable of binding a ligand molecule with high specificity, such as an antigen and an antibody specific for it. Basically, specific binding partners should bind with sufficient affinity to immobilize the duplicate analyte copy / complementary chain (in the case of capture samples) under the selected conditions. Specifically binding partners are known in the art and include, for example, biotin and aviadin or streptavidin, IgG and protein A, numerous known receptor-ligand pairs, and complementary polynucleotide chains. In the case of complementary polynucleotide binding partners, they are usually at least about 15 bases in length and can be at least 40 bases in length, and in addition, they have an F + C composition of at least about 40% and a maximum of about 60%. Polynucleotides can be represented by DNA, RNA, or synthetic nucleotide analogues.

 The term “bound”, as used here, refers to methods of attachment via covalent bonds or by strong non-covalent interactions (eg, hydrophobic interactions, hydrogen bonds, etc.). Covalent bonds can be, for example, from esters, ethers, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds and the like.

 The term “carrier” refers to any solid or semi-solid surface on which the desired binding partner can be anchored. Suitable substrates include glass, plastic, metal, polymer gels and the like, and may take the form of granules, holes, membranes and the like.

 The term "labeled", as used here, refers to any atom or part that can be used to provide a detectable (preferably quantitative) signal and which can be included in a polynucleotide or polypeptide.

 As used herein, the term “labeled probe” refers to an oligomer that includes a polynucleotide sequence for a target that is complementary to the target sequence that needs to be detected in the analyte polynucleotide. This complementary region is sufficient in length and complementary to the target sequence to allow the formation of a duplex consisting of a “labeled probe” and a “target sequence” that is detected by a label. The oligomer mates with the label either directly or indirectly through a set of ligand molecules with high specificity for each other. The sets of ligand molecules with high specificity are described above and also include multimers.

 The term "multimer", as described here, refers to linear or branched polymers of the same repeating single-stranded polynucleotide units or different single-stranded polynucleotide units. At least one of the units has a sequence, length, and composition that allows it to specifically hybridize to the first single-stranded nucleotide sequence of interest, usually an analyte or oligomer (e.g., labeled probe) linked to an analyte. In order to achieve this specificity and stability, this unit is usually at least 15 nucleotides long, typically no more than about 50 nucleotides long, and preferably about 30 nucleotides long, moreover, the F + C composition is normal at least about 40% and at most about 60% In addition to this unit (s), the multimer includes many units that are capable of specifically and stably hybridizing to the second single-stranded nucleotide of interest, usually a labeled polynucleotide or another multimer. These units are basically about the same size and composition with the multimers described above. When a multimer is intended for hybridization with another multimer, the first and second oligonucleotide units are heterogeneous (different) and do not hybridize with each other under the selected analysis conditions. Thus, multimers can be labeled samples or ligands that bind the label to the sample.

 As used here, the term "viral RNA", which includes HCV RNA, refers to the RNA of the viral genome, its fragments, its transcripts and mutant sequences derived from it.

 As used here, a “biological sample” refers to a sample of tissue or fluid isolated from an individual, including (but not limited to), for example, plasma, serum, cerebrospinal fluid, lymph, the skin, respiratory, intestinal and genitourinary tract, tears, saliva, milk, blood cells, tumors, organs and also in vitro cell culture samples (including, but not limited to, a specific medium obtained by cell growth in a cell culture medium, presumably virus-infected cells, ekombinantnye cells and cell components).

Description of the invention
The practice of the present invention uses, unless otherwise stated, generally accepted technologies of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are available to those skilled in science. Such methods are fully given in the literature. See, for example, Maniatis, Fitsch and Sambrook, Molecular cloning; A Laboratory manual (1982); DNA cloning, Volumes I and II (DN Glover ed. 1985); oligonucleotide synthesis (MIGaid ed., 1984); Nucleic acid hybridization (BDHames SIHiggins eds. 1984); Methods in enzymology Academic press series. Inc. ), especially Vol 154 and Vol 155 (Wu and Grossman, and Wu, eds., respectively). All patents, patent applications, and publications referred to both above and below are hereby incorporated by reference.

 The materials and processes used in the present invention make it possible to detect HSV as an etiological agent for BB-NANBV and provide a family of nucleotide sequences isolated from cDNA libraries that contain HCV cDNA sequences. These cDNA libraries were obtained based on nucleic acid sequences present in the plasma of HCV-infected chimpanzees. The construction of one of these libraries of the library "c" (ATCC N 40394) is described in BRO publication N 318216.

 Using the above-described HCV cDNA sequences, it is possible to construct oligomers that are used as reagents for the detection of viral polynucleotides in biological samples. For example, it is possible to synthesize DNA oligomers with a length of about 8-10 nucleotides or more, which are used as hybridization tests to detect the presence of HCV RNA, for example, in the blood of donors, blood fractions, serum of individuals suspected of carriage of the virus, or cell culture systems in which the virus is replicated. In addition, the new oligomers described herein allow further characterization of the HCV genome.

 Polynucleotide probes and primers derived from these sequences can be used to amplify the sequences present in cDNA libraries and / or screen cDNA libraries for additional overlapping cDNA sequences, which, in turn, can be used to obtain still overlapping sequences. As indicated below and in EPO publication N 318216, the HCV genome appears to be RNA, comprising mainly a large open reading frame (OPC), which encodes a large polyprotein.

 In addition to the above, the information below allows the identification of additional HCV strains and isolates. Isolation and characterization of additional HCV strains or isolates can be performed using techniques known to those skilled in the art, for example, by isolating nucleic acids from body components that contain viral particles and / or viral RNA, creating cDNAs and libraries using the oligomers described below, for screening libraries for clones containing the HCV cDNA sequences described herein and comparing HCV cDNA from the new isolates with cDNA described in EPO publication N 318216 and below. Strains and isolates that are suitable for HCV parameters are easily identified, as described in the definition section. Other methods for identifying strains are apparent to those skilled in the art based on the information provided herein.

Isolation of HCV cDNA sequences
The oligomers of this invention contain regions that form hybrid duplex structures with sequences for the target in HCV polynucleotides. HCV polynucleotide hybridizing regions of oligomers can be determined from the HCV cDNA sequence (s) described here and described in EPO publication N 318216. The HCV cDNA composition from HCV1, an HCV prototype, is shown in FIG. 18. The composite sequence is based on sequence information originating from a variety of HCV cDNA clones that have been isolated from a variety of HCV cDNA libraries, including the “c” library made in lambda-gt11 (ATCC No. 40394) and from human serum. HCV cDNA clones were isolated using the methods described in EPO publication N 318216. Briefly, most of the clones that were isolated contained sequences from the cDNA c library that was constructed using a chimpanzee serum kit with chronic HCV infection and containing a high titer of the virus, those. at least 10 ⁶ infectious doses of chimpanzee / ml (CID / ml). A pool of sera was used to isolate viral particles, nucleic acids isolated from these particles were used as a matrix in the construction of cDNA libraries of the viral genome. The initial clone, 5-1-1, was obtained by screening the “c” library with sera from infected individuals (with libraries). After isolation of the first clone, the remainder of the sequence was obtained by screening with synthetic polynucleotide probes, the sequences of which came from the 5'-region and 3'-region of the known HCV cDNA sequence / s.

 Description of cDNA sequence recovery methods is mainly of historical interest. The resulting sequences (and their complements) are given here, and sequences or any part thereof can be prepared using synthetic methods or by combining synthetic methods with partial sequence recovery using methods similar to those described in EPO publication N 318216.

Oligomeric tests and primers
Using both the HCV genome as a basis (as illustrated in FIG. 19) and / or the preferred conserved regions of the HCV genome, oligomers of approximately 8 nucleotides or longer can be prepared that hybridize with the positive chain (s) of HCV RNA or its complement, as well as with HCV cDNA. These oligomers can serve as probes for the detection (including isolation and / or labeling) of polynucleotides that contain HCV nucleotide sequences, and / or as primers for transcription and / or replication of the HCV sequences of the target. The oligomers contain a polynucleotide sequence for the target, which includes nucleotides complementary to the nucleotide sequence of the HCV target, as well as a sequence of sufficient length and complementary HCV sequence to form a duplex that is stable enough for the intended purpose. For example, if the goal is to isolate, through immobilization, an analyte containing the HCV sequence of the target, the oligomers will contain a polynucleotide region that is of sufficient length and complementarity with the HCV sequence of the target to provide sufficient stability of the duplex to immobilize the analyte on a solid surface through its binding to oligomers under conditions discharge. For example, if oligomers serve as primers for transcription and / or replication of target HCV sequences in an analyte polynucleotide, the oligomers will contain a polynucleotide region of sufficient length and complementarity to the target HCV sequence to allow the polymerizing agent to continue replication from the primers that form a stable complex with the target sequence under hybridization conditions. For example, if oligomers can also be used as labeled samples or linked to multimers, the polynucleotide region for binding to the target must be of sufficient length and complementarity to form stable hybrid duplex structures with labeled samples and / or multimers to allow duplex detection. Oligomers can contain at least about 4 adjacent nucleotides complementary to the HCV sequence of the target, typically oligomers contain at least about 8 adjacent nucleotides complementary to the HCV sequence of the target, and preferably contain at least about 14 nucleotides that are complementary to the HCV sequence of the target.

A suitable HCV nucleotide sequence for a target may include nucleotides complementary to nucleotides selected from HCV cDNA nucleotides (see end of description), which are shown in FIG. 18 (nn _x -nn _y indicate from approximately which nucleotide number x to which nucleotide y number).

 The oligomer, however, does not necessarily consist only of a sequence complementary to the HCV sequence of the target. It may additionally contain nucleotide sequences or other parts suitable for those purposes for which oligomers are used. For example, if oligomers are used as primers for amplification of HCV sequences through PCR, they may contain sequences that form, in a duplex state, restriction enzyme sites that facilitate the cloning of amplified sequences. For example, also if oligomers are to be used as “capture samples” in hybridization assays (described below), they will contain, in addition, a binding partner that binds to the oligomer containing a nucleotide sequence complementary to the HCV sequence of the target. Other types of motifs and sequences used in oligomers or mating with oligomers are sequences known in science and used in science and used for various purposes, including labeling of nucleotide samples.

 The preparation of oligomers is carried out by methods known in science, for example, methods that include excision, transcription or chemical synthesis. The sequences of the target and / or regions of the genome that are selected from oligomers that are complementary to the polynucleotides for the target depend on our goal. For example, if the goal is to screen biological samples for the presence of HCV (e.g., blood), preferred oligomers will be used as probes and / or primers and hybridize to conserved regions of the HCV genome. Some of the conserved regions of the HCV genome with which oligomers can bind are described herein, for example, regions comprising nucleotides numbered 5'-terminal at about 200, or from 4000 to about 5000, or from 8000 to 9040, as shown in FIG. 18, or preferably nucleotides 318 through 174, 4056 through 4448 and 4378 through 4902. Other regions of the genome that are conserved are easily identified by comparing nucleotide sequences of different HCV isolates, including the HCV prototype, HCV1.

 Methods for comparing genotypes to determine conservative and non-conservative regions are known in science, and examples of methods are given here.

 The hybridization analysis of nucleic acids is based on the hybridization of a single-stranded nucleic acid of the analyte (either DNA or RNA) with the nucleic acid of the sample and the resulting duplexes are detected. Samples for HCV polynucleotides (natural or derivative) have a length that allows the detection of unique viral sequences during hybridization. While the “working” length may be 6-8 nucleotides, sequences of 10-12 nucleotides are preferred, and about 20 nucleotides or more are apparently optimal. It is better that these sequences come from areas with a lack of heterogeneity. These samples can be done using routine methods, including the automatic method for synthesizing oligonucleotides. Among the samples used, for example, samples obtained from newly isolated clones, shown here as well as various oligomers used as samples with cDNA libraries are given below. Complement to any unique part of the HCV genome will be satisfactory. Complete complementarity is desired for use as samples, although this may not be necessary when the length of the fragment is increased.

 To use samples such as agents to detect the presence of HCV polynucleotides (for example, when screening for contaminated blood), a test sample, such as blood or serum, can be processed, if desired, to extract the nucleic acids contained therein. The nucleic acid obtained from the sample can be dispersed by gel electrophoresis or subjected to other size separation techniques; on the other hand, the nucleic acid sample can be transferred to the blot by dotting without size separation. To form hybrid duplexes with the sequence for the target of the sample, the region for the target nucleic acid of the analyte must be in single-stranded form. Where the sequence is naturally present in single chain form, denaturation is not required. However, where the sequence is present in double-stranded form, the sequence is denatured. Denaturation can be carried out by various techniques known in science. Following denaturation, the analyte nucleic acid and the sample are incubated under conditions that promote the formation of a stable hybrid of the target sequence in the sample and presumably the sequence for the target in the analyte, and the duplexes containing the sample (s) are detected.

 The detection of the resulting duplex or any other is usually carried out using labeled samples, alternatively the sample may be unlabeled, but detected by specific binding to a ligand that is labeled either directly or indirectly. Suitable labels and methods for labeling samples and ligands are known in the art and include, for example, radioactive labels that can be incorporated by known methods (e.g. nick translation or kinase), biotin, fluorescent groups, chemiluminescent groups (e.g. dioxitanes, especially trigerdioxitanes), enzymes, antibodies, and others.

 The sample region used to bind the analyte can be fully complementary to the HCV genome. Therefore, very stringent conditions (hybridization) are usually desirable in order to avoid false positive signals. However, stringent conditions should be used only if the samples are complementary to regions of the viral genome with no heterogeneity. The stringency of hybridization is determined by a number of factors during hybridization and during washing, including temperature, ionic strength, duration, concentration of formamide. These factors are noted, for example, in Maniatis, T (1982).

 Deviations from this basic pattern adopted in science, including those that facilitate the separation of duplexes that need to be detected from other materials, and / or those that amplify the signal from the labeled motif, can also be used. A number of these modifications are discussed, for example, y: Matthews and Kricka (1988), Analytical Biochemistry 169: 1, Landegreu et al. (1988), Science 242: 229, and Mittlin (1989), Clinical chem, 35: 1819. These and the following publications describing the breadth of analysis variations are hereby incorporated by reference. Samples suitable for detecting HCV in these assays include sequences that hybridize with the HCV polynucleotide sequences of the target to form duplexes with the analyte chain, where the duplexes are sufficiently stable for detection in a specific assay system.

 A suitable modification is described, for example, in US patent N 4868105, issued September 9, 1989, and EPO publication N 295807 (published June 16, 1987). These publications describe a method of hybridizing nucleic acids in a liquid phase in which the analyte nucleic acid is hybridized with a set of labeled samples with a set of capture samples. The sample – analyte complex binds during hybridization to an exciting sample on a solid support, which is complementary to a set of exciting samples. This allows the analyte nucleic acid to be removed from the solution as a solid phase complex. Having an analyte in the form of a solid-phase complex, this facilitates the subsequent stages of its separation in the analysis. A set of labeled samples is complementary to a labeled sample that binds through hybridization to a solid phase / analyte complex.

In most cases, genome sequences are expected to be present in the serum of infected individuals at relatively low levels, i.e., approximately 10 ² - 10 ³ infectious doses of chimpanzee (CID) per ml. This level may require the use of amplification techniques in hybridization assays. Such technologies are known in science. For example, the Enzo Biochemical Corporation "Bio-Bridge" system uses transferase of terminal deoxynucleotides to add unmodified 3'-poly-dT-tails to the DNA sample. The poly dT-terminal probe hybridizes to the nucleotide sequence of the target and then to biotin-modified poly-A. PCT publication 84/03520 and EP publication N 124221 describe a hybridization DNA analysis in which: 1) the analyte is annealed with a single stranded DNA sample complementary to an enzyme labeled oligonucleotide, and 2) the tail duplex obtained is hybridized with an enzyme labeled oligonucleotide, EPA 204510 describes a hybridization analysis of DNA wherein the analyte DNA is contacted with a probe that has a tail, such as a poly-dT tail, and an amplifier chain that has a sequence that hybridizes with the tail of the probe, such as a poly-A sequence, and which is capable of bind a lot of labeled strands. The type of hybridization assay described in EPO publication N 317 077 (published May 24, 1989) that detects sequences of approximately 10 ⁶ / ml uses nucleic acid multimers that bind to the analyte single stranded nucleic acid and which also bind to multiple single stranded labeled oligonucleotides. A particularly preferred technology may include amplification of the HCV sequences of the target in serum by approximately 10,000 times (i.e., approximately 10 ⁶ sequences / ml) as part of the hybridization system. Amplification can be accomplished by polymerase chain reaction (PCR) described by Saiki et al. (1986), Mullis, US patent N 4683195 and Mullis et al., US patent N 4683202. Amplification can precede or preferably follow prior to HCV purification by the target sequence. For example, amplification can be used in conjunction with the analysis methods described in US patent N 4868105, or if further amplification is desired, in combination with the hybridization system described in EPO publication N 317077. Preferred methods for detecting HCV sequences in an analyte polynucleotide chain are based on determination methods using the hybridization described in US patent N 4868105 and EPO publication N 317077. These methods are hybridization tests in the aqueous phase of the type of "sandwich", which use both exciting and labeled samples, which e hybridize with the sequences of the target in the analyte nucleic acid. When using these assays to screen biological samples for HCV, used samples bind to conserved regions of the genome. Exciting and labeled samples can be combined by linking them to the target sequence. Alternatively, in a preferred method, capture and labeled samples are available in the kits, and samples of one kit do not interfere with the samples of the other kit. And in the latter method, it is preferable that the set (s) of multiple capture samples be hybridized to the most conserved regions of the genome, while the set (s) of the set of labeled samples can be hybridized to areas that show slight divergence. For example, using the HCV1 prototype cDNA sequence shown in FIG. 18, one could use samples that hybridize to sequences in the nucleotide regions from about −318 to 174 and / or nucleotides in the region from about 4378 to about 4902, and / or nucleotides in the region from 4056 to 4448. Preferred samples hybridize to sequences in 5'-region of the HCV genome, since, as shown below, this region is apparently highly conserved. Thus, preferred samples can hybridize with either the positive chain in conserved regions and / or its complement depending on the purpose, for example, to detect viral genomic sequences, or to detect HCV cDNA sequences obtained by amplification, or to detect replicative intermediates to positive HCV RNA chain.

Detection of HCV RNA and polynucleotides originating therefrom using the HCV / c PCR method
A particularly useful method for detecting HCV PHK or related HCV RNA polynucleotides is the HCV / cPCR method, which is the subject of this application and which uses the polymerase chain reaction (PCR) technology described by Saiki et al. (1986), Mullis in US pat. N 4683195 and Mullis et al. In US patent N 4683202, HCV / cPCR method uses the primers and samples available in the information given here regarding the nature of the HCV genome.

 In most cases, short oligonucleotide primers are prepared using the PCR technique, which mate at the opposite ends of the desired sequence. The sequence between the primers does not need to be known. The polynucleotide sample is extracted and denatured, preferably by heating, and hybridized with oligonucleotide primers present in molar excess. The polymerization is catalyzed by sample and primer-dependent polymerase in the presence of deoxynucleotide triphosphates and nucleotide analogs (dNTPs). This leads to two “long products” that contain the corresponding primers as the 5-terminal TX, covalently linked to the newly synthesized complementary chains of the original chain. The replicated DNA is again denatured, hybridized with oligonucleotide primers, the hybridization conditions are restored, and the second cycle of replication begins. The second cycle provides two initial chains, two long products from cycle 1 and two “short products” replicated from long ones. Short products contain sequences (semantic or antisense) originating from the target sequence flanked at the 5 ′ and 3 ′ ends by primer sequences. In each additional cycle, the number of short replicating products increases exponentially. Thus, this process causes amplification of a specific target sequence.

 In this method, a sample suspected of containing HCV RNA or a fragment thereof is supplied. The sample is usually taken from an individual suspected of NANBH, however, other sources of the sample include, for example, a specific medium or cells from in vitro systems in which the virus is replicated. The sample, however, must contain the target nucleic acid sequence (s).

 This sample is then placed under conditions allowing conversion by reverse transcription of HCV RNA to HCV cDNA. The conditions for RNA reverse transcription are known to those skilled in the art and are described, for example, in Maniatis et al. (1982) and Methods in Enzymology. One preferred reverse transcription method utilizes reverse transcriptase from a variety of sources, including recombinant molecules and isolated from, for example, retroviruses, primarily avian myeloblastosis virus (AMV), and suitable conditions for transcription. HCV cDNA, the product of reverse transcription is RNA: DNA hybridomas that are obtained during the first round of reverse transcription, DNA: DNA hybrids are formed during two or more rounds of transcription.

 The HCV cDNA generated by reverse transcription is then subjected to PCR to amplify the target sequence. For amplification of HCV, cDNA is denatured and the separated strands hybridize with primers flanking the target sequence.

Chain separation can be obtained by any suitable denaturation method, including physical, chemical and enzymatic, known to those skilled in the art. A preferred method that is physical involves heating the nucleic acid until it is completely (99%) denatured. Typical thermal denaturation includes a range of temperatures from about 80 ^° C. to about 105 ^° C. and a time from 1 to 10 minutes.

 After hybridization of the HCV cDNA with primers, the HCV target sequences are replicated by polymerization, which uses any oligonucleotide primer to initiate synthesis of the replicative chain. Primers are selected so that they are complementary to the sequences of the HCV genome. Oligomeric primers complementary to the regions of the sense and antisense strands of the HCV cDNA can be composed (copied) from HCV cDNA sequences from the composite cDNA sequence shown in FIG. 18.

 The primers are selected so that their positions relative to the duplex sequence are such that the product being synthesized from one primer, when separated from the matrix (complement), serves as a template for building up another primer to give a replicative chain of a certain length.

 The primer is preferably single-stranded for maximum amplification efficiency, but may alternatively be double-stranded. If the primer is double-stranded, it is first heated to separate its chains before use to obtain chain extension products. It is better if the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of the buildup products in the presence of polymerization agents. The exact length of the primers will depend on many factors, including the temperature and source of the primer and how to use this method. For example, depending on the complexity of the target sequence, an oligonucleotide primer typically contains about 15-45 nucleotides, although it may contain more or less nucleotides. Short primer molecules generally require lower temperatures in order to efficiently form stable hybrid complexes with a matrix.

 The primers used here are selected so that they are “substantially” complementary to the different chains of each specific sequence that needs to be amplified. Therefore, the primers do not need to reflect the exact sequence of the matrix, but they must be sufficiently complementary to selectively hybridize with their respective chains. For example, a non-complementary nucleotide fragment can be attached to the 5-end of the primer, with the remainder of the primer sequence complementary to a particular strand. On the other hand, non-complementary bases or longer sequences can be introduced into the primer, provided that the primer is sufficiently complementary to one of the chains for amplification during hybridization with it and, as a result, for the formation of a duplex structure that can be expanded by polymerization. Non-complementary nucleotide sequences of primers may include restriction enzyme sites. Adding a restriction site to the end (s) of the target sequence would be particularly useful for cloning the target sequence.

 Keep in mind that a “primer,” as used herein, may refer to more than one primer, especially in the case where there is some ambiguity in the information regarding the terminal sequences (s) of the target region that are amplified. However, a “primer” includes a collection of oligonucleotide primers representing possible variations in a given sequence or includes nucleotides that allow for typical pairing. One of the oligonucleotide primers of this collection is homologous to the end of the target sequence. A specific case is shown in the examples where oligomeric sets of 44-mer and 45-mer were used to initiate amplification of a potentially variant region of the HCV genome.

 A wide variety of HCV strains or isolates are expected to have sequences with sequences different from HCV1 of the prototype strain. Therefore, in order to detect different strains, it is better to design primers that hybridize with conserved regions of the HCV genome. Conservative regions can be determined by comparing the nucleotide and amino acid sequences of several HCV strains / isolates. There are apparently at least three regions in the HCV genome of the conserved amino acids described above from which primers can originate. It is believed that such areas exist. The primers described below in the examples originate from the conserved regions of HCV, based on sequence homology with flavivirus sequences.

 Oligonucleotide primers can be prepared by any of the appropriate methods. Methods for producing specific sequence oligonucleotides are known in the art and include, for example, cloning and restriction enzyme digestion of suitable sequences and direct chemical synthesis. Chemical synthesis methods may include, for example, the phosphotether method described by Narang et al. (1979), the phosphodiester method disclosed by Brown et al. (1979), the diethyl phosphoamidate method disclosed by Beaucage et al. (1981), and the solid support method from the US patent N 4458066.

 Primers can be labeled, if desired, by inclusion methods (tags) detected spectroscopically, photochemically, biochemically, immunochemically or chemically.

 Matrix-dependent oligonucleotide primer / s buildup is catalyzed by polymerizing agents in the presence of adequate amounts of four deoxyribonucleotide triphosphates (dATP, dTTP and dGTP, dTTP) in a reaction medium that consists of suitable salts, metal cations and a buffered system with a certain pH. Suitable polymerizing agents are enzymes that are known to catalyze primer and matrix independent DNA synthesis. Known DNA polymerases include, for example, IE coli DNA polymerase or its Klenov fragment, T4 DNA polymerase and Tag DNA polymerase. Reaction conditions for catalysis of DNA synthesis with these DNA polymerases are known in the art.

 The products of this synthesis are duplex molecules, consisting of chains of matrices and chains of an expandable primer, which include the target sequence. These products, in turn, serve as matrices for yet another round of replication. In the second round of replication, the chain of the first cycle extended by the primer is annealed with its complementary primer, the synthesis gives a “short” product, limited at both the 5'- and 3'-ends of the primer sequences or its complements. Repeated cycles of denaturation, annealing of the primer and elongation lead to an exponential accumulation of the target region specified by the primers. A sufficient number of cycles is carried out to achieve the desired amount of polynucleotide containing the target nucleic acid region. The desired amount can vary and is determined by the task that the polynucleotide product serves.

 The PCR method can be performed on a variety of matrix sequences. For example, it can be carried out in stages, when new reagents are added after each stage, or in a way when all reagents are added at the same time, or partially in a stepwise manner, when fresh reagents are added after a certain number of stages.

 The preferred PCR reaction method is carried out as an automated process using a thermostable enzyme. In this process, the reaction mixture is carried out sequentially through the denaturation compartment, the primer annealing compartment, and the reaction compartment. You can use a machine that is specially adapted for use with a thermostable enzyme, which allows temperature changes without a manually controlled water system, since you do not need to add the enzyme in each cycle. This type of machine is a suitable commercial product of Perkin Elmer Cetus Corp.

After amplification by PCR, target polynucleotides are detected by hybridization with a polynucleotide probe, which forms a stable hybrid with the target sequence under severe or moderately stringent hybridization and washing conditions. If the samples are expected to be completely complementary (i.e., about 99% or higher) to the target sequence, stringent conditions are used. If several substitutions are expected, for example, if variant strains are expected that will result in the sample not being completely complementary, the stringency of hybridization can be reduced. However, conditions are selected which exclude non-specific / random binding. Conditions that affect hybridization and which are selected to exclude non-specific binding are known in the art and described, for example, by Maniatis et al. (1982). In general, lower salt concentration and high temperature increase the binding rigidity. For example, it is generally believed that stringent incubation conditions are in solutions that contain approximately 0.1 x SSC, 0.1% DDS at an incubation / washing temperature of about 65 ^° C, and moderately stringent conditions are incubation in solutions that contain approximately 1-2x SSC, 0.1% DDS and the washing / incubation temperature of about 50-65 ^o C. Low severity of conditions - 2x SSC and about 30-50 ^o C.

 Samples for target HCV sequences may be derived from the HCV cDNA sequence shown in FIG. 18 or from new isolates of HCV. HCV samples can be of any suitable size, which covers the region of the target, but includes primers and which gives specific hybridization with the region of the target. If there is complete complementarity, i.e. if the strain contains a sequence identical to the sample, since the duplex will be relatively stable under even severe conditions, the samples may be short, i.e. within about 10-30 nucleotide pairs. If a certain percentage of substitutions with a sample is expected, that is, if it is doubtful that the sample will hybridize with the variant region, the sample may be longer, since, apparently, the length somewhat softens the effect of unpaired substitutions. An example of this is given in the examples, which describe samples that bind to potential variants of HCV1. In this case, primers designed to bind HCV cDNA came from a hypothetically conserved region of the HCV genome, and one of the regions containing potential variations (based on the model of freviruses) was the target region. The sample used to detect target sequences contained approximately 268 nucleotide pairs.

 A sample nucleic acid having a sequence complementary to the target sequence can be synthesized using similar techniques described above to synthesize primer sequences. Optionally, a sample can be marked. Suitable labels are described above.

 In some cases, it may be desirable to determine the size of the PCR of the product detected by the sample. This can be especially important if variant HCV strains are suspected to contain deletions within the target region, or if they wish to confirm the PCR length of the product. In such cases, it is better to analyze the size of the products, as well as hybridization with the sample. Methods for determining the size of nucleic acids are known in the art and include, for example, gel electrophoresis, gradient deposition, and chromatography.

 The presence of a target sequence in a biological sample is detected by determining whether a hybrid is formed between the HCV polynucleotide probe and the nucleic acid subjected to PCR amplification technology. Methods for detecting hybrids formed by a probe and nucleic acid are known in the art. For example, it is generally accepted that an unlabeled sample can be transferred to the solid matrix with which it binds, and the binding sample is hybridized under conditions conducive to specific hybridization with a labeled sample, the solid matrix is then examined for the presence of a labeled sample. Alternatively, if the sample is labeled, the unlabeled sample is bound to the matrix and, after exposure under suitable hybridization conditions, the matrix is examined for the presence of the label. Other suitable hybridization methods are described above.

Determination of variant HCV sequences using PCR
In order to identify HCV strains and thereby designate samples for these variants, the HCV / cPCR method described above is used to amplify variant regions of the genome, so that the nucleic sequences of these variant regions of the target can be determined. Mostly variant types of HCV, as might be expected, are found in other geographical areas than those in which the HCV1 strain predominates, such as Japan, Africa, etc., or in various vertebrate species that are also infected with the virus. Variant HCV may also occur during passage in tissue culture systems or result from spontaneous or induced mutations.

 In order to amplify the variant region of the target, primers are designed to flank the suspicious region, and preferably complementary to conservative regions. Primers for two regions of HCV that are possibly conservative based on the flavivirus model are described in the examples. These primers and samples can be prepared using the sequence information of the HCV1 strain shown in FIG. 18.

 The nucleotide sequence analysis of the target region / s can be a direct analysis of amplified PCR products. The process of direct sequence analysis of amplified PCR products is described by Saiki et al. (1988).

 Alternatively, the amplified sequence / and targets can be cloned before sequence analysis. A method for direct cloning and sequence analysis of enzymatically amplified genomic fragments is described by Scharf (1986). In this method, the primers used in PCR technology are modified near their 5 ′ ends to give suitable restriction sites for cloning directly into, for example, the M13 sequence vector. After amplification, PCR products are digested with suitable restriction enzymes. Restriction fragments are ligated with the M13 vector and transformed, for example, host JM 103, seeded on plates and the resulting clones are screened by hybridization with a labeled oligonucleotide probe. Other methods of cloning and sequence analysis are known in the art.

Universal primers for flaviviruses and HCV
A study of the nature of the HCV genome, using samples derived from HCV cDNA, as well as the information contained in the HCV cDNA, suggests that HCV is a flavivirus. These studies are described in EPO publication N 318216, available to this Commissioner, which are hereby taken in their entirety. A comparison of the HCV cDNA sequence related to HCV cDNA clones with the known sequences of a number of flaviviruses shows that HCV contains sequences homologous to the conserved sequences of flaviviruses. These conservative sequences may allow the creation of primers that can be universal in application for amplification of the target regions of flaviviruses and HCV. These sequences are 16-mer and smaller sequences from the 3'-ends of the primers described in the examples. Then, species identification is performed using a sample specific to these species. The genomes of a number of flaviviruses are known in science and include, for example, Japanese encephalitis virus (Sumiyoshi et al., 1987), dengue type 2 virus (Hahn et al., 1988), yellow fever virus (Rice et al., 1985), dengue fever virus type 4 (Mackow, 1987), West Nile virus (Castle et al., 1986). The identification of HCV RNA is performed using a HCV-specific probe, the sequence of which can be determined from the HCV cDNA sequences provided herein.

 Alternatively, the use of sample kits designed to explain the degeneracy of codons and, therefore, the content of common flavivirus and HCV sequences, as determined by comparing HCV amino acid sequences with known flavivirus sequences, allows a basic detection system for these viruses.

Construction of the desired DNA sequences
Synthetic oligonucleotides can be prepared using an automatic oligonucleotide synthesizer, as described by Warner (1984). If desired, synthetic chains can be labeled with ³² P by treatment with polynucleotide kinase in the presence of ³² P-ATP using standard reaction conditions.

 DNA sequences, including those isolated from cDNA libraries, can be modified by known techniques, including, for example, site-directed mutagenesis, as described by Zoller (1982). Briefly, the DNA to be modified is packaged into a phage as a single-stranded sequence and converted into double-stranded DNA using DNA polymerase and as a primer, a synthetic oligonucleotide, complementary to the part of the modified DNA, and having the desired modification included in its own sequence. The resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of transformed bacteria that have replications of each phage chain are plated on agar to obtain plaques. Theoretically, 50% of new plaques contain a phage having a mutant sequence, and the remaining 50% retains the original sequence. Replicas from these plates hybridize with labeled synthetic samples at temperatures and conditions that allow hybridization with the desired chain, but not with an unmodified sequence. Identified by hybridization sequences are detected and cloned.

Whales for screening HCV related polynucleotides
Oligomers - samples and / or primers for amplification and / or screening of samples for HCV can be produced as whales (reagent kits). Whales for screening for HCV sequences include DNA oligomeric samples. Whales for amplification of HCV sequences may include oligomeric primers used for amplification. Typically, whales contain samples or primers in pre-measured or pre-determined amounts, as well as other suitable unpacked reagents and materials, in separate suitable containers necessary for a specific hybridization and / or description (s) of amplification. For example, a whale may contain standards, buffers, substrates, enzymes, substrates, labeled samples, binding partners, and / or test instructions.

Examples
Below are described examples of the present invention, which are provided for illustrative purposes only and do not limit the scope of the present invention.

Isolation and sequences of overlapping cDNA clones 13i, 26j, CA59a, CA84a, CA156e and CA167b
Clones 13i, 26j, CA59a, CA84a, CA156e, and CA167b were isolated from the lambda-gt11 library, which contains HCV cDNA (ATCC N 40394), the preparation of which is described in EPO publication N 318216 (published May 31, 1989) and WO 89/04669 ( published June 1, 1989). The library was screened with samples described below using the method described by Huynh (1985). The frequency of occurrence with corresponding samples of positive clones was about 1 in 50,000.

Isolation of clone 13i was performed using a synthetic sample derived from the sequence of clone 12f. The sequence of this test was:
5 'GAA CGT TGC GAT CTG GAA GAC AGG GAC AGG3'.

The isolation of clone 26j was performed using a sample derived from the 5'-region of clone K9-1. The sequence of the test was:
5'TAT CAG TTA TGC CAA CGG AAG CGG CCC CGA 3 '.

 The procedures for isolating clone 12f and clone k9-1 (also designated K9-1) are described in EPO publication N 318216 and their sequences are shown in FIG. 1 and 2, respectively. HCV cDNA sequences of clones 13i and 26j are shown in FIG. 4 and 5, respectively. The amino acids encoded by them are also shown, such as the overlap of clone 13i with clone 12f and the overlap of clone 26j with clone 13i. The sequences of these clones confirmed the sequence of clone K9-1. Clone K9-1 was isolated from another HCV cDNA library (see EPO publication N 218316).

Clone CA59a was isolated using a sample based on the sequence of the 5'-region of clone 26. The sequence of this sample was:
5 'CTG GTT AGC AGG GCT TTT CTA TCA CCA CAA 3'.

A sample derived from the sequence of clone CA59a was used to isolate clone CA84a. The sequence of the sample used for this selection was:
5 'AAG GTC CTG GTA GTG CTG CTG CTA TTT GCC 3'.

Clone CA156e was isolated using a sample derived from the sequence of clone CA84a. The sequence of the test was:
5 'ACT GGA CGA CGC AAG GTT GCA ATT GCT CTA 3'.

Clone CA167b was isolated using a sample derived from the sequence of clone CA156e. The sequence of the test was:
5 'TTC GAC GTC ACA TCG ATC TGC TTG TCG GGA 3'.

 The nucleotide sequences of HCV cDNA in clones CA59a, CA84a, CA156e and CA167b are shown in FIG. 6, 7, 8 and 9, respectively. The amino acid cells encoded by them in the same way as overlapping with the sequences of the respective clones are also shown in these figures.

Creating a "pi" HCV cDNA Library
The HCV cDNA library, the "pi" library, was constructed from the plasma of an infected chimpanzee of the same variety used to construct the lambda-gt11 HCV cDNA library (ATCC No. 40394) described in EPO publication No. 318216 and using mainly the same technology. However, when creating the pi library, a primer extension method was used in which the reverse transcription primer was based on the sequence of clone CA59a. The primer sequence was:
5 'GGT GAC GTG GGT TTC 3'.

Isolation and sequence of clone pi 14a
Screening for the "pi" HCV cDNA library described above with the sample used to isolate clone CA167b (see above) yielding clone pi 14a. This clone contains about 800 cDNA base pairs that overlap clones CA167b, CA156e, CA84a and CA59a that were isolated from the lambda-gt11 HCV cDNA library (ATCC No. 40394). Additionally, pi 14a also contains about 250 base pairs of DNA located above the HCV cDNA from clone CA167b.

Isolation and sequence of clones CA216a, CA290a and ag30a
Based on the sequence of clone CA167b, a synthetic sample was made having the following sequence:
5 'GGC TTT ACC ACG TCA CCA ATG ATT GCC CTA 3'.

 The above assay was used to screen for HCV sequences, resulting in clone CA216a shown in FIG. ten.

Another test was done based on the sequence of clone CA216a having the following sequence:
5 'TTT GGG TAA GGT CAT CGA TAC CCT TAC GTG 3'.

 Screening the lambda-gt11 library (ATCC No. 40394) with this assay gave clone CA290a, the HCV sequences of which are shown in FIG. eleven.

In a parallel approach, a primer cDNA library was made using nucleic acids extracted from the same infected plasma used in the original lambda-gt11 cDNA library described above. The primer used was based on the sequence of clones CA216a and CA290a:
5 'GAA GCC GCA CGT AAG 3'.

The cDNA library was made using methods similar to those described previously for the libraries used to isolate clones pi14a and K9-1. The sample used to screen this library was based on the sequence of clone CA290a:
5 'CCG GCG TAG GTC GCG CAA TTT GGG TAA 3'.

 Clone ag30a was isolated from the new library with the above assay and contained about 670 base pairs of the HCV sequence. See FIG. 12. Part of this sequence overlaps with the HCV sequence of clones CA216a and CA290a. About 300 base pairs of the ag30a sequence, however, lie above the sequence of clone CA290a. The non-overlapping sequence shows the start codon (*) and stop codons, which may indicate the start of the HCV OPC. In FIG. 12 also indicates presumably small encoded peptides (#), which possibly play a role in the regulation of translation in the same way as the presumably first amino acid of the putative polypeptide (/) and the underlying encoded amino acids.

Isolation and sequence of clone CA205a
Clone CA205a was isolated from the lambda-gt11 library (ATCC No. 40394) using a synthetic probe originating from the sequence of clone CA290a (Fig. 11). The sequence of the test was:
5 'TCA GAT CGT TGG TGG AGT TTA CTT GTT GCC 3'.

 The HCV cDNA sequence in CA205a shown in FIG. 13 overlaps with cDNA sequences of both clone ag30a and CA290a. The overlap of this sequence with CA290a is shown by a dashed line above the sequence (the drawing also shows the putative amino acids encoded by this fragment).

 As can be seen from the HCV cDNA sequences, in clones CA205a and ag30a, the putative HCV polyprotein appears to start at the ATG start codon, and the HCV sequences of both clones contain two adjacent stop codons (TGATAGs) forty-two nucleotides higher than this ATG. OPC appears to start after these stop codons and extends to at least 8907 nucleotides (see the HCV cDNA compound shown in FIG. 18).

Isolation and sequence of clone 18g
Based on the sequence of clone ag30a (see FIG. 12) and the overlapping clone from the original lambda-gt11 library (ATS No. 40394) CA230a, a synthetic sample was made having the following sequence:
5 'CCA TAG TGG TCT GCG GAA CCG GTG AGT ACA 3'.

 Screening of the original lambda-gt11 HCV cDNA library with the sample gave clone 18g, the HCV cDNA sequence of which is shown in FIG. 14. Also shown in the drawing is overlapping with clone ag30a and putative polypeptides encoded in HCV cDNA.

 The cDNA in clone 18g (C18g or 18g) overlaps with clones 30a and CA205a described above. The C18g sequence also contains the double stop codon region observed in clone ag30a. The polynucleotide region above these stop codons is thought to represent part of the 5'-region of the HCV genome, which possibly contains short OPC and which can be confirmed by direct sequencing of the purified HCV genome. These alleged small coded petids may play a regulatory role in translation. The HCV region of the genome above the region represented by C18g can be isolated for sequence analysis using essentially the technology described in EPO publication N 318216 to isolate cDNA sequences lying above the HCV cDNA sequence in clone 12f. Essentially small synthetic polynucleotide reverse transcriptase primers that are based on the C18g sequence are synthesized and used to bind the corresponding sequence in HCV genomic RNA. The primer sequences are proximal to the known 5'-end of C18g, but to a large extent lie below to allow the creation of sequences of probes above the sequences of primers. Use standard methods of priming and cloning. The resulting cDNA libraries are screened with sequences above the prime sites (as determined from the clarified C18 sequence). HCV genomic RNA is obtained either from plasma or from liver samples of individuals with NANBH. Since HCV appears to be a flavipoise virus, the 5 ′ end of the genome can be modified with a “cap” structure. The flavivirus genomes are known to contain 5-terminal “cap” structures (yellow fever virus, Rice et al. (1988), dengue virus, Hahn et al. (1988), Japanese encephalitis virus (1987)).

Isolation and sequence of clones from beta-HCV cDNA library
Clones containing cDNA representing the 3'-terminal region of the HCV genome were isolated from a cDNA library constructed from a plasma pool of an infected chimpanzee, which was used to create the HCV cDNA of the lambda-gt11 library (ATCC No. 40394) described in EPO publication No. 318216. In order to create a DNA library, the extracted RNA from plasma was equipped with a poly A tail using poly (pA) polymerase, and cDNA was synthesized using oligo (dT) _12-18 as a reverse transcription primer. The resulting RNA: DNA hybrid was digested with RNase H and converted into double-stranded HCV cDNA. The resulting HCV cDNA was cloned into lambda-gt10, using essentially the technology described by Huynh (1985), which gave a beta (or b) HCV cDNA library. The procedures used were as follows.

An aliquot (12 ml) of plasma was treated with proteinase K and extracted with an equal volume of phenol saturated with 0.05 M Tris-HCl, pH 7.5, 0.05% (v / v) beta-mercaptoethanol, 0.1% (w / volume) hydroxy, 1 mM EDTA. The resulting aqueous phase was reextracted with a mixture of phenol, followed by 3 extraction with a 1: 1 mixture containing phenol and chloroform: isoamyl alcohol (24: 1), followed by 2 extraction with a mixture of chloroform and isoamyl alcohol (1: 1). Following the reduction of the aqueous phase to 200 mM in NaCl, nucleic acids in the aqueous phase were precipitated overnight at -20 ^° C. with 2.5 volumes of cold absolute alcohol. The precipitates were collected by centrifugation at 10,000 rpm for 40 minutes, washed with 70% ethanol containing 20 mM NaCl and 100% cold ethanol, dried for 5 minutes in a desiccator and dissolved in water.

The nucleic acids isolated from the plasma pool of infected chimpanzees were supplied with a poly-pA tail using poly-A polymerase in the presence of human placenta ribonuclease inhibitor (HPRI) (purchased from Amersham Corp.) using MS2 RNA as a carrier. Isolated nucleic acids equivalent to 2 ml of plasma were incubated in a solution containing TMN (50 mM Tris-HCl, pH 7.9, 10 mM MgCl ₂ , 250 mM NaCl, 2.5 mM MnCl ₂ , 2 mM dithiothreitol (DTT)) . 40 micromoles alpha ³² P ATP, 20 units of HPRI (Amersham Corp.) and about 9-10 units of RNAse-free poly-A polymerase (BRL). The incubation was carried out at 37 ^° C. for 10 minutes and the EDTA reaction was stopped (final concentration of about 250 mM). The solution was extracted with an equal volume of phenol-chloroform and an equal volume of chloroform, and nucleic acids were precipitated overnight at -20 ^° C. with 2.5 volumes of ethanol in the presence of 200 mM NaCl.

Isolation of clone b5a
The beta HCV cDNA library was screened by hybridization using a synthetic probe that had a sequence based on the HCV cDNA sequence of clone 15e. The isolation of clone 15e is described in EPO publication N 318216, and its sequence is shown in FIG. 3. The sequence of the synthetic sample was:
5 'ATT GCG AGA TCT ACG GGG CCT GCT ACT CCA 3'.

 Screening of the library gave clone beta-5a (b5a), which contains an HCV cDNA region of approximately 1000 base pairs. The 5'-region of this cDNA overlaps with clones 35f, 19g, 26g and 15e (these clones are described above). The region between the 3'-terminal poly-A sequence and the 3'-sequence that overlaps clone 15e contains approximately 200 base pairs. This clone identifies the region of the 3'-terminal sequence of the HCV genome.

 The b5a sequence is contained within the HCV cDNA sequence of clone 16jh (described below). Moreover, this sequence is also present in CC34a isolated from the original lambda-gt11 library (ATCC No. 40394). (The original lambda-gt11 library is called the "C" library here).

Результирующую кДНК подвергали амплификации путем PCR, используя два праймера:

Праймер JH32 содержал 2o нуклеотидные последовательности, гибридизующиеся с 5'-концом района мишени в кДНК с определенной T_m 66^oC. JH11 происходил из части олиго дТ-праймер-адаптера, таким образом он специфичен к 3'-концу кДНК при T_m 64^oC. Оба праймера были сделаны так, чтобы иметь рестрикционный сайт для фермента рестрикции, NotI, на 5'-конце, для использования в последующем клонировании амплифицированной HCV кДНК.Isolation and sequence of clones created by amplification of the 3'-region of the HCV genome
Multiple cDNA clones were created containing nucleotide sequences originating from the 3'-region of the genome. This was accomplished by amplifying the target region of the genome by a polymerase chain reaction described by Saiki et al. (1986) and Saiki et al. (1988), which was modified as described below. The amplified HCV RNA was obtained from the original pool of infected chimpanzee plasma, which was used to create the lambda-gt11 cDNA HCV library (ATCC No. 40394) described in EPO publication No. 318216. The HCV RNA was isolated as described above. E. coli poly-A polymerase was added to the isolated RNA at the 3'-end of ATP as described by Sippel (1973), except that the nucleic acids isolated from chimpanzee sera were replaced with a substrate, nucleic acid, RNA with poly-A was then subjected to reverse transcription to obtain cDNA using reverse transcriptase using the oligo dT primer adapter to a large extent as described by Han (1987), except that the components and sequence of the primer adapter were:

The resulting cDNA was amplified by PCR using two primers:

Primer JH32 contained 2o nucleotide sequences hybridizing to the 5'-end of the target region in a cDNA with a specific T _m 66 ^o C. JH11 came from a portion of the oligo dT primer adapter, so it is specific for the 3'-end of the cDNA at T _m 64 ^o C. Both primers were designed to have a restriction site for the restriction enzyme, NotI, at the 5'-end, for use in subsequent cloning of amplified HCV cDNA.

The PCR reaction was carried out by suspending cDNA and primers in 100 microliters of the reaction mixture containing four deoxyribonucleoside triphosphates, buffer salts and metal ions, and thermostable DNA polymerase isolated from Thermus aquaticus (Tag polymerase), which is in the Perkin Elmer Cetus kit (N 801- 0043 or N 801-0055). The PCR reaction was carried out in 35 cycles in a Perkin Elmer Cetus DNA Thermal Amplifier. Each cycle consisted of a 1.5-minute denaturation step at 94 ^° C, annealing step at 60 ^° C, 2 min, a primer elongation step at 72 ^° C, 3 min. PCR products were subjected to Southern blot analysis using a 3-nucleotide probe JH34, the sequence of which was based on the sequence of the 3'-terminal region of clone 15e. JH34 sequence:
5 'CTT GAT CTA CCT CCA ATC ATT CAA AGA CTC 3'.

 PCR products detected by HCV cDNA breakdown have a size range of from about 50 to about 400 nucleotide pairs.

 In order to clone amplified HCV cDNA, PCR products were digested with NotI and size-separated by polyacrylamide gel electrophoresis. DNA of more than 300 pairs of nucleotides (bp) was cloned into the NotI site of PUC18S. The PUC18S vector is constructed by incorporating the NotI polylinker cloned between the EcoRI and SalI sites of FIG. 18. Clones were screened for cDNA using a JH34 assay. A number of politic clones were obtained and sequenced. The nucleotide sequence of the HCV cDNA insertion of one of these clones, 16 jh, and the amino acids encoded by it are shown in FIG. 15. The drawing shows the nucleotide heterogeneity found in the HCV cDNA sequence in clone 16 jh compared to another clone of this region.

Isolation and sequence of clone 6K
Based on the sequence of clone 16 jh and clone b5a (see above), a synthetic test was made having the following sequence:
5 'TCT TCA ACT GGG CAG TAA GAA CAA AGC TCA 3'.

Screening of the original lambda-gt11 HCV cDNA library (described in EPO publication N 318216) with the sample gave clones
with a frequency of approximately 1 out of 10 ⁶ , one of them was named clone 6K (also called C6K), the HCV cDNA sequence of which is shown in FIG. 16. The drawing also shows overlapping with clone 16jh and putative polypeptides encoded by HCV cDNA. HCV cDNA sequence information in clone 6K was obtained from only one strand. Information on this clone is given below, where the clone is listed as lambda-gt11 C6K. The conformation of the C6K sequence as part of the OPC encoding the HCV1 polypeptide was obtained by sequencing other overlapping clones.

Isolation and sequence of clone p131jh
A clone containing a sequence from the 3'-region of the HCV genome and which contains a stop codon in a frame was isolated as described above to isolate clones formed by PCR by amplification of the 3'-region of the genome, except that HCV1 RNA was converted to cDNA, using an oligonucleotide
5 'AAT TCG CGG CCG CCA TAC GAT TTA GGT GAC ACT ATA GAA T ₁₅ 3'.

This cDNA was then amplified by PCR reaction using primers
5 'TTC GCG GCC GCT ACA GCG GGG GAG ACA T 3'
and
5 'AAT TCG CGG CCG CCA TAC GA 3'.

After amplification, PCR products were precipitated with spermine, NotI digested and extracted with phenol. The purified products were cloned into the NotI site of puc 18S and positive clones were selected using the oligonucleotide:
The cDNA of one clone designated p131jh is shown in FIG. 17. This clone contains a large stop frame codon (OPC) contained in the HCV genome.

Isolation and sequence of clone 5'-clone 32
A clone containing a sequence from the 5'-region of the HCV genome, above the sequence of clone b114a, was isolated and the nucleotide sequence was determined by modifying the method of isolation and sequence determination of clones created by PCR amplification of the 3'-region of the genome described in US N 456637, which is made in the links. Basically, the target region of the genome was amplified by PCR technology described by Saiki et al. (1986) and Saiki et al. (1988). The amplified HCV RNA was obtained by extraction of human serum (US Clinical Isolate, HCV 27) using the cold Gaunidin thiocyanate method described by Han et al. (1987). The extracted RNA was converted to single-stranded cDNA by reverse transcriptase using a primer, JH94, which is complementary to -250 nucleotides at -223 of the HCV genome (see FIG. 18). JH94 sequence
5 'CCT GCG GCC GCA CGA CAC TCA TAC TAA 3'.

Полученную кДНК подвергали амплификации PCR, используя два праймера, JH94 (описанный выше) и JH11, который имеет следующую последовательность:

PCR реакцию проводили суспендируя кДНК и праймеры в 100 микролитрах реакционной смеси, содержащей четыре дезоксинуклеозид трифосфата, буферные соли и ионы металлов, и термостабильную ДНК-полимеразу, выделенную из thermus aquaticus (Taq-полимеразу), которая есть в Perkin Elmer Cetus ДНК термальном ките (N 801-0043 или N 801-0055). PCR реакцию выполняли за 35 циклов в термальном циклизаторе Perkin Elmer Cetus. Каждый цикл состоял из 1,5-минутной стадии денатурации при 94^oC, стадии отжига при 60^oC, 2 мин, и стадии элонгации праймера при 72^oC, 3 мин.The conversion of single- to double-stranded PCR cDNA was accomplished by adding a tail of approximately 20-50 dA residues to the DNA using terminal deoxynucleotidyl transferase (Sambrook et al. (1989), Molecular cloning) and replicating the resulting molecule using the following oligo-dT a primer adapter that contains the NotI site and the sp6 promoter:

The resulting cDNA was subjected to PCR amplification using two primers, JH94 (described above) and JH11, which has the following sequence:

The PCR reaction was carried out by suspending cDNA and primers in 100 microliters of the reaction mixture containing four deoxynucleoside triphosphates, buffer salts and metal ions, and thermostable DNA polymerase isolated from thermus aquaticus (Taq polymerase), which is in the Perkin Elmer Cetus DNA thermal whale ( N 801-0043 or N 801-0055). The PCR reaction was performed in 35 cycles in a Perkin Elmer Cetus thermal cyclizer. Each cycle consisted of a 1.5-minute denaturation step at 94 ^° C, annealing step at 60 ^° C, 2 min, and a primer elongation step at 72 ^° C, 3 min.

PCR products were digested with NotI and cloned into puc 18S. Clones containing HCV nucleotide sequences were obtained by screening with a sample. Alex 90, which was derived from -312 to -283 of the HCV1 genome and has the sequence:
5 'ACC ATG AAT CAC TCC CCT GTG AGG AAC TAC 3'.

 The cDNAs of the isolated clones were sequenced by the dideoxy-termination method (Sanger et al. (1977)). The cDNA sequence of one of the selected clones, 5-clone 32, covers the region of nucleotides -224 to -341 in FIG. 18.

В этой структуре прерывистые линии указывают возможные водородные связи между комплементарными нуклеотидами.Analysis of the nucleotide sequence of HCV cDNA showed that the replica of the HCV RNA chain contains an HZ-rich region, which is possibly capable of forming a stable hairpin structure:

In this structure, dashed lines indicate possible hydrogen bonds between complementary nucleotides.

 A search in the computer database of Genebank found that there were no homologous sequences from known viral sequences. Thus, this sequence is possibly unique at the 5'-end of the HCV genome.

 The hairpin structure can serve as a transcriptase recognition signal and / or it can contribute to RNA stability at the 5'-end.

Compiled HCV cDNA Sequences
The HCV cDNA sequence was assembled from a series of overlapping clones originating from various HCV cDNA libraries described here and in EPO publication N 318216. The clones of FIG. 18 are clones 5'-32, b114a, 18g, ag30a, CA205a, CA290a, CA216a, pi14a, CA167b, CA156e, CA84a, CA59a, K9-1, 26j, 13i, 12f, 14i, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g, 39c, 35f, 19g, 26g, 15e, b5a, 16jh, C6K and p131jh.

 Methods for isolating these clones as well as their sequences are described here in EPO publication N 318216, which are incorporated herein by reference. In FIG. 18, the three bars above the sequence indicate the position of the putative methionine initiation codon.

 Clone b114a overlaps with clones 18g, ag30a and CA205a, except that clone b114a contains an additional two nucleotides above the sequence in clone 18g (i.e., 5'-CA). These two additional nucleotides are included in the HCV genomic sequence shown in FIG. 18.

 It should be noted that although several of the clones described above were obtained from libraries other than the original HCV cDNA lambda-gt11 C library (ATCC No. 40394), these clones contain cDNA sequences that overlap with the HCV cDNA sequences of the original library. Thus, essentially the entire HCV sequence originates from the original lambda-gt11 C library (ATCC No. 40394), which was used to isolate the first HCV clone cDNA (5-1-1). Isolation of clone 5-1-1 is described in EPO publication N 318216, which is incorporated herein by reference.

 Also shown is the putative sequence of a large HCV polyprotein encoded in the HCV1 composite cDNA. The first amino acid in the sequence is the suspected large OPC initiator methionine. Variant amino acids due to the heterogeneity of the clones are shown above sequence. Since the lambda-gt11 library was created from serum obtained from one individual (see EPO publication N 318216), the results suggest that variant viral sequences (both nucleotide and amino acid) are present in this individual.

 A study of the composite HCV cDNA sequence shows that in addition to the large OPC, it has a number of OPCs above the OPC encoding polyproteins, and within the sequence encoding polyproteins there are a large number of smaller OPCs in two other translational frames. OPC above HCV polyprotein are shown in table 1.

 The reading frame, position and size of the OPC below the sequence encoding the presumably initiating MET polyprotein are shown in Table 2. A large polyprotein is the one transmitted from reading frame 2.

 In addition to the above, a study of a sequence that is complementary to the HCV RNA genomic chain also showed that it contains several small OPCs. One of these OPCs, which is complementary to -341 to +837 nucleotides in the HCV RNA sequence, encodes a 385 amino acid polypeptide.

Comparison of sequences of 5'-regions obtained from HCV isolates from different geographical areas
The nucleotide sequences of the 5'-regions of HCV isolates from the USA (HCV18, HCV27), from Italy (HCVI1, HCVI24) and Korea (HCVK1) were compared.

Амплификацию HCV дцДНК делали методом PCR, используя JH93 и JH52 как 5'- и 3'-праймеры соответственно, HCV последовательность в JH93 происходила из HCV нуклеотидов с -317 по -296, в JH52 с -93 по -117, номер нуклеотида указан в скобках внизу последовательности. В JH52 подчеркнутый динуклеотид был мутирован для создания NotI сайта. Последовательности этих праймеров следующие

После амплификации продукты PCR расщепляют NotI и клонируют в puc 18S. HCV кДНК секвенировали или непосредственно после амплификации PCR или, альтернативно, секвенировали клонированные HCV кДНК путем наращивания праймера или дидезокси методом. Наращивание праймера и дидезокси метод сиквенса выполнялись как описано выше для последовательности 5'-клона 32.Isolation of HCV cDNA sequences was essentially carried out as described above to isolate the 5'-clone 32, except as follows. The extracted RNA was back transcribed into cDNA using either JH51 or r16 as primers that are complementary to HCV nucleotides from -90 to -73 and from 366 to 383, respectively. The sequences of these primers are as follows

Amplification of HCV dsDNA was done by PCR using JH93 and JH52 as 5'- and 3'-primers, respectively, the HCV sequence in JH93 came from HCV nucleotides from -317 to -296, in JH52 from -93 to -117, the nucleotide number is listed in brackets at the bottom of the sequence. In JH52, the underlined dinucleotide was mutated to create a NotI site. The sequences of these primers are as follows

After amplification, PCR products cleave NotI and clone in puc 18S. HCV cDNA was sequenced either immediately after PCR amplification or, alternatively, the cloned HCV cDNA was sequenced by primer extension or dideoxy method. The primer extension and dideoxy sequence method were performed as described above for the sequence of the 5'-clone 32.

The PCR method for the direct sequence used Alex 90 (see sequence above) as a 5'-primer and r25 as a 3'-primer. Alex 90 was derived from HCV nucleotides from -312 to -283 and r25 was derived from nucleotides from 365 to 342 (see FIG. 18). R25 sequence:
5 'ACC TTA CCC AAA TTG CGC GAC CTA 3'.

 A comparison of the sequences of the 5 'region' of HCV27, HCVK1, HCV1 24, HCVI1 and HCV18 with the sequence of the prototype HCV, HCV1 showed the following. The 5 'study region is highly conserved among 5'HCV isolates. Apparently, these sequences are identical, with the exception of one nucleotide deleted at position 171 in HCV 124, and ambiguity in four nucleotides at positions -222 through -219 in the HCVK1 isolate.

 High levels of sequence conservatism in this region may reflect the role of this region in viral replication and / or transcription and / or translation.

Variations in the sequence of HCV isolates from different individuals
HCV isolates that contain sequences that are derived from CDC / HCV1 have been identified in human individuals, some of which were serologically positive for anti-C100-3 antibodies (EC10 was negative). The identification of these new isolates was carried out by cloning and sequencing segments of the HCV genome that were amplified by PCR technology using CDC / HC1 sequences. Amplification was performed essentially based on the HCV / cPCR method. This method uses primers and probes based on the cDNA sequences described herein. The first stage of the method is the synthesis of cDNA, or the HCV genome, or its replicative intermediate, using reverse transcriptase. After synthesis of HCV cDNA and before amplification of the RNA, the sample is degraded using technologies known in science. The desired segment of HCV cDNA is then amplified using appropriate primers. The amplified sequences are cloned and clones containing the amplified sequences are detected with a probe that is complementary to the sequence lying between the primers but which does not overlap the primers.

HCV isolates isolated from people in the USA
Blood samples that were used as a source of HCV varions were obtained from American Red Cross in Charlotte, North Carolina, and from the Community Blood Center of Kansas, Kansas City, Missous. Samples were screened for antibodies to the HCV C100-3 antigen using ELISA? as described in EPO publication N 318216, and are subject to additional Western analysis using polyclonal goat anti-human HPR to measure anti-HCV antibodies. Two samples, # 23 and # 27, from American Red Cross and from the Community Blood Center of Kansas, respectively, were determined to be HCV positive in these analyzes.

Viral particles present in the serum of these samples were isolated by ultracentrifugation under the conditions described by Bradley et al. (1985). RNA was extracted from the particles by digestion with proteinase K and DDS at a final concentration of 10 micrograms / ml proteinase K and 0.1% DDS, digestion was performed for 1 hour at 37 ^° C. Viral RNA was then purified by extraction with chloroform-phenol as described in EPO publication N 318216.

 HCV RNA in RNA preparations was back transcribed into cDNA essentially as described in EPO publication N 318216, except that the JHC 7 oligonucleotide, which corresponds to the cDNA sequence of 1958-1939 and which has the following sequence, was used as a primer for the reverse transcription reaction.

 JHC 7: CCA GCG GTG GCC TGG TAT TG.

After synthesis of both cDNA strands, the resulting cDNA was then amplified by PCR, essentially as described above, to isolate clones created by PCR amplification, except that the nucleotide primers used, i.e., JHC6 and ALX80, were designed to amplify the 1080 nucleotide segment HCV genome from CDC / HCV1 nucleotides 673 to 1751. In addition, the primers were designed to introduce a restriction site at the 3 ′ end of the PCR product and the blunt 5 ′ end. Primer Sequences:
ALX 80: TTT GGG TAA GGT CAT CGA TAC CCT TAC GTG;
and
JHC 6: ATA TGC GGC CGC CTT CCG TTG GCA TAA.

 ALX 80 corresponds to nucleotides 673-702 of the CDC / HCV1 sequence, JHC6 corresponds to nucleotides 1752-1738 of HCV1 (in addition, there are 12 extranucleotides that encode the NotI site). The designation of nucleotides in JHC 6, i.e. number from position, indicates a place in the antisense chain.

 After PCR amplification from the above primers, the blunt ends were converted to the NotI site as follows. A 15 dG homopolymer tail was attached to the PCR product using terminal deoxynucleotide transferase, and the products were again amplified by PCR using JHC6 and JHC13 as primers. The last primer, JHC13, the sequence of which follows, is made to introduce the NotI site in addition to the Sp6 phage promoter. (Sp6 promoter described in Genetic engineering, J. Setlow Ed. (1988)).

 JHC 13: AAT TCG CGG CCG CCA TAC GAT TTA GGT GAC ACT ATA GAA CCC CCC CCC CCC CCC.

 In order to clone amplified HCV cDNA, PCR products were digested with NotI, besieged with spermine to remove free oligonucleotides (Hoopes et al. 1981), and cloned at the NotI site of puc 18S (see section IV.A. 34). The HCV cDNA of the three clones originating from each HCV isolate was subjected to sequence analysis. The analysis was essentially carried out in accordance with the method described by Chem and Seeburg (1985).

 The consensus sequences of clones originating from HCV sample 23 are shown in FIG. 39.

 FIG. 34 and 35 show a comparison of the row-wise positive chain of the nucleotide sequence (FIG. 34) and putative amino acid sequences (FIG. 35) of samples 23, 27 and HCV1. The amino acid sequence of HCV1 in FIG. 34 represents amino acid numbers 129-467 of an HCV polyprotein encoded by a large OPC HCV genomic RNA. The study of FIG. 39 shows that there are variations in the sequences of the three selected clones. Variations in the sequences at the nucleotide level and at the amino acid level are summarized in Table 3. In Table 3, the polypeptide designated S and NS1 represents amino acids numbers 130 through ~ 380, and 380 through ~ 470, respectively. The numbering comes from the alleged initiating methionine. S and NS1 terminology is based on the positions of sequences encoding polypeptides using the flavivirus model. As discussed above, however, recent evidence suggests that there is no complete correlation between HCV and flaviviruses for viral polypeptide domains, especially in putative E / NS1 domains. Indeed, HCV polypeptides and their coding domains may exhibit a significant deviation from the flavivirus model.

 Although there are abnormalities in the newly isolated HCV sequences, the cloned sequences from samples 23 and 27 (named HCV 23 and HCV 27) each contain 1019 nucleotides, indicating the absence of deletions and other mutants in the region in isolated clones. The sequences in FIG. 34 and 35 also show that the selected sequences are not rearranged in this region.

 A comparison of the consensus sequences for HCV1 and for other HCV isolates is summarized in Table 3. The variations in the sequences of the HCV1 and HCV chimpanzee isolate isolated from humans are almost the same as those observed between HCV from humans.

 Interestingly, sequence variations from the two putative domains are not the same. The sequence of the putative S region is apparently relatively constant and randomly scattered throughout the genome. In contrast, the putative NS1 region has a higher degree of variability than the entire sequence and variations, apparently, there are about 28 amino acids in the hypervariable pocket, which are located about 70 amino acids below the putative N-terminus of the putative polyprotein.

 Although it can be argued that the detected variations were introduced during the amplification process, it does not appear that all variations came from this. It has been estimated that Tag polymerase introduces errors into a sequence of approximately one per 10 thousand pairs of nucleotides of the template DNA per cycle (Saiki et al., 1988). Based on this assessment, up to 7 errors could be introduced during PCR amplification of 119 bp DNA fragment. However, the three subclones of HCV 23 and HCV 27 gave 29 and 14 variant nucleotides, respectively. The following data suggests that these variations occur naturally. About 60% of nucleotide substitutions are silent mutations that do not alter the amino acid sequence. Variations introduced by Tag polymerase during PCR amplification are to be expected to occur by chance, however, the results show that variant sequences form a cluster in at least one specific region. Moreover, consensus sequences occurred during the sequence of many different clones from PCR amplified products.

HCV isolates from patients in Italy and the USA
The HCV RNA segments present in various isolates were amplified by the HCV / c PCR method. These segments span the region of ~ 0.6 kb, ~ 1.6 kb lower from methionine encoding the startcodon of the putative HCV polyprotein. Isolates from biological samples obtained from HCV infected individuals. In more detail, isolate HCV # 18 from human plasma of an individual in the USA, EC1 and EC10 from an Italian patient’s liver biopsy, and Th from an African patient’s peripheral blood fraction of mononucleocytes. Comparable segments of HCV RNA are isolated from chimpanzees.

RNA was extracted from samples of human plasma using phenol: CHCl ₃ : isoamyl alcohol extraction. Either 0.1 ml or 0.01 ml of plasma was diluted to a final volume of 1.0 ml of TENB proteinase K / DDS solution (0.05 M Tris-HCl, pH 8.0, 0.001 M EDTA, 0.1 M NaCl, 1 mg / ml proteinase K and 0.5% DDS) containing 10-40 micrograms / ml polyadenylinic acid and incubated at 37 ^° C. for 60 minutes. After this proteinase treatment, the fractions of the obtained plasma were deproteinized by extraction with TE (50 mM Tris-HCl, pH 8.0, 1 mM EDTA) saturated phenol, pH 6.5. The phenolic phase was separated by centrifugation and re-extracted with TENB containing 0.1% DDS. The resulting aqueous phases from each extraction were combined and extracted twice with an equal volume of phenol / chloroform / isoamyl alcohol (1: 1 99: 1) and then twice with an equal volume of 99: 1 mixture of chloroform / isoamyl alcohol. This was followed by phase separation by centrifugation and the aqueous phase was brought to a final concentration of 0.2 M Na acetate, and nucleic acids were precipitated by the addition of two volumes of ethanol. Precipitated nucleic acids were isolated by ultracentrifugation in a SW 41 rotor at 38 thousand rpm for 60 minutes at 4 ^° C or in a microcentrifuge for 10 minutes at 10 thousand rpm.

 RNA extracted from a liver biopsy was supplied from Dr. F. Bonino, Ospedale Maggiore di S. Giovanni Battista, Torino, Italy.

The mononucleocyte fraction was obtained by sedimentation of an individual aliquot of blood through Ficoll-Paque ^® (Pharmacia Corp), using the manufacturer's instructions. Total RNA was extracted from the fraction using the guanidine thiocinate procedure described in EPO publication N 318216 (see also Choo et al., 1989).

Synthesis of HCV cDNA from samples was performed using reverse transcriptase and primers derived from clones 156e and K91. These primers, which are antisense with respect to genomic RNA, have the following sequences
156e 16B: 5 'CGA CAA GAA AGA CAG A 3'
and
K91 / 16B 5 'CGT TGG CAT AAC TGA T 3'.

Following ethanol precipitation, the precipitated RNA or nucleic acid fraction was dried and resuspended in DERS-treated distilled water. The secondary structures of nucleic acids were destroyed by heating to 65 ^° C. for 10 minutes and the samples were immediately cooled in ice. cDNA was synthesized using from 1 to 3 micrograms of total RNA from the liver and from nucleic acids, or RNA extracted from 10 to 100 microliters of plasma. Reverse transcriptase was used in the synthesis and the reaction was carried out in 25 microliters using the manufacturer's description, BRL. All cDNA synthesis mixtures contained 23 units of an RNase inhibitor, RNASIN ^m (Fisher / promega). After cDNA synthesis, the reaction mixtures were diluted with water, boiled for 10 minutes, and quickly cooled in ice.

 Each set of samples was subjected to two rounds of amplification. The reaction primers were selected so as to amplify the regions designated "EnvL" and "EnvR". The "EnvL" region contains nucleotides 669-1243 and putative amino acids 117-308, the "EnvR" region contains nucleotides 1215-1629 and encodes putative amino acids 300-408 (putative amino acids are numbered starting from the putative methionine initiator codon). The relationship of these regions with respect to the putative polyprotein encoded by HCV cDNA and polypeptides encoded by the flavivirus model is shown in FIG. 40.

Primers for the first round of PCR reactions were derived from HCV cDNA sequences of either clone ag30a, clone 156e, or clone K9-1. The primers used to amplify the region were 156e16B (shown above) and ag30a16A for the sense strand. Amplification of the EnvR region used primer K92 / 16B (shown above) and 156e16a for the sense strand. The sequences of the primers of the sense strand are as follows
For EnvL, ag30a16A: 5 'CTC TAT GGC AAT GAG G 3'
and
For EnvR, 156e16A: 5 'AGC TTC GAC CTC ACA T 3'.

PCR reactions were carried out largely according to the manufacturer's instructions (Cetus-Perkin-Elmer), except that 1 microgram of RNase A was added. The reactions were carried out in a final volume of 100 microliters. PCR was performed in 30 cycles using a regime of 94 ^° C (1 min), 37 ^° C (2 min) and 72 ^° C (3 min) with a 7 minute elongation at 72 ^° C in the last cycle. Samples were then extracted with phenol: CHCl ₃ , precipitated with ethanol 2 times, suspended in 10 mM Tris-HCl, pH 8.0, and concentrated using Centricon-30 (Amicon) filtration. This procedure essentially removes oligonucleotides smaller than 30 nucleotides in size, thus removing primers from the first round of PCR amplification.

Centricon-30 concentrated samples were then subjected to a second round of PCR amplification using samples obtained from clones 202a and 156e for the EnvL region and from 156e and 59a for the EnvR region. EnvL region amplification primers have the following sequences
202aEnv41a: 5 'CTT GAA TTC GCA ATT TGG GTA AGG TCA TCG ATA CCC TTA CG 3'
and
156e38B ': 5' CTT GAA TTC GAT AGA GCA ATT GCA ACC TTG CGT CGT CC 3 '.

The primers for amplification of the EnvR region in RNA derived from humans have the following sequences
156e38A ': 5' CTT GAA TTC GGA CGA CGC AAG GTT GCA ATT GCT CTA TC 3 '
and
59aEnv39C: 5 'CTT GAA TTC CAG CCG GTG TTG AGG CTA TCA TTG CAG TTC 3'.

PCR amplification was performed in 35 cycles using a regime of 94 ^° C (1 min), 60 ^° C (1 min), and 72 ^° C (2 min) with 7 min. elongation at 72 ^o C in the last cycle. Samples were further extracted with phenol: CHCl ₃ , precipitated twice, and digested with EcoRI. PCR reaction products were analyzed by electrophoresis separation in 6% polyacrylamide gels. DNA of approximately the estimated length of the expected PCR product was electroeluted from the gels and subcloned into either pGEM-4 plasmid vector or lambda-gt11. The expected size product for EnvL and EnvR after the first round of amplification is 615 bp and 683 bp, respectively, after the second round of amplification, the expected product sizes for EnvL and EnvR are 414 bp and 575 bp, respectively. Plasmids containing amplified products were used to transform host cells, pGEM-4 plasmid was used to transform DH5 alpha, and lambda-gt11 was used to transform C600 delta-HFL. Clones of transformed cells that either hybridized with suitable HCV probes (described below) or those that inserted the correct size. The insertions were then cloned into M13 and sequenced.

Samples for all HCV / cPCR products consisted of ³² P-labeled sections of HCV cDNA prepared by PCR amplification of clone 216 (using CA216a16A and 216a16B as primers) and clone 84 (using CA84a16A and CA84a16B or CA84a16C as primers), ³² P were included in the products PCR nick translation. Samples for the first and second round of EnvL amplification were from clone 216. The samples for the first round of EnvR amplification were 84 (i.e., CA84a16A and CA84a16B), for the second round of EnvL amplification were CA84a16A and CA84a16C. These samples do not overlap with the primers used in the HCV / cPCR reactions. The primer sequences for PCR amplification of the samples are shown in table 4.

 Information on the sequence of variants in the EnvL region was obtained from 3 clones from HCT # 18, 2 clones from TH, 3 clones from their EC1 and HCV1 clones described in EPO publication N 318216 and above. A comparison of the composite nucleotide sequence of each isolate originating from these clones is shown in FIG. 41. In the drawing, each sequence is shown from 5 ′ to 3 ′ for the sense strand of the EnvL region and these sequences were combined line by line. The vertical lines and capital letters indicate the homology of the sequence, the absence of the line and the capital letters indicate the absence of homology. Sequences not shown in lines are as follows: line 1, Thorn; line 2, EC1; line 3, HCT # 18; line 4, HCV1.

 Sequence information for variants in the EnvR region was obtained from two EC10 clones and from HCV1 clones described in EPO publication N 318216 and above. A comparison of the nucleotide sequences of EC10 (clone 2) and the composite HCV 1 sequences is shown in FIG. 42, each sequence is represented by 5 ′ and 3 ′ for the sense strand of the EnvR region and the sequences are aligned line by line. Double dashes between sequences indicate sequence homology.

 A comparison of the amino acid sequences encoding EnvL (amino acids # 117-308) and EnvR region (amino acids # 300-438) for each isolate is shown in FIG. 43 and 44, respectively. These drawings include the sequences of isolates JH23 and JH27 described above. Sequences from the Japanese isolate are also indicated; these sequences were supplied from Dr. Miyamura, Japan. The drawings show the amino acid sequence for this region in its entirety for HCV1 and non-homologous amino acids in various isolates are indicated.

 As shown in FIG. 43, in the EnvL region as a whole, about 93% homology between HCV1 and other isolates. HCT18, Th and EC1 have about 97% homology with HCV1, JH23 and JH27 have about 96% and 95% homology, respectively, with HCV1. FIG. 44 shows that the homology in the EnvR region is much less than in the EnvL region, moreover, it seems that one sub-region is hypervariable (i.e. amino acids 383-405). These data are summarized in table 5.

Serum 5'-HCV RNA positive and negative detection
HCV 27 RNA isolated from serum was analyzed for the presence of positive and negative chains using the PCR method. The PCR method was performed to a large extent as described above, with the exception of the following. The extracted HCV27 RNA was back transcribed into single stranded cDNA using either Alex 90 or JH52 as a primer (see above for sequences). The sequence of Alex 90 mates with it at positions -312 to -283 of the positive chain of HCV RNA, while JH52 mates with nucleotides of -117 to -93 of the negative chain. The resulting single-stranded HCV cDNAs were each individually amplified by PCR using Alex 90 and JH52. Amplified products were detected by Southern blotting using Alex 89 as a sample. Alex 89 mates with nucleotides of numbers -203 to -175 HCV RNA. Sequence Alex 89:
5 'CCA TAG TGG TCT GCG GAA CCG GTG AGT ACA 3'.

 The analysis showed that by this method the signals of the amplified products of both RNA chains were of equal intensity. These results suggest that HCV RNA in the 5 'region may exist as double-stranded RNA.

Sandwich Hybridization Samples for HCV
This example provides sets of labeled and capture samples useful for detecting HCV RNA in biological samples, using essentially the analysis described in US patent N 4868105. This method is a sandwich hybridization in the aqueous phase, which uses both capture and and labeled samples that hybridize to target sequences in the analyte nucleic acid. When screening biological samples for HCV, the used samples bind to conserved regions of the HCV genome, and HCV binding regions are selected for their uniqueness in the HCV genome. Areas that bind to the binding partner of the capture sample or part of the labeled sample that binds to the labeled motif (or to the amplified multimer, if used as described in EPO publication N 317077) are selected such that they do not bind to any of the known sequences in databank or in HCV, and which have a suitable G + C composition to allow the formation of stable duplexes with their complements under selective conditions. Exciting and labeled samples are in sets 1 - 3 (see the end of the description) and samples of one set are not mixed with samples of another set. These samples consist of HCV sequences of the coding chain of the HCV cDNA prototype sequence shown in FIG. 18. In the above sets, each sample contains, in addition to sequences complementary to HCV sequences, the following sequence below the HCV sequence (ie, at the 3-end): 5 'CTT CTT TGG AGA AAG TGG TG3'. This sequence, common to each capture sample, is complementary to the sequence of the binding partner (s), so that after hybridization, the duplex can be captured by fixing it on the solid phase.

 Also, in each set, each labeled sample contains, in addition to sequences complementary to HCV sequences, the following sequence below the HCV sequence: 5 'TTA GGC ATA GGA CCC GTG TC3'.

 If the method described in EPO publication N 317077 is used, the sequence common to each labeled sample is complementary to the sequence of the multimer in order to allow the formation of hybrid duplexes with this multimer.

 The sample sequences of the above sets are shown in FIG. 19.

Detection of HCV polynucleotide sequences using PCR amplification
In a commonly used method for amplifying HCV RNA with PCR, it is contemplated that the RNA strand is a virion or mRNA strand, which is a sense strand. However, it also seems possible that replicative intermediate forms can also be detected that will be "antisense", in which case the primer will be "semantic". The sense strand of RNA containing the target region hybridizes with an antisense primer that primifies the synthesis of the replicative strand containing the target. cDNA on an RNA template is synthesized by primer and sample dependent reverse transcriptase. cDNA in the resulting RNA: DNA hybrid is freed by denaturation and RNase treatment. Primers are annealed with cDNA and are expanded with primer and matrix independent DNA polymerase. The products are denatured, annealed again with primers and a second round of synthesis is carried out. A certain number of cycles is carried out until the amplified product appears, containing the target region in the desired amount, which at least corresponds to the detected level.

Detection of amplified HCV nucleic acid sequences derived from HCV nucleic acid sequences in the liver and plasma samples from chimpanzees with NANBH
HCV nucleic acids present in the liver and plasma of chimpanzees with NANBH and not present in control chimpanzees were amplified using the essentially polymerase chain reaction described by (PCR) Saiki et al. (1986). Primer oligonucleotides were derived from HCV cDNA sequences of clone 81 (FIG. 21) or clones 36 (FIG. 22) and 37b (FIG. 24). Amplified sequences were detected by gel electrophoresis and a modified Southern blotting method, using a suitable cDNA oligomer or nick-translated cDNA sequence as a sample with a sequence from this region between, but not including, two primers. RNA samples containing HCV sequences in order to study them in the amplification system were isolated from liver biopsies of three chimpanzees with NANBH and from two control chimpanzees. The polyA + RNA fraction was isolated by the guanidine thiocyanate procedure described by Maniatis et al. (1982).

RNA samples that were examined by the amplification system were also isolated from the plasma of two chimpanzees with NANBH and from one control chimpanzee in the same way as from the plasma pool of control chimpanzees. One infected chimpanzee had a titer equal to or greater than 10 ⁶ CID / ml, and another infected chimpanzee had a titer equal to or greater than 10 ⁵ CID / ml.

Nucleic acids were extracted from plasma as follows. Either 0.1 ml or 0.01 ml of plasma was diluted to a final volume of 1.0 ml TENB (Proteinase K / DDS / 0.05 M Tris-HCl, pH 8.0, 0.01 M EDTA, 0.1 M NaCl, 1 mg / ml proteinase K and 0.5% DDS) containing 10 micrograms / ml polyadenyl acid, and incubated at 37 ^° C. for 60 minutes. After this treatment with proteinase K, the obtained plasma fractions were deproteinized by extraction of TE (10.0 mM Tris-HCl, pH 8.0, 1 mM EDTA) with saturated phenol. The phenolic phase was separated by centrifugation and re-extracted with TENB containing 0.1% DDS. The resulting aqueous phases from each extraction were collected and extracted twice with an equal volume of phenol / chloroform / isoamyl alcohol (1: 1 (99: 1)), and then twice with an equal volume of 99: 1 mixture of chloroform / isoamyl alcohol. After phase separation by centrifugation, the aqueous phase was brought to a final concentration of 0.2 M Na acetate and nucleic acids were precipitated by the addition of two volumes of ethanol. Precipitated nucleic acids were collected by ultracentrifugation in a SW 41 rotor at 38 thousand rpm for 60 minutes at 4 ^o C.

In addition to the above, a high chimpanzee plasma titer and control pool plasmas were alternatively extracted with 50 micrograms of poly-A carrier using the Chomcyrski and Sacchi (1987) procedures. This procedure used guanidine thiocyanate extraction. RNA was recovered by centrifugation at 1000 rpm for 10 minutes at 4 ^o C in an Eppendorf centrifuge.

 In two cases, before cDNA synthesis in the PCR reaction, nucleic acids extracted from plasma by the K / DDS / phenolic method were further purified by binding and elution from S and S columns. This procedure was carried out in accordance with the manufacturer's Elutip-R instructions.

The cDNA used as a template for the PCR reaction was derived from nucleic acids (or total nucleic acids or RNA) prepared as described above. After ethanol precipitation, the precipitated nucleic acids were dried and resuspended in distilled water treated with DEPC. The secondary nucleic acid structures were destroyed by heating at 65 ^° C. for 10 minutes and the samples were immediately cooled in ice. cDNA was synthesized using 1 to 3 micrograms of total chimpanzee RNA from the liver or plasma microliters. In the synthesis, reverse transcriptase was used and it was carried out in 25 microliters of the reaction mixture, using the manufacturer-specific description, BRL. Primers for cDNA synthesis were those that were also used in the PCR reaction described below. All cDNA synthesis mixtures contained 23 units of an RNase inhibitor, RNASIN ^m (Fisher / promega). After cDNA synthesis, the reaction mixtures were diluted with water, boiled for 10 minutes, and quickly cooled in ice.

PCR reactions were performed essentially as directed by the manufacturer (Cetus-Perkin-Elmer) with the exception of adding 1 microgram of RNase A. The reactions were carried out in a final volume of 100 microliters. PCR was performed in 35 cycles using 37 ^° C (2 min), 72 ^° C (3 min) and 94 ^° C (1 min).

 Primers for cDNA synthesis and for PCR reactions were derived from HCV cDNA sequences of either clone 81, clone 36, or clone 37b (HCV cDNA sequences of clones 81, 36 and 37b are shown in Figures 21, 22 and 23, respectively).

The sequences of the two 16-dimensional primers originating from clone 81 were:
5 'CAA TCA TAC CTG ACA G 3'
and
5 'GAT AAC CTC TGC CTG A 3'.

The primer sequence from clone 36 was
5 'GCA TGT CAT GAT GTAT 3'.

The primer sequence from clone 37b was:
5 'ACA ATA CGT GTG TCA C 3'.

 In PCR reactions, primer pairs consist of either two 16-mer originating from clone 81 or 16-mer from clone 36 and a 16-mer from clone 37b.

PCR reaction products were analyzed by alkaline gel electrophoresis, followed by Southern blotting and detection of amplified HCV cDNA sequences with ³² P-labeled internal oligonucleotide probe obtained from the HCV cDNA region, which does not overlap with primers. Mixtures from the PCR reaction were extracted with phenol / chloroform and nucleic acids precipitated from the aqueous phase with salt and ethanol. Precipitated nucleic acids were collected by centrifugation and dissolved in distilled water. Aliquots of the samples were electrophoresed on a 1.8% alkaline agarose gel. Single-stranded DNAs of 60, 108, and 161 nucleotides in size were run together on gels as molecular weight markers. After electrophoresis, DNA in gels was transferred onto Biorad Zeta probe ^m paper. The conditions of prehybridization and hybridization and washing were those specific to the manufacturer.

 The samples used for hybridization detection of amplified HCV cDNA sequences were as follows. When a pair of PCR primers came from clone 81, the sample was 108-mer with a sequence corresponding to the sequence that is located in the region between the sequences of the two primers. When a pair of PCR primers came from clone 36 and 37b, the sample was nick-translated HCV cDNA insertion originating from clone 35, the nucleotide sequence of which is shown in FIG. 31. The primers were derived from nucleotides 155-170 of insertion clone 37 and 206-268 from insertion clone 36. The 3'-end of the HCV insertion cDNA in clone 35 overlaps the nucleotides 1-186 of the insertion in clone 36, and the 5'-end of the insertion clone 35 overlaps nucleotides 207-269 insertion of clone 37b. (Compare FIGS. 23, 34, and 24). Thus, the cDNA insertion of clone 35 covers part of the region between the sequences from clone 36 and 37b of the resulting primers and is used as a probe for amplified sequences that include these primers.

 RNA analysis from liver samples was carried out in accordance with the above procedure, using both sets of primers and samples. DNA from the liver of three NANBH chimpanzees gave positive hybridization results for amplifiable sequences of the expected size (161 and 586 nucleotides for 81 and 36 and 37b, respectively), while control chimpanzees gave negative hybridization results. The same results were achieved by repeating this experiment three times.

 The analysis of nucleic acids and RNA from plasma was carried out in accordance with the above procedure, using primers and a sample from clone 81. Plasmas were from two chimpanzees with NANBH, from a control chimpanzee and collected plasmas from control chimpanzees (combined). Both plasma NANBHs contained nucleic acids / RNAs that gave positive results in PCR amplification assays, while both control plasmas gave negative results. These results are successively obtained several times.

 Defective viruses are known to be found among RNA viruses. Using PCR technology, it is possible to construct primers for amplification of the HCV genome sequences. When analyzing amplified products, it is expected that it is possible to identify defective versions of the viral genome in the same way as wild-type viral species. Accordingly, using two primers, based on the known HCV sequence, it is possible to predict exactly the expected size of the PCR product. Any large species observed during gel electrophoresis and hybridization analysis could represent potential variant genomes. Alternatively, any smaller species observed in a similar manner may represent defective agents. Assays of these types would be useful in confirming the exact origin of a known HCV sequence, whether it is truly a wild-type viral sequence or a defective genome. The technologies and methods for these analyzes are well known in science, described previously. This methodology will enable those skilled in this field to receive related (wild-type or defective) forms of the viral genome.

Detection of sequences in capture particles that, when amplified by PCR, hybridize to HCV cDNA originating from clone 81
RNA in capture particles obtained as described below. Sequence analysis that hybridizes to HCV cDNA originating from clone 81 was performed using the PCR amplification procedure as described above, except that the hybridization probe was a canase-labeled oligonucleotide originating from clone 81 of the cDNA sequence. The results showed that the amplified sequences hybridize with a cDNA probe.

Particles were captured from HCV-infected chimpanzee plasma using polystyrene beads coated with immunopurified antibodies directed against the polypeptide encoded
in clone 5-1-1. The procedure for the preparation of an immuno-purified antibody preparation is described in EPO publication N 318216, which is generally the property of the present representative and which is incorporated herein by reference. Briefly, the HCV polypeptide encoded by clone 5-1-1 was expressed as a superoxide dismutase (SOD) fusion polypeptide. This was accomplished by subcloning a clone of 5-1-1 insertion cDNA into the pSODcf1 expression vector (Steimer et al. (1986)). DNA isolated from pSODcf1 was treated with BamHI and EcoRI, and the following linker was ligated with the linear DNA formed by these restriction enzymes:
5 'GAT CCT GGA ATT CTG ATA AGA CCT TAA GAC TAT TTT AA 3'.

After cloning, a plasmid containing the insert was isolated. The insertion containing plasmid was digested with EcoRI. HCV cDNA insertion in clone 5-1-1 was excised with EcoRI and ligated into linearized EcoRI plasmid DNA. The DNA mixture was used to transform E. coli strain D1210 (Sadler et al., (1980)). Recombinants with 5-1-1 cDNA in the correct orientation for OPC expression were determined by restriction mapping and nucleotide sequencing. Recombinant bacteria of one clone were induced to express the SOD-NANB _5-1-1 polypeptide by growing bacteria in the presence of IPTG. The fused polypeptide was purified from recombinant E. coli by various extraction of cell extracts with urea, followed by chromatography on an anion-cation exchange column. Purified SOD-NANB _5-1-1 polypeptide was attached to a nitrocellulose membrane. Antibodies in HCV samples of infected sera were adsorbed with a matrix-linked polypeptide. After washing to remove non-specifically bound materials and unbound materials, the bound antibody was freed from the bound polypeptide.

cPCR method for detecting HCV RNA in the liver and serum of individuals with NANBH
Reliability and profitability of the modified form of PCR analysis, i.e. cPCR analysis to detect HCV infection was determined by performing analysis on total liver RNA and serum from infected individuals. In cPCR analysis, the putative viral RNA in the sample was back transcribed into cDNA by reverse transcriptase, the segment of the resulting cDNA was then amplified using a modified version of the technology described by Saiki et al. (1986). Technology primers were derived from HCV RNA, which can be identified by the HCV cDNA family provided here. An amplified product corresponding to HCV RNA was detected using a sample originating from the HCV cDNA family shown here.

 The cPCR / HCV analysis used in these studies was performed using the following methods for preparing RNA, reverse transcribing RNA into cDNA, amplifying specific segments of PCR cDNA, and analyzing PCR products.

 RNA was extracted from the liver using the guanidine isothiocyanate total RNA preparation method described by Maniatie et al. (1982).

In order to isolate total RNA from plasma, the plasma was diluted five to ten times with TENB (0.1 M NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA) and incubated with K / DDS proteinase solution (0, 5% DDS, 1 mg / ml proteinase K, 20 micrograms / ml poly A carrier) for 60-90 minutes at 37 ^° C. Samples were extracted once with phenol (pH 6.5), the resulting organic phase was re-extracted once with TENB, containing 0.1% DDS, and the aqueous phases of both extractions were combined and extracted twice with an equal volume of phenol / CHCl ₃ / isomyl alcohol (1: 1 (99: 1)). The resulting aqueous phases were extracted with an equal volume of CHCl ₃ / isomyl alcohol (99: 1) twice and precipitated with ethanol using 0.2 M sodium acetate, pH 6.5 and 2.5 volumes of 100% ethanol, precipitation overnight at -20 ^o C.

The cDNA used as a sample for the PCR reaction was prepared using the copied samples to prepare the corresponding cDNA. Each RNA sample containing either 2 micrograms of total heat-denatured RNA from chimpanzee liver, RNA from 2 microliters of plasma, or 10% RNA extracted from a 10 mm x 4 mm cylinder of human liver biopsy was incubated in 25 microliters of the reaction mixture containing 1 micromole of each primer , 2 mmol of each deoxyribonucleotide triphosphate (dNTP), 50 mmol of Tris-HCl, pH 8.3, 5 mmol of MgCl ₂ , 5 mmol of dithiothritol (DTT), 73 mmol of KCl, 40 units of RNase inhibitor (RNASIN) and 5 units of reverse transcriptase AMV . Incubation was carried out for 60 minutes at 37 ^o C. After the synthesis of cDNA, the reaction mixture was diluted with 50 microliters of deionized water (DIW), boiled for 10 minutes and cooled in ice.

Amplification of the HCV cDNA segment was performed using two synthetic oligomeric 16-dimensional primers whose sequences were derived from HCV cDNA clones 36 (antisense) and 37b (sense). The primer sequence from clone 36 was:
5 'GCA TGT CAT GAT GTA T 3'.

The primer sequence from clone 37b was:
5 'ACA ATA CGT GTG TCA C 3'.

Primers were used at a final concentration of 1 micromole each. In order to amplify the HCV cDNA segment, which is flanked by primers, cDNA samples were incubated with 0.1 micrograms of RNase A and PCR perkin Elmer Cetus PCR kit reagents (N 801-0043 or N 801-0055) in accordance with the manufacturer's instructions. The PCR reaction was performed either in 30 cycles or in 60 cycles in a Perkin Elmer Cetus DNA thermal cyclizer. Each cycle consisted of 1 min. denaturation step at 94 ^° C, annealing step 2 min at 37 ^° C, elongation step 3 min at 72 ^° C. However, the elongation step in the final cycle (30 or 60) was 7 minutes, not 3 minutes. After amplification, the samples were extracted with an equal volume of phenol / chloroform (1: 1), followed by extraction with an equal volume of chloroform, and then the samples were precipitated with ethanol containing 0.2 M sodium acetate.

CPCR products were analyzed as follows. The products were electrophoresed on 1.8% alkaline agarose gels according to Murakawa et al. (1988) and transferred onto Zeta ^m Probe paper (Biokad Corp.) by gel blotting overnight in 0.4 M NaOH. Blots were neutralized in 2 x SSC (1 x SSC contains 0.15 M NaCl, 0.015 M sodium citrate), prehybridized in 0.3 M NaCl, 15 mM sodium phosphate buffer, pH 6.8, 15 mM EDTA, 1.0% DDS, 0.5% skim milk (Cornation Co.) and 0.5 mg / ml of ultrasound-destroyed denatured DNA from salmon sperm. Blots that had to be analyzed for HCV cDNA fragments were hybridized with a ³² P-labeled probe formed by nick translation of the HCV insertion cDNA sequence in clone 35 described in EPO publication N 318216. After hybridization, the blots were washed in 0.1 x SSC (1 x SSC contains 0.15 M NaCl, 0.01 M Na citrate) at 65 ^° C, dried and autoradiographed. The expected product size is 586 nucleotides in length; products that hybridize with the sample and migrate in gels at this size are considered positive for viral RNA.

As a control, cPCR primers designed to amplify alpha-1 antitryptosine mRNA were used to check for the presence of RNA in each sample analyzed. The coding region of the alpha-1 antitryptosin gene is described by Rosenberg et al. (1984). Synthetic oligomeric 16-dimensional primers designed to amplify the 365 nucleotide fragment of the alpha-1 antitryptosin coding region were derived from nucleotides 22-37 (sense) and nucleotides 372-387 (antisense). PCR products were detected using a ³² P-nick-translated sample that lies between and does not include sequences with DNA / PCR primers.

Due to the exceptional sensitivity of the PCR reaction, all samples were run at least three times. All false positive signals were eliminated during the following precautions: 1) elimination of aerosols by using screw caps on test tubes with rubber O-shaped gaskets-insulators, 2) digging out Ranin Micromem ^m pipettes with droppers with freely moving pistons / capillaries and 3) selection of oligonucleotides sequences for cDNA and RNA primers from two non-adherent cDNA clones.

Detection of HCV RNA in liver samples by cPCR
cPCR analysis was performed on total RNA isolated from the liver of three chimpanzees infected with a NANBH agent in a laboratory, and from liver biopsies of Italian patients diagnosed with chronic NANBH.

FIG. 24 shows the results of cPCR analysis using 1 microgram of each total liver RNA preparation. RNA was isolated from chimpanzee liver samples in the chronic phase of infection (910) (line 1), two chimpanzees in the acute phase of infection (1028 and 508) (lines 2 and 3, respectively). PCR was performed on samples of lanes 1-3 for 30 cycles, and autoradiograms of blots containing these tracks were exposed for 5 hours. cDNA from 1 microgram of total RNA of an infected animal in the acute phase (lane 4) 1028 and three infected chimpanzees (lanes 5-7) were amplified for 60 cycles, and autoradiograms containing those tracks were exposed for 7 days. ³² P-labeled digested MspI pBR322 DNA served as a marker on all autoradiograms. It can be seen from these results that the cDNA corresponding to HCV RNA is visible only in chimpanzee samples with NANBH, whether acute or chronic (lanes 1, 3, and 4). CPCR products on these lanes migrated between marker fragments of 527 and 622 nucleotides (not shown).

 FIG. 24B shows the results of cPCR analysis using 10% RNA extracted from 10 mm x 4 mm liver biopsy cylinders from 15 patients with chronic NANB (lanes 1-15), one patient with cryptogenic liver disease (lane 16) and one control sample from a patient with chronic hepatitis B (lane 17). PCR amplification was performed in 30 cycles and autoradiograms of the blots were exposed for 4 days, except that lane 1 was exposed for 15 hours. As can be seen from the results, 9/15 (60%) of human samples are positive for HCV RNA (lanes 1, 2, 4, 6, 7, 10-13). One patient diagnosed with cryptogenic liver disease (lane 16) and one patient with chronic HBV infection (lane 17) are relatively negative in cPCR analysis.

Comparison of HCV / cPCR analysis of biopsies from human liver and RIA from serum using HCV C100-3 polypeptide
The SOD / HCV C100-3 polypeptide (also called C100) is a recombinant fusion polypeptide that contains 363 viral amino acids. The polypeptide is used to detect antibodies to HCV (see Kuo et al., 1989). The preparation method of C100 is described in EPO publication N 318216.

Radioimmunoassay using C100 was performed on serum collected from the same 17 patients whose liver samples were subjected to HCV / cPCR analysis as described above. Serum was collected on the same day as the liver biopsin. The analysis was performed essentially as described in EPO publication N 318216, which is wholly owned by the authorized person and is incorporated herein by reference. Briefly, microtiter plates (Immulon 2 Removeawell strips) were coated with 0.1 micrograms of purified C100. The plates were incubated for 1 hour at 37 ^° C with serum samples (100 microliters dilution 1: 100) or appropriate controls. After incubation, unbound material was removed, the plates were washed, and human C100 antibody complexes were detected by incubation with ¹²⁵ I-labeled anti-human sheep immunoglobulin. Radioactivity was determined in each well.

 RIA results showed that sixty-seven percent of these samples were positive for anti-C100 antibodies. Serum from a patient diagnosed with cryptogenic liver disease was positive for anti-C100 antibodies, although viral RNA levels were undetectable in the patient's liver in this sample. The correlation between the presence of anti-C100 antibodies and HCV RNA was seventy percent, two patients negative for RIA antibodies had significant levels of HCV RNA in their liver (data not shown).

 The results indicate that the virus is often present in the liver of patients with circulating anti-C100 antibodies, and confirms the claim that the presence of anti-C100 antibodies accurately reflects exposure to HCV. Moreover, taken together, these results indicate that this type of HCV is responsible for NANBH in at least 75% of the patients in this study, and that the main HCV strains in Italy appear to be closely related to the strains prevailing in the United States.

HCV / cPCR analysis of sera: detection of viral RNA in chimpanzees with acute phase infection
A temporary relationship between the detection of liver damage, the presence of HCV RNA, and the presence of anti-HCV antibodies was performed in serum from two chimpanzees infected with NANBH in the laboratory (pos. 771 and 910). Liver damage was determined by alanine aminotransferase (ALT) levels; the presence of HCV RNA was determined by HCV with the PCR analysis described above, antiHCV antibodies were detected using C100 RIA.

 HCV / cPCR analysis was performed on RNA extracted from 1 microliter of chimpanzee plasma. Sera were taken from 771 chimpanzees 25, 32, 70 and 88 days after infection, PCR was performed in 30 cycles, and the autoradiogram was exposed for 18 days. Sera were taken from 910 chimpanzees on days 11, 23, and 67 after infection, PCR was performed in 60 cycles, and the autoradiogram was exposed for 5 days.

 The analysis results are shown in FIG. 25A for chimpanzees 771 and FIG. 25B for chimpanzees 910. From a comparison of FIG. 25A and 25B, it appears that an early, well-defined peak of ALT values during the acute phase of hepatitis correlates with the presence of viral RNA in the infected individual.

 These data also indicate that the presence of HCV RNA, which indicates the state of time, precedes the presence of anti-HCV antibodies. Chimpanzee 771 (FIG. 25A) shows an early-detected acute episode of blood transfusion with NANBH in 28 days, as characterized by the first peak in ALT levels. HCV RNA was detected in sera collected on days 25 and 32. However, anti-HCV antibodies were absent during this acute phase. In contrast, on day 70, HCV RNA was below the experimental level of detection and anti-HCV antibodies increased. On day 88, HCV RNA remained undetectable, while anti-HCV antibodies significantly increased over 70 days.

 The results obtained from chimpanzee 910 serum were somewhat similar according to the Pattern, although the time of induction of HCV antibodies during infection was not detected during the acute phase of the disease, which increased to at least 67 days, anti-HCV antibodies detected by RIA on day 11 were caused by passive immunization animal 910 antibodies from plasma used to inoculate the animal. Anti-HCV antibodies were found in chimpanzee 910, in serum during the late, chronic phase of infection (data not shown).

It should be noted that low plasma ALT values from individuals with chronic NANBH do not necessarily correlate with weak virus production. A pool of 17 different plasma samples taken from 910 chimpanzees more than two to three and a half years after inoculation was examined for HCV RNA levels. ALT values of the samples did not exceed 45 honey / ml, however, titration indicated high titers of HCV (3 x 10 ⁶ STD / ml). cPCR was performed in 30 cycles and the autoradiogram was exposed for 15 hours, PCR analysis clearly showed the presence of viral RNA (data not shown).

HCV / cPCR serum analysis: detection of viral RNA in the acute phase of infection in humans
Plasma from surgical patients collected during early acute NANBH was tested for HCV RNA and anti-HCV antibodies using HCV / cPCR analysis with C100-RIA, respectively. HCV / cPCR analysis was performed using 1 microliter of plasma from a patient and out of four controls in people with known pedigrees, cPCR was performed in 30 cycles, and after hybridization and washing, autoradiography was exposed for eight hours.

The results showed that sera from surgical patients during acute infection contained high levels of viral RNA, and that anti-HCV antibodies were not detected by C100-RIA (data not shown). Plasma in the acute phase of surgical patients has been known to have a high titer of NANBH infectious agents (10 ^6.5 CID / ml, as determined by Feinstone et al. (1981), Feinstone et al., 1983). It should be noted, however, that this patient was indeed gray-secretive with C100-RIA anti-HCV antibodies for approximately 9 months after infection. Sera from control plasmas of pedigree patients were negative in both the HCV / cPCR assay and the C100-RIA.

Sensitivity HCV / cPCR Analysis
The sensitivity of the HCV / cPCR analysis was determined by analysis of ten-fold serial dilutions of a known titer plasma pool. Chimpanzee plasma had a titer of ~ 3 • 10 ⁵ CID / ml, and RNA was extracted from ten-fold dilutions of 1 microliter of plasma. cPCR was performed in 30 cycles and after hybridization and washing, the autoradiogram was exposed for 15 hours. The cPCR products obtained by amplification of ~ 300, ~ 30, and ~ 3 CID HCV genomes are shown in lanes 1 - 3, respectively, in FIG. 28. Samples in lanes 1 and 2 were detected on autoradiograms exposed for 2 hours.

 Since the full titer of HCV in infected individuals is believed to be between approximately 100 and 10,000 CID / ml plasma, these data suggest that HCV / cPCR analysis may be clinically useful.

HCV / cPCR analysis of variant HCV strains
Primers consisting of a set of oligomeric 44-mers and a set of oligomeric 45-mer were designed to amplify HCV strains similar or identical to the HCV isolate from which the cDNA sequence in FIG. 18 is happening. The premise underlying the creation of these primers is our discovery that HCV is a flavivirus. Compared to HCV, members of the Feaviviridae family have two major conserved sets of amino acid sequences. TATPPG and QRRGR, in the putative NS3 region of these viruses. Several other smaller sets can be seen, for example, in the case of GDD in the putative NS5 region. Other kits were determined by comparing known amino acid sequences with HCV sequences. This information was deduced from the sequences for several members of Flaviviridae that have been described, including Japanese encephalitis virus (Sumiyoshi et al. 1987), yellow fever virus (Rice et al., 1985), dengue virus type 2 (Hahn et al., 1988) , dengue virus type 4 (Mackow, 1987) and West Nile virus (Castle et al., 1986). Conservative amino acid sequences and the frequency of codon usage are presented in Table 6 (the primer sequence is chosen so as to minimize the number of degenerate nucleotides at the 3-end of the primer sequence and maximize the number of nucleotides at the 3'-end of each primer that exactly match any of the possible nucleotide sequences or their complements encoding conservative amino acids).

44-mer and 45-mer oligonucleotide primers are designed such that sequences encoding these amino acids are included within the primer. Moreover, they contain degeneracy at the 3 ′ end of each primer and originate from two different regions of the genome that are present in clone 40 ⁶ (see FIG. 27) and which originate from the region encoding presumably HCV NS3. The formula for oligonucleotide primers in these kits is:
5 'GAC TGC GGG GGC GAG ACT GGT TGT GCT CGC ACC GCC ACC CCX CC 3',
where X is A, T, G or G; and
5 'TCT GTA GAT GCC TGG CTT CCC CCT GCC AGT CCT GCC CCG AGT YTG 3',
where Y is T or C.

 HCV / cPCR analysis was performed using these primers to amplify the HCV RNA of chimpanzee 910 plasma. The method of this analysis was essentially carried out as described in the section above, except that 44- and 45-dimensional sets of oligomeric primers were replaced with primers originating from clones 36 and 37b. In addition, amplified HCV cDNA was detected by hybridization with a sample originating from clone 40a, the sequence of which is shown in FIG. 29.

A sample was prepared by amplification of a segment of clone 40a using the PCR method described above and 18-dimensional primers containing the following sequences:
5 'GAG ACA TCT CAT CTT CTG 3'
and
5 'GAG CGT GAT TGT CTC AAT 3'.

After amplification of the drug, samples were labeled with ³² P-nick translation.

FIG. 30 shows autoradiography of Southern blots hybridized to a sequence originating from clone 40a. ³² P-labeled MspI digested DNA fragments of pBR 322 served as markers (lane 1). The predicted size of the PCR product obtained by amplification using these primers is 490 nucleotides (pt). Repeated reactions are shown in lanes 2 and 3.

Analysis of HCV 5'-region variants
Based on the flavivirus model, the 5'-region of the HCV cDNA, which is flanked by the regions represented in clones ag30a and K9-1, encodes a segment of the supposedly protein (s) of the membrane and / or matrix. (E / M). Chimpanzee sera from which the HCV cDNA c library was derived were designed to be analyzed by HCV / cPCR to determine if variants are present within this region of the target.

 HCV / cPCR analysis was performed essentially as described above to isolate clone 5'-32, with the exception of the primers and samples used. FIG. 32 shows the relationship of primers and samples (and clones from which they originated) with primers and samples of the HCV cDNA target region. One set of PCR primers, ag30a16A and K91Env16B, came from clones ag30a and K9-1, which lie above and below, respectively, the target sequence. The expected length of the PCR product primed with ag30a16A and K91Env16B is 1.145 bp based on the confirmed sequence of HCV cDNA. Two other sets of PCR primers covering the region amplified using ag30a16A and K91Env16B and overlapping each other were also used to amplify serum HCV RNA. Thus, in this case, PCR reactions were carried out using both one set of primers ag30a16A and CA156e16B, and a second set of primers CA156e16A and 91Env16B. The sizes of the expected PCR product for these pairs were 615 nucleotides (NT) and 683 NT, respectively. Lists of primers, clones from which they originate, and primer sequences are shown in Table 7.

Samples for all three HCV / cPCR products consisted of ³² P-labeled portions of HCV cDNA that were prepared by PCR amplification of clone 216 (using CA216a16A and 216a16B as primers) and clone 84 (using CA84a16A and CA84a16B as primers), ³² P were included in PCR products by nick translation. These samples did not overlap with the primers used in the HCV / cPCR reactions.

FIG. 33 shows an autoradiography of a Southern blot in which HCV / cPCR products are hybridized with ³² P-labeled samples. HCV / cPCR products stacked with ag30a16A and K91Env16 primers (lane 1) were approximately 1.1 kb, no other PCR products were observed at 15-hour exposure. The products stacked with the ag30a15A (CA156e16B) primer sets (lane 2) and CA156e16a / KEnv16B (lane 3) were approximately 625 nt and 700 nt, respectively. The size of the PCR products was determined by comparison with the relative migration of the fragments obtained by digesting PBR 22 MspI and PhiX 174 digested with HalIII (lane 5).

 The above study detects insertions or deletions up to approximately 20 nt-50 nt and DNA rearrangements that alter the size of the target DNA. The results in FIG. 33 confirm that there is only one major cDNA species originating from the E / M region of HCV in chimpanzee sera.

Amplification to clone HCV cDNA sequences using PCR and primers originating from conserved regions of flavivirus genomic sequences
Our discovery that HCV is a flaviform virus has allowed us to use the strategy for cloning uncharacterized HCV cDNA sequences using PCR technology and primers originating from regions encoding conserved amino acid sequences of flaviviruses. In general, one of the primers comes from a specific HCV genomic sequence, and the other primer, which flanks the region of non-sequenced HCV polynucleotide, comes from a conservative region of the flavivirus genome. Flavivirus genomes are known to contain conserved sequences within the NS1 and E polypeptides, which are encoded in the 5 ′ region of the flavivirus genome. Thus, to isolate cDNA sequences originating from presumably comparable regions of the HCV genome, the upstream primer is designed to come from conserved sequences within these flavivirus polypeptides. The downstream primer originates from the upstream end of a known portion of the HCV cDNA.

 Due to the degeneracy of the code, substitutions are possible in flavivirus samples compared to the corresponding HCV genomic sequence. Therefore, they use a strategy similar to that described by Lee (1988). The Lee procedure uses mixed oligonucleotide primers complementary to the reverse translation products of the amino acid sequence, mixed primer sequences take into account each degenerate codon in a conserved amino acid sequence.

 Three sets of primer mixtures are based on amino acid homology found in several flaviviruses, including dengue 2,4 (D-2,4), Japanese encephalitis virus (JEV), yellow fever (YF), and West Nile (WN). The primer mixture derived from the most upstream conserved sequence (5'-I) is based on the amino acid sequence gly-trp-gly, which is part of the conserved sequence asp-arg-gly-trp-gly-aspN, found in E protein D-2 , JEV, YF, and WN. The following primer mixture (5'-2) is based on the right located sequence in the E protein phe-asp-gly-asp-ser-tyr-ileu-phe-gly-asp-ser-tyr-ilen and is derived from phe-gly-asp , this conserved sequence is present in D2, JEV, YF, and WN. The third primer mixture (5'-3) is based on the amino acid sequence arg-ser-cys, which is part of the conserved sequence cys-cys-arg-ser-cys in NS 1 protein D-2, D-4, JEV, YF and WN . The individual primers that form the 5'-3 mixture are shown in FIG. 45. In addition to the varying sequences originating from the conserved region, each primer in each mixture also contains a constant region at the 5'-end that contains a sequence encoding the sites for the restriction enzymes HindIII MboI and EcoRI.

The ssc5h20A primer located to the right comes from the nucleotide sequence of clone 5h, which contains cDNA with sequences that overlap the sequences of clones 14i and 11b. Ssc5h20A sequence:
5 'GTA ATA TGG TGA CAG AGT CA 3'.

You can also use an alternative primer, ssc5h34A. This primer originates from the sequence of clone 5h and additionally contains nucleotides at the 5'-end that create a restriction site, thereby facilitating cloning. Ssc5h34A sequence:
5 'GAT CTC TAG AGA AAT CAA TAT GGT GAC AGA GTC A 3'.

The PCR reaction, which was originally described by Saiki et al., 1986, is carried out essentially as described by Lee et al., 1988, except that the cDNA sample in RNA is isolated from the liver of an infected HCV chimpanzee or from virus particles isolated from serum HCV infected chimpanzee. In addition, the annealing conditions are less stringent in the first round of amplification (0.6 M NaCl and 25 ^° C.), since the portion of the primer annealed with the HCV sequence has only 9 nucleotides and can be replaced. Moreover, if ssc5h34A is used, additional sequences not originating from the genome tend to destabilize the primer-matrix hybrid. After the first round of amplification, the annealing conditions can be more stringent (0.066 M NaCl and 32-37 ^° C), since the amplified sequences now contain regions that are complementary or duplicate of the primers. In addition, the first 10 amplification cycles are carried out with a Klenov fragment 1 under suitable conditions for this enzyme. After completion of these cycles, the samples are extracted and reacted with Tag polymerase according to the instructions of the whale as determined by Cetus / Perkin - Elmer.

After amplification, amplified HCV cDNA sequences are detected by hybridization using a probe derived from clone 5h. This sample originates from sequences lying above the sequences used in the primer and does not overlap with sequences from clone 5h derived primers. The sequence of this sample
5 'CCC AGC GGC GTA CGC GCT GGA CAC GGA GGT GGC CGC GTC GTC TGG CGG TGT TGT TCT CGT CGG GTT GAT GGC GC 3'.

Industrial application
The method described here, as well as oligomers, both samples and primers derived from HCV cDNA, and whales containing them, are useful for accurate, relatively simple and economical determination of the presence of HCV in biological samples, especially in blood used for blood transfusion and in individuals suspicious of HCV infection. Moreover, these methods and oligomers may be useful for detecting HCV infections at earlier stages than immunological assays based on the use of recombinant HCV polypeptides. Hybridization analysis of the amplified polynucleotide also detects HCV RNA in random samples that are anti-HCV negative. Thus, the samples and primers described herein can be used amplified in hybridization assays in combination with immunoassays based on HCV polypeptides to better identify infections caused by HCV and HCV-infected samples, including blood.

 The information provided here allows you to have primers and / or samples that originate from conserved regions of the HCV genome. The provision of these primers and samples makes possible the main method by which variant HCV strains are detected and which will be used for screening blood and its products.

 If the primers used in the method originate from conserved regions of the genome, the method should be aimed at the detection and / or identification of variant HCV strains. This, in turn, should lead to additional immunological reagents for the detection and diagnosis of HCV, as well as to the creation of polynucleotide reagents for the detection and / or treatment of HCV.

 In addition, sets of primers and samples obtained from regions of conserved amino acid sequences of flaviviruses and HCV allow the use of a universal detection method for these infectious agents.

 The following listed materials (listed at the end of the description) are deposited in terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Dr. Rockiville Maryland 20852 and are named by the following Accession numbers.

Claims (44)

 1. The polynucleotide used to detect hepatitis C virus (HCV), corresponding to the conservative 5 'region of the hepatitis C virus genome from nucleotide at position - 340 to nucleotide at + 200 or complementary to it, as shown in Fig. 18.  2. A fragment of the polynucleotide described in claim 1 of the formula containing at least 8 nucleotides capable of hybridizing with the conserved 5 ′ region of the genome of the virus.  3. A fragment of the polynucleotide according to claim 2, characterized in that it contains nucleotides from position - 318 to position + 174, as shown in Fig. 18.  4. The polynucleotide fragment according to claim 2, characterized in that it contains nucleotides from position - 340 to position - 320, as shown in Fig. 18.  5. A fragment of the polynucleotide according to claim 2, characterized in that it contains nucleotides from position 330 to 320, as shown in Fig. 18.  6. A fragment of the polynucleotide according to claim 2, characterized in that it contains nucleotides from position - 313 to position - 282, as shown in Fig. 18.  7. The fragment of the polynucleotide according to claim 2, characterized in that it contains nucleotides from position - 203 to position - 173, as shown in Fig. 18.  8. The fragment of the polynucleotide according to claim 2, characterized in that it contains nucleotides from position 252 to position 221, as shown in Fig. 18.  9. The polynucleotide used to detect hepatitis C virus corresponding to the 3 'region of the hepatitis C virus genome from nucleotide at position 8867 to nucleotide at position 9060 or complementary to it, as shown in Fig. 18.  10. A fragment of the polynucleotide described in claim 9 of the formula containing at least 8 nucleotides capable of hybridizing with the conserved region of the genome of the virus.  11. The polynucleotide fragment of claim 10, characterized in that it contains nucleotides from position 8870 to 8880, as shown in Fig. 18.  12. The polynucleotide fragment of claim 10, characterized in that it contains nucleotides from position 8880 to 8890, as shown in Fig. 18.  13. The polynucleotide fragment of claim 10, characterized in that it contains nucleotides from position 8890 to 8900, as shown in Fig. 18.  14. The fragment of the polynucleotide of claim 10, characterized in that it contains nucleotides from position 8900 to 8910, as shown in Fig. 18.  15. A fragment of the polynucleotide of claim 10, characterized in that it contains nucleotides from position 8910 to 8920, as shown in Fig. 18.  16 A fragment of a polynucleotide according to claim 10, characterized in that it contains nucleotides from position 8920 to 8930, as shown in Fig. 18.  17. A fragment of the polynucleotide according to claim 10, characterized in that it contains nucleotides from position 8930 to 8940, as shown in Fig. 18.  18. A fragment of a polynucleotide according to claim 10, characterized in that it contains nucleotides from position 8940 to 8950, as shown in Fig. 18.  19. A fragment of a polynucleotide according to claim 10, characterized in that it contains nucleotides from position 8950 to 8960, as shown in Fig. 18.  20. A fragment of the polynucleotide according to claim 10, characterized in that it contains nucleotides from position 8960 to 8970, as shown in Fig. 18.  21. The fragment of the polynucleotide according to p. 10, characterized in that it contains nucleotides from position 8970 to 8980, as shown in Fig. 18.  22. A fragment of the polynucleotide according to claim 10, characterized in that it contains nucleotides from position 8980 to 8990, as shown in Fig. 18.  23. A fragment of a polynucleotide according to claim 10, characterized in that it contains nucleotides from position 8990 to 9000, as shown in Fig. 18.  24. A fragment of a polynucleotide according to claim 10, characterized in that it contains nucleotides from position 9000 to 9010, as shown in Fig. 18.  25. A fragment of the polynucleotide according to claim 10, characterized in that it contains nucleotides from position 9010 to 9020, as shown in Fig. 18.  26. A fragment of the polynucleotide according to claim 10, characterized in that it contains nucleotides from position 9020 to 9030, as shown in Fig. 18.  27. A fragment of the polynucleotide according to claim 10, characterized in that it contains nucleotides from position 9030 to 9040, as shown in Fig. 18.  28. A fragment of a polynucleotide according to claim 10, characterized in that it contains nucleotides from position 9040 to 9050, as shown in Fig. 18.  29. A fragment of the polynucleotide according to claim 10, characterized in that it contains nucleotides from position 9050 to 9060, as shown in Fig. 18.  30. A polynucleotide corresponding to a conserved region of the genome of hepatitis C virus, containing nucleotides from position 4000 to position 5000 or complementary nucleotides, as shown in Fig. 18.  31. A polynucleotide fragment according to claim 30, containing at least 8 nucleotides.  32. A fragment of the polynucleotide according to p. 31, characterized in that it contains nucleotides from position 4056 to position 4448, as shown in Fig. 18.  33. A fragment of the polynucleotide according to p. 31, characterized in that it contains nucleotides from position 4378 to position 4902, as shown in Fig. 18.  34. A fragment of the polynucleotide according to p. 31, characterized in that it contains nucleotides from position 4042 to position 4059, as shown in Fig. 18.  35. A fragment of the polynucleotide according to p. 31, characterized in that it contains nucleotides from position 4456 to position 4470, as shown in Fig. 18.  36. The polynucleotide corresponding to the 3 'conservative region of the hepatitis C virus genome containing nucleotides from position 8000 to position 9040 or complementary nucleotides, as shown in Fig. 18.  37. A fragment of the polynucleotide according to clause 36, characterized in that it contains at least 8 nucleotides.  38. A fragment of the polynucleotide according to clause 37, containing nucleotides from position 8209 to position 8217, as shown in Fig. 18.  39. A primer for DNA synthesis representing a fragment of a polynucleotide according to any one of claims 2 to 8, 10 to 29, 31 to 35, 37 or 38.  40. A labeled probe carrying a detectable label representing a polynucleotide fragment according to any one of claims 2 to 8, 10 to 29, 31 to 35, 37 or 38.  41. A capture probe capable of binding HCV nucleic acids to a solid support, representing a polynucleotide fragment according to any one of claims 2 to 8, 10 to 29, 31 to 35, 37 or 38.  42. A method for detecting HCV nucleic acid in a sample, comprising incubating a polynucleotide fragment with the specified sample under conditions that ensure the formation of hybridized duplexes between the polynucleotide reagent and HCV nucleic acid, detecting the formed duplexes, characterized in that the polynucleotide according to any one of any of the following claims 1, 9, 30 or 36, or a polynucleotide fragment according to any one of claims 2 to 8, 10 to 29, 31 to 35, 37 or 38, a labeled probe according to claim 40, or a capture probe according to claim 41.  43. The method for detecting HCV nucleic acid in a sample according to claim 42, characterized in that prior to incubating the polynucleotide reagent with the specified sample, the nucleic acid in the sample is further amplified by the polymerase chain reaction using the primer according to claim 39.  44. A kit for detecting HCV nucleic acid containing a diagnostic reagent and a suitable container, characterized in that as a diagnostic reagent it contains a polynucleotide according to any one of claims 1, 9, 30 or 36, or a polynucleotide fragment according to any one of claims 2 - 8, 10 - 29, 31 - 35, 37 or 38.  45. A method for testing blood for HCV content, characterized in that the analyzed nucleic acid is obtained from a blood sample suspected of containing HCV nucleic acid, incubated with a polynucleotide fragment according to any one of claims 2 to 8, 10 to 29, 31 to 35, 37 or 38 as a polynucleotide reagent under conditions allowing the formation of hybridized duplexes between HCV nucleic acid and said polynucleotide reagent, and the presence or absence of hybridized duplexes is taken into account when determining the results of tests. ation. Priority on points:
05/18/89 for PP. 42 to 45;
04.04.90 PP 1 - 41.

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