Polypeptide having antigenic properties of hepatitis c virus and its fragment (options), diagnostic reagent (options), set for detection of antibodies (options) , method of detection antibody (options)

 

(57) Abstract:

The invention relates to materials and methods of curbing the epidemic of hepatitis neither a nor B (hepatitis C virus). The proposed polypeptide having antigenic properties of hepatitis C virus and their fragments. Proposed diagnostic reagent for the detection of antibodies against hepatitis C virus, kit for detection of antibodies against hepatitis C virus, comprising a diagnostic reagent, and a method for detecting antibodies against hepatitis C. the Invention allows to obtain reliable diagnostic and prognostic tools for the determination of nucleic acids, antigens and antibodies associated with hepatitis C. 17 C. and 13 C.p. f-crystals, 47 ill., 19 table.

The invention relates to materials and methods of curbing the epidemic of hepatitis neither A nor B. More specifically it concerns a diagnostic polypeptides, diagnostic reagents, diagnostic kits and diagnostic methods for the detection of the etiologic agent of hepatitis virus neither A nor B, i.e., hepatitis virus C.

Bibliographic references in the description to the application

Barr et al. in 1986, Biotechniques 4:428.

Botstein (1979), Gene 8:17.

Brinth (1981) in: Molecular Biology of the Yeast Saccharomyces. Vol. 1, p.445, Cold Spring Harbor Press.

Broach et al. (1983), Meth. Enz. 101:307.

Chang et al. (1977) Nature 198:1056.

Chirgwin et al. (1979), Biochemistry 18:5294.

Chomczynski and Sacchi (1987), Analytical Biochemistry 162:156.

Clewell et al. (1969), Proc. Natl. Acad. Sci. USA 62:1159.

Clewell (1972), J. Bacteriol. 110:667.

Cohen (1972), Proc. Natl. Acad. Sci. USA 69:2110.

Cousens et al. (1987), Gene 61:265.

De Boer et al. (1983), Proc. Natl. Acad. Sci. USA 292:128.

Dreesman et al. (1985), J. Infect. Disease 151:761.

Feinstone, S. M. and Hoofnagle, J. H. (1984), New Engl. J. Med. 311:185.

Fields & Knipe (1986), Fundamental Virology (Raven Press, N. Y.).

Fiers et al. (1978) Nature 273:113.

Gerety, R. J. et al., in Viral Hepatitis and Liver Disease (Vyas, B. N., Dienstag, J. L. , and Hoofnagle, J. H., eds, Grune and Stratton, Inc., 1984) pp. 23-47.

Goeddel et al. (1980), Nucleic Acids Res. 8:4057.

Graham and Van der Ed (1978), Virology 52:546.

Grunstein and Hogness (1975), Proc. Natl. Acad. Sci. USA 73:3961.

Grych et al. (1985) Nature 316:74.

After Gubler and Hoffman (1983), Gene 25:263.

Hammerling et al. (1981), Monoclonal Antibodies and T-cell Hybridomas.

Hess et al. (1968), J. Adv. Enzyme Reg 7:149.

Hinnen et al. (1978), Proc. Natl. Acad. Sci. 75:1929.

Hitzeman et al. (1980), J. Biol. Chem. 255:2073.

Holland et al. (1978), Biochemistry 17:4900.

Holland (1981), J. Biol. Chem. 256:1385.

Houghton et al. (1981), Nucleic Acids Res. 9:247.

Hunyh, T. V. et al. (1985) in DNA Cloning Techniques, A Practical Approach (D. Glover, Ed., IRL Press, Oxford, U. K.), pp. 49-78.

Lee et al. (1988), Science 239:1288.

Maniatis, T. et al. (1982) Molecular Cloning; A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N. Y.).

Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London).

Maxam et al. (1980), Methods in Enzymology 65:499.

MacNamara et al. (1984), Science 226:1325.

Messing et al. (1981), Nucleic Acids Res. 9:309.

Messing (1983), Methods in Enzymology 101:20-37.

Methods in Enzymology (Academic Press).

Michelle et al. Int. Symposium on Viral Hepaatitis. Monath (1986) in The Viruses: The Togaviradae and Flaviviridae (Series eds. Fraenkel-Conrat and Wagner, vol. eds. Schlesinger and Schlesinger, Plenum Press), p.375-440.

Nagahuma et al. (1984), Anal. Biochem. 141:74.

Neurath et al. (1984), Science 224:392.

Nisonoff et al. (1981), Clin. Immunol. Immunopathol. 21:397-406.

Overby, L. R. (1985), Curr. Hepatol. 5:49.

Overby, L. R. (1986), Curr. Hepatol. 6:65.

Overby, L. R. (1987), Curr. Hepatol. 7:35.

Peleg (1969) Nature 221:193.

Pfefferkorn and Shapiro (1974), in Comprehensive Virology, Vol. 2 (Fraenkel-Conrat & Wagner, eds. Plenum, N. Y.) pp. 171-230.

Prince, A. M. (1983), Annu. Rev. Environ. 37:217.

Rice et al. (1986) in The Viruses: The Togaviridae and Flaviviridae (Series eds. Fraenkel-Conrat and Wagner, vol. eds. Schlesinger and Schlesinger, Plenum Press), p. 279-328.

Roehrig (1986) in The Viruses: The Togaviridae and Flaviviridae (Series eds. Fraenkel-Conrat and Wagner, vol. eds. Schlesinger and Schlesinger, Plenum Press).

Sadler et al. (1980), Gene 8:279.

Saiki et al. (1986) Nature 324:163.

Sanger et al. (1977), Proc. Natl. Acad. Sci. USA 74:5463.

Shimatake et al. (1981) Nature 292:128.

Steimer et al. (1986), J. Virol. 58:9.

Stollar (1980), in The Togaviruses (R. W. Schlesinger, ed. Academic Press, N. Y.), pp. 584-622.

Taylor et al. (1976), Biochem. Biophys. Acta 442:324.

Towbin et al. (1979), Proc. Natl. Acad. Sci. USA 76, 4350.

Tsu and Herzenberg (1980) in Selected Methods in Cellular Immunology (W. H. Freeman and Co.) pp. 373-391.

Vytdehaag et al. (1985), J. Immunol. 134:1225.

Valenzuela, P., et al. (1982) Nature 298:344.

Valenzuela, P., et al. (1984), in Hepatitis B (Millman, I. et al., ed. Plenum Press) pp. 225-236.

Warner (1984), DNA 3:401.

Wu and Grossman (1987), Methods in Enzymology Vol. 154, Pecombinant DNA, Part E.

Wu (1987), Methods in Enzymology, Vol 155, Pecombinant DNA, Part f

Zoller (1982), Nucleic Acids Res. 10:6487.

References to patents

U.S. patents NN 4341761, 4399121, 4427783, 4444887, 4466917, 4472500, 4491632 and 4493890.

Prerequisites for creating inventions

Hepatitis neither A nor B is transmitted illness of a family of diseases induced, apparently, virus, and which differ from other forms caused by the virus of liver diseases, including those diseases that are caused by a known hepatitis viruses, i.e. hepatitis A virus, hepatitis B and Delta virus-hepatitis b, and hepatitis caused by cytomegalovirus or virus Estein-Barr. Hepatitis neither A nor B was Perse testify, that hepatitis neither A nor B is due portable infectious agent or agents. However, a portable pathogen responsible for hepatitis neither A nor B, so far not identified, and the number of agents that cause disease, is unknown.

Epidemiological data suggest that there may be three types of hepatitis neither A nor B: epidemic type associated with water; epidemic type, portable blood or needle; and sporadically appearing (community acquired) type of hepatitis. However, a number of pathogens that can cause hepatitis neither A nor B is unknown.

Clinical diagnosis and identification of hepatitis neither A nor B were primarily made by exclusion of other viral markers. Among the methods used to determine the suspected antigen and antibodies to hepatitis neither A nor B, agar-gel diffusion, radioimmunoelectrophoresis, immunofluorescence microscopy, immunoelectron microscopy, radioimmunoassay tests fermentopathy immunosorption test. However, none of these methods of analysis have not shown sufficient sensitivity, specificity and reproducibility, to be used as a diagnostic test for hepati and specificity of antigen systems - antibody-related pathogens hepatitis b neither A nor B. This is due, at least partially, primary or co-infection with hepatitis b virus hepatitis b neither A nor B patients and the known complexity of soluble and particulate antigens associated with hepatitis B virus, as well as the integration of the DNA of hepatitis b virus in the genome of liver cells. In addition there is the possibility that hepatitis neither A nor B is called more than one infectious agent, as well as the possibility that hepatitis neither A nor B is diagnosed incorrectly. In addition, it is unclear what determines the serological tests in the serum of patients with hepatitis neither A nor B. Was accepted without proof that the agar-gel diffusion and radioimmunoelectrophoresis analyses determine an autoimmune reaction or interaction of non-specific proteins, which sometimes occur between serum samples, and that they do not represent specific reactions antigen - antibody. Immunofluorescence, fermentopathy immunosorbent and radioimmunological assays, apparently, determine low levels of material similar to rheumatoid factor, which is often present in the serum of patients with hepatitis neither A nor B, as well as in patients with other the military examination, may be antibody to a specific host cytoplasmic antigens.

There are several candidates for hepatitis neither A nor B. See, for example, reviews of Prince (1983), Feinstone and Hoofnagle (1984) and Overby (1985, 1986 and 1987) and article Iwarson (1987). But there is no evidence that any of these candidates is an etiologic agent of hepatitis neither A nor B.

The need for sensitive, specific methods of detection and identification of carriers of hepatitis b neither A nor B and virus-infected neither A nor B blood and blood products is very significant. Post-transfusion hepatitis appear in about 10% of cases of patients with transfusion and up to 90% of these cases, there are patients with hepatitis neither A nor B. the Main problem in this disease is the frequent development of chronic liver disease (25-55%).

The treatment of the patient, as well as the prevention of transmission of hepatitis b neither A nor B with blood or blood products, or through close contact with ill require a reliable diagnostic and prognostic tools to identify nucleic acids, antigens and antibodies associated with hepatitis neither A nor B. In addition there is a need for effective vaccines and immunotherapeutics concerns the isolation and characterization of recently discovered the etiologic agent of hepatitis neither A, neither B, hepatitis C. More specifically, the invention relates to the collection of replicas circular DNA parts of the genome of the hepatitis virus C. These replica ring DNA were selected by a method that includes a new operation of filtration products in the expression of their pools circular DNA created from particles of the agent (agent) in infected tissue with sera of patients with hepatitis neither A nor B, for determination of newly synthesized antigens derived from the genome to the highlighted and non-characterized viral agent, and the selection of clones that produce products, immunological interacting only with sera of infected individuals compared to uninfected persons.

Studies of the nature of the genome of hepatitis C virus using samples obtained from the circular DNA of hepatitis C virus and the sequence information contained in the circular DNA of hepatitis C virus, suggests that the hepatitis C virus is flaviviruses or flaviopolis virus.

Part of the base sequence of circular DNA derived from hepatitis C virus, are useful as samples to determine the presence of virus in samples and to isolate naturally occurring variants of the virus. This FR is encoded in the genome (genome) of the hepatitis C virus, and allow the production of polypeptides which are useful as standards and reagents in diagnostic tests and/or as components of vaccines. Antibodies, both polyclonal and monoclonal, directed against epitopes of hepatitis C virus contained within these polypeptide sequences are also useful for diagnostic tests, as therapeutic tools for screening of antiviral agents for isolation (separation) of the pathogen virus neither A nor B, which received these pieces of circular DNA. In addition, when using samples obtained from these parts of the circular DNA, it is possible to select and evaluate a sequence of other parts of the genome of hepatitis C virus, thus generating additional samples and polypeptides are useful for diagnosis and/or treatment, both as a preventive and therapeutic effect on hepatitis neither A nor B.

Aspects of the invention are purified hepatitis C virus; preparation of polypeptides of purified hepatitis C virus; the purified polypeptide of hepatitis C virus; a purified polypeptide containing the epitope immunologically identifiable epitope contained in the hepatitis C virus

Included aspects of sedevacantist, derived from the genome of the hepatitis C virus or circular DNA hepatitis C virus; recombinant polypeptide containing the epitope of hepatitis C virus and condensed polypeptide, polypeptide fusions, containing polypeptide of hepatitis C virus

Aspects of the invention relating to sets of diagnostic tools, the following: samples for analysis for the presence of polynucleotides derived from hepatitis C virus, comprising a sample containing a nucleotide sequence of hepatitis C virus about 8 or more nucleotides, in suitable packaging; samples for analysis for the presence of the antigen of the hepatitis C virus containing antibody directed against the antigen of the virus C, be definition, in suitable packaging; samples for analysis for the presence of antibodies directed against the antigen of the hepatitis C virus, comprising a polypeptide containing an epitope of hepatitis C virus present in the antigen of the hepatitis C virus, in appropriate packaging.

Other aspects of the invention are polypeptide containing antipop of hepatitis C, fixed on a solid substrate, and the antibody to the epitope of hepatitis C virus, fixed on a solid substrate.

The following aspects of izobretatel master, transformed expression vector containing a sequence encoding the polypeptide containing the epitope of hepatitis C virus under conditions that permit expression of the above-mentioned polypeptide, and the polypeptide containing the epitope received this metol.

The invention also includes a method of identifying nucleic acids in the sample containing reactive nucleic acid sample with a sample of polynucleotide of hepatitis C virus under conditions that allow the formation of a polypeptide of the duplex between the sample and the nucleic acid of hepatitis C virus sample, and determining polynucleotide duplex that contains the sample.

Immunological studies also included in the scope of the invention. These include immunological assay to detect antigen of the hepatitis C virus, comprising the incubation of the sample, podozriteljnogo on the content of the antigen hepatitis C antibody tests directed against the antigen of the hepatitis C virus, subject to determination under conditions that allow the formation of complex antigen - antibody; and determining the complex antigen - antibody containing antibody samples. An immunological assay for the detection of antibodies directed against the antigen is Titus C, with the polypeptide sample, which contains the epitope of hepatitis C virus, under conditions that allow the formation of a system of the antibody - antigen, and the detection system of the antibody - antigen containing the antigen of the sample.

In the scope of the invention also includes vaccines for the treatment of infection of hepatitis C virus, comprising the immunogenic peptide containing the epitope of hepatitis C virus, or an inactivated preparation of hepatitis C virus, or attenuated preparation of hepatitis C virus

Another aspect of the invention is a tissue culture of growing cells infected with hepatitis C virus

Still another aspect of the invention is a method of obtaining antibodies to hepatitis C virus, including the introduction of the patient selected immunogenic polypeptide containing an epitope of hepatitis C virus in a quantity sufficient to produce an immune response.

And another aspect of the invention is a method of separation of circular DNA derived from the genome of unidentified infectious agent, comprising: (a) obtaining host cells transformed with expression vectors contained in the pool circular DNA prepared from nucleic acids isolated from tissue infected by the agent, and b) the interaction of the products of expression of circular DNA containing the antibody component of the patient's body, infected referred to an infectious agent under conditions that allow the immune response, and determination of complexes of the antibody - antigen formed by the interaction; (c) growing the host cell carrying the expression of polypeptides that form complexes of the antibody - antigen operations (b) under conditions that allow the cultivation of individual clones, and the allocation of the above-mentioned clones; (d) culturing cells from the clones obtained in the operation (C) under conditions that allow expression of the polypeptide(s) encoded in the circular DNA, and the interaction of the products of expression of the antibody components of body of individuals, other than the patient for surgery (a), infected with an infectious agent, and control by persons not infected by the agent, and the determination of the complexes of the antibody - antigen formed by the interaction; (e) culturing host cells that carry the expression of polypeptides that form complexes of the antibody - antigen containing the antibody components of the body infected individuals and entities suspected infection, but not with the components of the persons in the control group, under conditions that allow both the development of individual clones, the AI (e).

In Fig. 1 shows donacina the nucleotide sequence of circular DNA virus hepatitis C, inserted in clone 5-1-1, and the putative amino acid sequence of the polypeptide encoded therein.

In Fig. 2 presents the homologues of overlapping sequences circular DNA virus hepatitis C in clones 5-1-1, 81, 1-2, and 91.

In Fig. 3 shows a complex sequence of bases circular DNA virus hepatitis C received from overlapping clones 81, 1-2, and 91, and the sequence of amino acids encoded therein.

In Fig. 4 shows the sequence dunacity nucleotides circular DNA virus hepatitis C, inserted in clone 81, and the putative amino acid sequence encoded in it.

In Fig. 5 shows the sequence of the bases of circular DNA virus hepatitis C in clone 36, the segment which overlaps the circular DNA of hepatitis b neither A nor B in clone 81, and the sequence of the polypeptides encoded in clone 36.

In Fig. 6 presents the combined openly readable structure of circular DNA virus hepatitis C in clones 36 and 81 and the polypeptides encoded therein.

In Fig. 7 shows the sequence of bases number of the CLASS="ptx2">

In Fig. 8 shows the sequence of the bases of circular DNA virus hepatitis C in clone 35, the segment which overlaps clone 36, and the encoded polypeptide her.

In Fig. 9 presents the combined openly readable structure of circular DNA virus hepatitis C in clones 35, 36, 81 and 32 and encoded polypeptide her.

In Fig. 10 shows the sequence of bases of circular DNA virus hepatitis C in clone 37b, the segment which overlaps clone 35, and the encoded polypeptide her.

In Fig. 11 shows the sequence of the bases of circular DNA virus hepatitis C in clone 33b, the segment which overlaps clone 32, and the encoded polypeptide her.

In Fig. 12 shows the sequence of the bases of circular DNA virus hepatitis C in clone 40b, the segment which overlaps clone 37b, and the encoded polypeptide her.

In Fig. 13 shows the sequence of the bases of circular DNA virus hepatitis C in clone 25c, the segment which overlaps clone 33b, and the encoded polypeptide her.

In Fig. 14 shows the nucleotide sequence and encoded polypeptide her openly readable structure that extends through colleenhaney circular DNA virus hepatitis C in clones 40b and 33c, and the encoded amino acids in it.

In Fig. 16 shows the sequence of bases (circular) DNA of hepatitis C virus in the clone 8h, the segment which overlaps clone 33c, and the encoded amino acids in it.

In Fig. 17 shows the sequence of the bases in the circular DNA of hepatitis C virus in the clone 7E, the segment which overlaps clone 8h, and the encoded amino acids in it.

In Fig. 18 shows the sequence of the bases of circular DNA virus hepatitis C in clone 14c, the segment which overlaps clone 25c, and the encoded amino acids in it.

In Fig. 19 shows the sequence of bases in the circular DNA of hepatitis C virus in the clone 8f, the segment which overlaps clone 14c, and the encoded amino acids in it.

In Fig. 20 shows the sequence of the bases of circular DNA virus hepatitis C in clone 33f, the segment which overlaps clone 8f, and the encoded amino acids in it.

In Fig. 21 shows the sequence of the bases of circular DNA virus hepatitis C in clone 33g, the segment which overlaps clone 33f, and the encoded amino acids in it.

In Fig. 22 shows the sequence of the bases ring DN is data in it amino acids.

In Fig. 23 shows the sequence of bases of circular DNA virus hepatitis C in clone 11b, the segment which overlaps the sequence of bases in the clone 7f, and the encoded amino acids in it.

In Fig. 24 shows the sequence of the bases of circular DNA virus hepatitis C in clone 14i, the segment which overlaps the sequence of bases in clone 11b, and the encoded amino acids in it.

In Fig. 25 shows the sequence of bases of circular DNA virus hepatitis C in clone 39c, the segment which overlaps the sequence of bases in the clone 33g, and the encoded amino acids in it.

In Fig. 26 presents a complex sequence of bases circular DNA virus hepatitis C, obtained from the combination of circular DNA in the clones 14i, 11b, 7f, 7e, 8h, 33C, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f and 33g, and presents the amino acid sequence of the polypeptide encoded in extended openly readable structure in the obtained base sequence.

In Fig. 27 shows the sequence of the bases of circular DNA virus hepatitis C in clone 12f, the segment which overlaps clone 14i, and the encoded amino acids in it.

On Phi is ecrivait clone 39c, encoded in its sequence of amino acids.

In Fig. 29 shows the sequence of the bases of circular DNA virus hepatitis C in clone 19g, the segment which overlaps clone 35f, and the encoded amino acids in it.

In Fig. 30 shows the sequence of bases in the clone 26g, the segment which overlaps clone 19g, and the encoded amino acids in it.

In Fig. 31 shows the sequence of bases in the clone 15e, the segment which overlaps clone 26g, and the encoded amino acids in it.

In Fig. 32 shows the sequence of bases in the complex circular DNA, which is obtained by combining clones from 12f to 15e in the direction 5' to 3', amino acids, encoded on a continuous openly readable structure.

In Fig. 33 shows a photograph of the Western spots obtained on the condensed protein, peroxydisulfate - neither A nor B5-1-1serum chimpanzees infected with hepatitis neither A nor B, A and B.

In Fig. 34 shows a photograph of the Western spots obtained on the condensed protein, peroxydisulfate - neither A nor B5-1-1serum human, infected with hepatitis neither A nor B, VI is substantial evidence of vector raw.

In Fig. 36 shows the putative amino acid sequence of carboxykinase condensed polypeptide C100-3 and is encoded in its sequence of nucleotides.

In Fig. 37A shows a photograph of the painted blue dye polyacrylamide gel, which identifies the polypeptide C100-3, expressed in yeast.

In Fig. 37B presents Western blot polypeptide C100-3 serum infected with a virus neither A nor B person.

In Fig. 38 presents autoradiography Northern blots of RNA isolated from the liver of an infected virus BB-neither A nor B chimpanzees studied virus BB-neither A nor B circular DNA of clone 81.

In Fig. 39 presents autoradiography nucleic acid of the virus neither A nor B, treated with ribonuclease A or desoksiribonukleaza I investigated and circular DNA virus BB-neither A nor B clone 81.

In Fig. 40 presents autoradiography nucleic acids extracted from virus particles neither A nor B caught in the infected anti-neither A nor B5-1-1plasma, has been labeled with phosphorus-32 circular DNA virus neither A nor B from clone 81.

In Fig. 41 presents autoradiogram filters containing videolecture of circular DNA virus nor A no B in clone 81.

In Fig. 42 presents homologues between the polypeptide encoded in the circular DNA of hepatitis C and NS-protein flavivirus Dangle.

In Fig. 43 presents a histogram of the distribution of infection of hepatitis C virus in randomly selected samples as determined by ELISA analysis.

In Fig. 44 presents a histogram of the distribution of infection of hepatitis C virus in random samples, obtained using two configurations mates immunoglobulin - enzyme in the ELISA analysis.

In Fig. 45 shows the sequence in the primary mixture obtained from the stored sequence of NS1 flaviviruses.

In Fig. 46 presents the sequence of bases of circular DNA virus hepatitis C in clone k9-l, the segment which overlaps the circular DNA of Fig. 26, and the encoded amino acids.

In Fig. 47 presents the sequence of bases of complex circular DNA obtained in connection clones k9-1 to 15e in the direction 5' to 3'; presents amino acids encoded in the open reading extended structure.

Ways of carrying out the invention

I. local etiological agent nor A neither B. Therefore, used in the description of the "hepatitis C virus" for the causative agent of hepatitis neither A nor B, which previously belonged to the virus neither A nor B and/or BB-neither A nor B. the Terms "hepatitis C virus, hepatitis neither A nor B and hepatitis BB-neither A nor B" are used here interchangeably. As an extension of this terminology, the disease was called the hepatitis C virus, formerly called the hepatitis neither A nor B, hepatitis C. the Term hepatitis neither A nor B and hepatitis C can be used here as equivalent.

The term "hepatitis C virus", as used in the description means the types of viruses that cause hepatitis neither A nor B, and weakened strains and defective viral particles derived from these strains. As will be shown below, the genome of hepatitis C virus consists of RNA. It is known that RNA-containing viruses have relatively high levels of spontaneous mutations, i.e., according to published data, in the order of 10-3-10-4the nucleotide (Fields and Knipe, 1986). Therefore, there are multiple strains within a species of hepatitis C, defined below. The compositions and methods described herein make possible the reproduction, identification, detection and isolation of different related viruses. Moreover, they also allow to obtain the diagnosis is ekologicheskogo use, because they inhibit the replication of hepatitis C virus

The information contained herein, while obtained on the same strain of hepatitis C, called hereinafter CDC/HCV1 is sufficient for viral taxonomist identified other strains, which belong to this kind of viruses. As described already, we discovered that the hepatitis C virus belongs to flavivirus or plagiodontia virus. The morphology and composition of the particles flavivirus known and described Brinton (1986). In General, taking into account morphology, flavivirus contain Central nucleocapsid surrounded by a lipid declaim. The virions are spherical and have a diameter of about 40-50 nm. Their nuclei have a diameter of about 25-30 nm. Along the outer surface of the shell of the virion are outgrowths of approximately 5-10 nm with integral protuberances with a diameter of about 2 nm.

The hepatitis C virus encodes an epitope which is immunologically identical to the epitope in the genome of the hepatitis C virus, from which originate the circular DNA, recorded therein. The epitope is unique to hepatitis C when compared with other known flaviviruses. The uniqueness of the epitope may be determined by its immunological reactivity with hepatitis C virus and the Russian reactivity known in the field, for example radioimmunological analysis, ELISA analysis, hemagglutination, and some examples of suitable methods of analysis are given in the description.

In addition to the above applicable the following parameters, either individually or in combination with identification of the strain as hepatitis C. Since the strains of the hepatitis C virus evolutionary related, then you should expect that the total homology of the genomes at the nucleotide level will be about 40% or more, preferably about 60% or more, and even more preferably about 80% or more; and in addition that will have a corresponding close sequence at least about 13 nucleotides. The correspondence between the genomic sequence of the proposed hepatitis C virus and the sequence of bases of circular DNA virus hepatitis C CDC/CHl can be determined by methods known in this field. For example, they can be determined by direct comparison of sequential information polynucleotide from the estimated hepatitis C virus and the sequence(s) circular DNA virus hepatitis C, recorded therein. For example, they can be thus determined by hybridization polynucleotide must be used before fermentation with subsequent fermentation odnorazovoe specific nuclease(s), followed by determining the amount of fermentation fragments.

Because of the evolutionary relatedness of strains of hepatitis C virus estimated strains of the hepatitis C virus is identical to its homology to the polypeptide level. In General, strains of hepatitis C virus by more than 40% are homologous, preferably more than 60% of the homologues and even more preferably more than 80% of the homologues at the polypeptide level. Methods for determining homology amino acid sequence known in the field. For example, the amino acid sequence can be determined directly and compared with sequences provided for this in advance. For example, the nucleotide sequence of genomic material intended hepatitis C virus can be identified (usually through an intermediate circular DNA): is encoded in its sequence of amino acids can be identified and appropriate sites compared.

As used in the description, polynucleotide, "derived from" (derived from) the specified sequence, for example, circular DNA virus of hepatitis C, particularly those listed in the examples in Fig. 1-32, or from the genome of the virus is at least 6 nucleotides, is preferably at least a sequence of about 8 nucleotides and more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides, respectively, i.e., homologous or complementary to a segment sequence of nucleotides. Preferably, the sequence of the plot, which is polynucleotide was homologous or complementary sequences, which are unique to the genome of hepatitis C virus whether or not this sequence is unique to the genome of the hepatitis C virus can be determined by methods known to experts in this field. For example, the sequence can be compared with sequences in the data Bank, i.e. the Bank of genes, to determine whether she uninfected host or in other organisms. The sequence can also be compared with known sequences of other viral agents, including those that are known as causative agents of hepatitis, i.e. hepatitis A virus, hepatitis B virus and hepatitis D, and with other members of the family of flaviviruses. Compliance or noncompliance taken sequence to other sequences of possible sequences of nucleic acids by hybridization are known in this area and are discussed below. Cm. also, for example, Maniatis and others (1982). In addition inconsistencies double polynucleotides formed by hybridization, it is possible to determine the known methods, including, for example, the fermentation of a nuclease such as S1, which is specifically sprayway odnorazovye sites in double polynucleotide. Areas from which can be "produced" a typical DNA sequence include, but are not limited to, for example, the region coded by specific epitopes, as well as retranscription and/or netransliruemye area.

The derived nucleotide is not necessarily physically derived from the shown sequence of nucleotides, and can be obtained in any manner, including, for example, chemical synthesis or DNA replication or reverse transcription or transcription, which are based on information given by the sequence of bases in the area (areas), which is polynucleotide. In addition, the combination of sections corresponding to those of known sequence can be modified in ways known in the field corresponding to the intended use.

Similarly, the sequence of the polypeptide or amino, presented on Fig. 1-32, or from the genome of the hepatitis C virus, refers to a polypeptide having an amino acid sequence identical to the sequence of the polypeptide encoded in the sequence or in part, with this part contains at least 3-5 amino acids, and more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is immunologically identical to a polypeptide encoded in the sequence.

A recombinant or derived polypeptide is not necessarily translated from a known nucleic acid sequences, for example sequences shown in Fig. 1-26, or from the genome of hepatitis C virus: it can be obtained in any other way, for example by chemical synthesis or by expression of a recombinant expression system, or isolation from the mutated virus of hepatitis C.

The term "recombinant polynucleotide" used in the description, referring to polynucleotide genomic, semisynthetic, synthetic origin, or derived from circular DNA, and by its origin and processing: (1) is not associated with all or part of polynucleotides, with which it is associated in nature.

The term "polynucleotide" used in the description refers to a polymeric form of nucleotides of any length as ribonucleotides and deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes dantcathy and admonitory DNA and dantcathy and admonitory RNA. It also includes modified, for example by methylation and/or coating, and unmodified forms of polynucleotide.

Used in the description of the term "hepatitis C virus containing a sequence corresponding to circular DNA, means that the hepatitis C virus contains a polynucleotide sequence that is homologous or complementary sequences in the corresponding DNA; the degree of homology or complementarity to a circular DNA will be approximately 50% or more and preferably at least about 70% and even more preferably is at least about 90%. Sequence that is relevant, will consist of at least 70 nucleotides, preferably at least about 80 nucleotides and even more preferably at least about 90 nucleotides in DISTNAME in this area, including, for example, a direct comparison of material sequence described circular DNA or hybridization and fermentation ednonachalie a nuclease with subsequent determination of the amount of fermentation fragments.

The term "purified viral polynucleotide" refers to the genome of the hepatitis C virus, or its fragment, which is substantially free, i.e. contains less than 50%, preferably less than 70% and even more preferably less than 90% of the polypeptide, which viral polynucleotide associated in nature. The technology of purification of viral polynucleotides from viral particles known in the field and includes, for example, the gap particles chao genotype agent and separation of polynucleotide(s) and polypeptides ion exchange chromatographia, chromatography on affinity and sedimentation according to density.

The term "purified viral polypeptide" refers to a polypeptide of hepatitis C virus, or its fragment, which is substantially free, i.e. contains less than 50%, preferably less than 70% and even more preferably less than 90% of the cellular components with which the viral polypeptide is naturally associated. Methods for purification of viral polypeptides are known in this area and examples of these mleok", "cell culture" and other such terms signify microorganisms or line higher Karioth cells grown as single-celled forms that apply to cells that can be or have been, used as recipients for recombinant vector or other DNA breaks, and include the progeny of the initial cells that were infected with the viral nucleic acid. It should be understood that the progeny of single parent cell may not necessarily be completely identical in morphology or in genomic or total complement of DNA as the original parent, due to accidental or deliberate mutation. The descendants of the parental cell that are sufficiently similar to the parent, must be characterized by the relevant property, such as the availability of nucleotide sequence that encodes a desired protein, is included in the offspring, fall under this definition and are included in the scope of the above mentioned term.

"Replicon" is any genetic element, i.e.

a plasmid, chromosome, virus that behaves as an Autonomous unit of polynucleotide replication within a cell, i.e., capable of replication under its own control.

"Vector" is replicase (synthesis) of the attached segment.

"Control sequence" refers to polynucleotide sequences which are necessary to implement the expression of the encoded sequences with which they are associated. The nature of such control sequences are different depending on the host organism: in prokaryotes, such control sequences generally include promoter, the binding site of the ribosome and terminators; in eukaryotes, generally, such control sequences include promoters, terminators and, in some cases, genes amplifiers. The term "control sequence" refers to the inclusion, as a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example key sequence.

"Operable linked" refers to the position at which the components described are in a relationship, allowing them to perform the function intended. A control sequence "operable associated with the coding sequence, are connected in such a way that expression of the coding sequence is achieved under conditions compatible with the control poletnosti, which encodes the polypeptide; this area may be part of the coding sequence and (total) of the entire coding sequence.

"Coding sequence" is a sequence of polynucleotides, which is transcribed into messenger RNA and/or translated into a polypeptide, being placed under the control of the corresponding regular sequences. The boundaries of the coding sequence defined triggers the broadcast of the codon at the 5'-terminal and stopping the broadcasting of the codon at the 3'-terminal. The coding sequence includes, but is not limited to, messenger RNA, circular DNA and recombinant polynucleotide sequence.

"Immunologically identical with/as" refers to the presence of epitope(s) and polypeptide(s) that are also present and are unique to specific polypeptide(s), usually to proteins of hepatitis C virus Immunological identity can be determined by the binding of the antibody and/or competition in binding; these methods are well known to the average person skilled in the art and are explained below. The uniqueness of the epitope may be determined by computer studies known what anenemy amino acid sequence with other known proteins.

Used in the description, the term "epitope" refers to an antigenic determinant of a polypeptide: the epitope must contain three amino acids in a spatial conformation which is unique to the epitope, usually epitope contains at least five amino acids and more usually contains at least 8-10 amino acids. Methods of determining spatial conformation of amino acids known in the field and include, for example, x-ray crystallography and nuclear magnetic resonance in two dimensions.

The polypeptide is immunologically reactive with an antibody, if it binds to the antibody through the detection antibody specific epitope contained in the polypeptide. Immunological reactivity may be determined by the binding of an antibody, more specifically kinetics of binding antibodies, and/or competitive binding using as a competitor(s) known polypeptide(s) containing the epitope against which the antibody directed. Methods of determining whether a polypeptide reactive with the antibody, known in this field.

Used in the description, the term "immunogenic polypeptide containing an epitope of hepatitis C virus" includes what they means, for example, by chemical synthesis or expression of the polypeptide in a recombinant organism.

The term "polypeptide" refers to a molecular chain of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term does not apply to postexplosion modifications of the polypeptide, such as glycolization, acetylation, phosphorylation and the like.

The term "transformation" used in the description refers to the insertion of exogenous polynucleotide into the host cell, irrespective of the method used for the introduction of, for example, direct uptake, transduction, or f-crossing. Exogenous polynucleotide can be saved as a at vector such as a plasmid, or alternatively may be integrated into the host genome.

"Processing" used in the description refers to the prevention and/or therapy.

The term "individual" used in the description refers to a vertebrate, in particular species of mammal, and includes, but is not limited to, domestic animals, sports animals, primates and humans.

The term "plus-strand (thread) nucleic acids, the use of the inhabitants of the sequence, complementary to the sequence "plus " thread".

The term "positive filamentous genome of the virus used in the description, is such, in which the genome, or DNA or RNA is ednonachalie and which encodes a viral polypeptide(s). Examples of the positive filamentous RNA viruses include togavirus, corona viruses, retroviruses, picornaviruses and caliciviruses. Also included flavivirus that previously were classified as togavirus. See Fields and Knipe (1986).

Used in the description, the term "antibody containing body component" refers to a component body of the individual that is the source of the antibodies of interest. Antibody containing components of the body, known in the field and include, but are not limited to, for example, plasma, serum, cerebral spinal fluid, lymph and external secretions of the respiratory, gastrointestinal and urogenital tracts, as well as tears, saliva, milk, white blood cells and myeloma.

Used in the description, the term "purified hepatitis C virus" refers to a preparation of hepatitis C virus, which is separated from cellular components with which it is usually associated virus, and other types of viruses that can prisutstvie, centrifugation and affinity chromatography; methods for obtaining purified hepatitis C virus described below.

II. Description of the invention

When implementing the present invention will be used, unless specifically stated, the conventional methods of molecular biology, Microbiology, recombinant DNA and immunology, known to specialists in these areas. Such methods are fully explained in the literature. See the following sources: Maniatis, Fitsch & Sambrook, Molecular Cloning; A Laboratory Manual (1982); DNA Cloning, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); reduced and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the series. Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London), Scopes, (1987), Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N. Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

All patents, patent applications and publications mentioned above and below, are included in the bibliographic list.

Useful materials and processes solenoceridae, isolated from a pool of circular DNA derived from sequences of nucleic acids that are present in the plasma of infected with hepatitis C, a chimpanzee. This family of nucleotide sequences is not from a human or a chimpanzee, because it produces hybrids genomic DNA from uninfected individuals, neither man nor chimpanzees, because the nucleotides of this family of sequences are present only in the plasma and liver of chimpanzees from infection of hepatitis C virus and because the sequence is not present in the gene Bank. In addition, the family sequence shows no significant homology with sequences contained in the genome of hepatitis C.

The sequence of one member of the family contained in clone 5-1-1, has one continuous openly readable structure that encodes a polypeptide with approximately 50 amino acids. Sera from persons infected with hepatitis C virus, contains antibodies that are associated with the polypeptide, whereas serum from uninfected person does not contain antibodies to this polypeptide. And finally, while serum from uninfected chimpanzees does not contain antibodies to this polypeptide, antithetic not defined in chimpanzees and humans, people with hepatitis a and hepatitis B. According to these criteria, the sequence is circular DNA to viral sequences, the virus causes or is associated with hepatitis b neither A nor B; this sequence is shown in Fig. 1. As discussed above, the sequence of circular DNA in clone 5 - 1-1 differs from the sequences of other separate circular DNA that it contains 28 extra base pairs.

The other identified members of the family of circular DNA, which were selected using as a sample of the synthetic equivalent to the fragment of circular DNA in clone 5-1-1 shown in Fig. 3. Member of a family of circular DNA, which was isolated using a synthetic sequence derived from circular DNA in clone 81, shown in Fig. 5, and the composition of this sequence with the sequence of clone 81 shown in Fig. 6. Other members of the family of circular DNA, including those present in the clone 12f, 14i, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f and 33g, 39c, 35f, 19g, 26g, and 15e are described in section IV.A. The composition of the circular DNA of these clones described in section IV.A.19 and shown in Fig. 32. The composition of the circular DNA shows that is by agreement with the assumption, above that the hepatitis C virus is flaviviruses or flaviopolis virus.

The presence of this family of circular DNA, shown in Fig. 1-32 inclusive, allows the preparation of sample DNA and polypeptides useful in the diagnosis of hepatitis neither A nor B is caused by infection with the hepatitis C virus, and for mass testing of donor blood and blood recipients and blood products for HIV. For example, sequences it is possible to synthesize DNA oligomers with 8-10 nucleotides or more, are useful as hybridization assays to detect the presence of viral genome, for example in the serum of persons suspected of carrying the virus, or to check the patient's blood for the presence of virus. The family of sequences circular DNA also allows for the design and preparation of specific polypeptides of hepatitis C virus, are useful as diagnostic tools to detect the presence of antibodies formed by the hepatitis neither A nor b Antibodies to purified polypeptides derived from circular DNA, can also be used to detect viral antigens in infected individuals and in the blood.

The knowledge of these sequences circular DNA makes possible the Rus hepatitis C, and also to generate antibodies, which in turn can be used to protect from disease and/or treatment of people with hepatitis C individuals.

In addition, the family of sequences circular DNA allows further definition of the properties of the genome of hepatitis C virus Polynucleotide sample derived from these sequences, can be used to test pools circular DNA on additional overlapping sequences circular DNA, which in turn can be used to obtain sequences overlapped genetic code. Despite the fact that the genome is divided into segments and the segments lacking in the overall sequence, this method can be used to obtain the sequence of the whole genome. However, if the genome is divided into segments, other segments of the genome can be obtained by repeating the procedure of serological screening of a lambda gt11 used for selection of clones described here circular DNA, or alternative allocation of the genome of the treated particles of hepatitis C virus

The family of sequences circular DNA and polypeptides derived from these sequences, as well as A(s) BB no A, no B. for Example, antibodies directed against epitopes of hepatitis C virus contained in the polypeptides obtained from circular DNA can be used in methods based on affinity chromatography for isolation of the virus. Alternative antibodies can be used to identify viral particles, selected by other methods. Viral antigens and genomic material selected viral particles can then be investigated further to determine their characteristics.

Information obtained from further studies of the genome(s) of the hepatitis C virus, as well as further characteristics of the antigens of the hepatitis C virus and characteristics of the genome allow the design and synthesis of additional samples and polypeptides, and antibodies that can be used for diagnosis, prevention and treatment of hepatitis neither A nor B, induced by hepatitis C virus, and for screening of infected blood and related blood products.

The availability of samples for hepatitis C, including antigens and antibodies, and polynucleotides derived from the genome, from which it received the collection of sequences of circular DNA, allows the development of tissue culture systems, which will be the main apologymove treatment, based on antiviral compounds, which mainly inhibit replication or infection of hepatitis C virus

The method used for identification and isolation of the etiologic agent of hepatitis neither A nor B is new, and it can be applied for identification and/or selection is still non-characterized agents that contain a genome that are associated with various diseases, including diseases caused by viruses, viroids, bacteria, fungi and parasites. In this method, a pool of circular DNA was generated from the nucleic acids present in the infected tissue of an infected individual. Pool was created in a vector that allows the expression of polypeptides encoded in circular DNA. Clones of host cells containing the vector, which produces an immunologically reactive fragment of the polypeptide etiological agent were selected immunological screening products of expression of pool with the antibody containing body component from another individual previously infected suspected pathogen. The operation method of the immunological screening included the interaction of the products of expression vectors containing circular DNA antibody containing comp what product(s) expression and antibodies of the second infected individual. The selected clones were subjected to further immunological screening interaction of their products expression with the antibody contained in the components of the body other individuals infected with the putative agent, and with control individuals, uninfected alleged agent, and determined the formation of complexes of antigen - antibody with antibodies from infected individuals; and allocated vectors containing circular DNA, which encode polypeptides immunologically interacting with antibodies from infected individuals and individuals suspected infection, but not with control individuals. Infected individuals used to build a pool of circular DNA and immunological screening does not need to be the same species.

Selected circular DNA as a result of this method and the products of their expression and antibodies directed against the products of expression, are useful for characterizing and/or capture the etiologic agent. As described in more detail below, this method is successfully used for the selection of a family of circular DNA derived from the genome of hepatitis C virus

II.A. Obtaining a sequence to the second high titer virus, i.e., at least 106infectious doses chimpanzees/ml (IDS/ml) was used for isolation of viral particles; nucleic acids isolated from these particles, was used as matrix when designing a pool of circular DNA to viral genome. The process of separating particles of the proposed hepatitis C virus and design of the pool circular DNA in lambda gt11 described in section IV.A.1. Lambda gt11 is a vector, which was specially developed for the expression of the built-in circular DNA as (condensed) polypeptides merge with beta-galactosidase and for screening a large number of recombinant phage specific anticorodal derived against a specific antigen. Pool lambda gt11 circular DNA produced from a pool of circular DNA containing circular DNA with an average size of approximately 200 base pairs, researched encoded epitopes that need to communicate specifically with sera obtained from patients who have had previous domestic hepatitis neither A nor B. Huynh, T. V. et al., (1985). Was sifted about 106phages and identified five positive phages purified and then tested for specificity of binding with sera from different people and chimpanzees, before INFI is a crank man. This link seemed election for sera of patients with previous infection with hepatitis b neither A nor B as seven sera of normal donor blood showed no such link.

The sequence of circular DNA in recombinant phage 5-1-1 was determined and is shown in Fig. 1. The polypeptide encoded by this clone circular DNA, which is in the same translational structure as N-terminal beta-galactosidase part of the molecule (condensed) polypeptide fusions, shown above the nucleotide sequence. This translational openly readable structure, therefore, encodes the epitope(s), specifically recognized by serum of patients after infection hepatitis neither A nor B.

The presence of circular DNA in recombinant phage 5-1-1 allowed the selection of other clones containing additional segments and/or alternative segments of circular DNA to viral genome. The pool is circular DNA lambda gt11, described above, was investigated using synthetic polynucleotide derived from the sequence of clonal circular DNA 5-1-1. This screening gave the other three clones that were identified as 81, 1-2, 91; circular DNA containing the four independent clones are shown in Fig. 2, in which homologues are indicated by vertical lines. The sequence of nucleotides present unique clones 5-1-1, 81 and 91, shown in small letters.

Clonal circular DNA present in the recombinant phage in the clones 5-1-1, 81, 1-2, and 91 are vysokomolochnye and differ only in two sites. First, the nucleotide number 67 in the clone 1-2 is a thymidine, while the other three clones contain sitively residue in this position. This substitution, however, does not change the nature of the encoded amino acids.

The second difference between the clones is that the clone 5-1-1 contains 28 base pairs in its 5'-terminal, which are not present in the other clones. Additional sequence can be 5'-end cloning artifact; the 5'-end cloning artifacts commonly observed in the products of the methods associated with the circular DNA.

Synthetic sequence derived from the 5'-region and the 3'-section of the ring DNA of hepatitis C virus in clone 81 was used for screening and selection of circular DNA from a pool of circular DNA virus hepatitis neither A nor B lambda gt11 that overlap the circular DNA of clone 81. (Section IV.A.5). Sequence VO2">

Similarly, synthetic polynucleotide based on the 5'-part of the clone 36 was used for screening and selection of circular DNA from a pool of circular DNA virus hepatitis neither A nor B lambda gt11, which overlaps the circular DNA of clone 36 (section IV.A.8). Purified recombinant clone containing the phage circular DNA, which was hybridisierung synthetic polynucleotide sample, was named clone 35, and a sequence of circular DNA virus neither A nor B in this clone is shown in Fig. 8.

When using the selection method of overlapping sequences circular DNA were obtained clones containing additional ascending and descending sequence of circular DNA virus of hepatitis C. the Selection of these clones are described below in section IV.A.

Analysis of nucleotide sequences of circular DNA virus hepatitis C in selected clones shows that composite circular DNA encoded in the selected clones, contains one long section openly readable patterns. In Fig. 26 shows the sequence composition of DNA from these clones together with the polypeptide of the proposed hepatitis C virus encoded in it.

Description of the method of rehabilitationist (and their complements) are presented in the description, and sequence, or any part of them can be obtained by synthetic methods or combination of methods of synthesis with the restoration of partial sequences using methods similar to the methods described herein.

Strains lambda gt11, replicated from the pool of circular DNA virus hepatitis C and clones 5-1-1, 81, 1-2, 91, deposited in the American collection of type cultures (ATSS) N 12301 Parklawn Dr., Rockville, Maryland 20852, and assigned an incrementing number (in terms of Budapest Treaty) are presented in table. A.

In the adoption and registration of the application as a patent of the United States all restrictions on the availability of these deposits will be certainly eliminated and access to the specified depot will be provided in consideration of said application face that is defined for this authorized, specified in 37 CFR 1.14 and 35 USC 1.22. In addition, these deposited strains will be kept for 30 years from the date of Deposit, or five years after the last treatment for the deposited cultures, or within the time of validity of a U.S. patent, if not longer, these and other deposited materials mentioned in the description are only for convenience, not because they are required for the implementation of this Ivanych materials are listed in the bibliographic reference.

The above description of "transition" of the genome in the allocation of overlapping sequences circular DNA from a pool of lambda gt11 of hepatitis C virus provides one method, which can be selected circular DNA corresponding to the entire genome of the virus of hepatitis C. However, in addition to contained in the description information, there are other methods of extraction circular DNA that is obvious to the experts in this field. Some of these methods are described in section IV.A below.

II.B. Getting viral polypeptides and their fragments

The presence of ring sequences of DNA or isolated using DNA sequences of Fig. 1-32, as discussed below, as well as sequences of circular DNA in these figures enables the construction of expression vectors encoding antigenic active sites of the polypeptide encoded in one of the threads. These antigenic active areas can be obtained from an external cover or membrane antigens, or core antigens, including, for example, polynucleotides proteins, polynucleotide polymerase(s) and other viral proteins required for replication and/or Assembly of viral particles. Fragments encoding the desired polypeptides derived from konektory can for example, to contain part of the sequences of the merger, such as beta-galactosidase or peroxydisulfate, preferably peroxydisulfate. Methods and vectors useful for obtaining polypeptides containing sequences merge peroxydisulfate described in the publication of the European patent office N 0196056 from 1 October 1986. Vectors, encoding the polypeptides of the fusion (synthetic polypeptides) peroxydisulfate and polypeptides of hepatitis C virus, i.e., neither A nor B5-1-1neither A nor B81and C100-3, which is encoded in composite circular DNA virus hepatitis C, described in section IV.B.1, IV. B. 2 and IV.B.4, respectively. Any desired portion of the circular DNA of hepatitis C virus containing a publicly readable structure in one of the sensitive filaments can be obtained as a recombinant polypeptide, such as a Mature protein, or fusion protein; alternatively, the polypeptide encoded in the circular DNA can be obtained by chemical synthesis.

DNA encoding the desired polypeptide or fused or Mature form, containing or not containing a signal sequence to allow the secretion may be Legerova in the expression vectors, suitable for any traditional hose the peptides, and briefly some of the General control system and line of host cells are described in section III. A below. Then polypeptide isolated from lysed cells or culture medium and purified to the extent necessary for its subsequent use. Cleaning can be carried out is known in the field of methods, for example, salt fractionation, chromatography on ion-exchange resins, centrifugation and the like. See, for example, "Methods in Enzymology for different ways of purification of proteins. Such polypeptides can be used as diagnostic tools, or those that contribute to neutralizing antibodies, can be used to prepare vaccines. Antibodies generated against these polypeptides, can also be used as a diagnostic tool or for passive immunotherapy. In addition, as described below in section II.J, antibodies to these polypeptides are useful in the isolation and identification of particles of hepatitis C virus

The antigens of the hepatitis C virus can be isolated from virions of hepatitis C virus Virions can be grown in infected with hepatitis C virus cells in tissue culture or in the infected host.

II.C. Getting animal - typically 8-10 amino acids or less in length. Fragments of at least five amino acids can characterize the antigenic site. These segments may correspond to sites of antigen hepatitis C. Therefore, using circular DNA virus hepatitis C as a basis, you can get the encoded deoxyribonucleic acid short segments of polypeptides of hepatitis C virus recombinant expression, either as fusion proteins or as separate polypeptides. In addition, short amino acid sequences can be conveniently obtained by chemical synthesis. When synthesized, the polypeptide has the exact configuration to give the correct epitope, but is too small to be immunogenic, the polypeptide may be associated with a suitable carrier.

Several methods of receiving such communications known in this field, including education bisulfide linkages using N-Succinimidyl-3-(2 - pyridylthio)-propionate and Succinimidyl-4-(N - maleimidomethyl)-cyclohexane-1-carboxylate supplied by Pierce Company, Rockford, Illinois (if the peptide lacks a sulfhydryl group, this can be achieved by adding a cysteine residue). These reagents form bisulfide the connection between them and pepti the th amino group in another protein. The diversity of these disulfide/ameloblastoma means known. See, for example, Immun. Rev., (1982), 62:185. Other bifunctional linking agents to form thioethers rather than bisulfide communication. Many of these tiefenrausch tools are commercially available and include reactive esters of 6-multimediaphoto acid, 2-bromoxynil acid, 2-todokanai acid, 4-(N-maleimidomethyl) -cyclohexane-1 - carboxylic acid, and the like. Carboxyl groups can be activated by combining them with succinimido or with the sodium salt of 1-hydroxyl - 2-nitro-4-sulfonic acid. Listed above list is not exhaustive, and can be used in the modification shown in the list of connections.

Can be used with any carrier that does not itself causes the formation of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, such as functionalized latex of sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids, such as polyglutamine acid, polylysine and the like; copolymers of amino acids and inactive viral casticin, the immunoglobulin molecules, thyroglobulin, ovalbumin, titanosilicates and other proteins known to specialists in this field.

II. D. Obtaining hybrid particles immunogens containing epitopes of hepatitis C virus

The immunogenicity of the epitopes of the hepatitis C virus can also be enhanced by getting their systems mammals or enzyme systems, merged or collected from forming particles of proteins, such as, for example, the system associated with the surface antigen of hepatitis B. Received it epitope of the virus neither A nor B is directly connected to form particles of the protein-coding sequence, producing hybrids that are immunogenic with respect to the epitope of hepatitis C. In addition, all received vectors include epitopes that are specific to the hepatitis B virus, with various degree of immunogenicity, such as, for example, pre-S peptide. Thus, particles built of forming particles of the protein, which include sequences of hepatitis C virus, immunogenic in relation to hepatitis C and hepatitis B.

The surface antigen of hepatitis was found, was formed and assembled into particles in S. cerevisiae (P. Valenzuela et al., 1982), as well as AET immunogenicity Monomeric subunit. The Assembly may also include immunodominant epitope of the surface antigen of hepatitis B containing 55 amino acids pripoverhnostnogo (pre-S) plot. (Neurath et al., 1984). The Assembly of particles of pre-S surface antigen hepatitis B, synthesized in yeast, as described in published European patent office N 174444 on March 19, 1986; hybrids comprising heterologous viral sequences for enzyme expression, as described in published European patent office N 175261 dated March 26, 1986, Both applications are owned by the present applicant and described in the bibliographic reference. These assemblies of particles can also be carried out in mammalian cells such as Chinese hamster egg using SV 40-digidrofolyatreduktazy vector (Michelle et al., 1984).

In addition, the parts forming the particles of the protein coding sequence can be replaced with codons encoding the epitope of hepatitis C virus In the substitution sites are not required to mediate the aggregation of elements (items) for the formation of immunogenic particles in yeast or mammals, can be erased, thereby eliminating additional antigenic localization of hepatitis B virus from competition with epee immunogenic polypeptides, obtained from circular DNA virus of hepatitis C, as well as from sequences of circular DNA in Fig. 1-32 or from the genome of hepatitis C virus to which they relate. The observed homology between hepatitis C virus and flavivirus gives information related polypeptides that are most likely suitable for vaccines in their effectiveness, as well as regions of the genome in which they are encoded. The overall structure of the genome flavivirus described by Rice et al., 1986 RNA genome flavivirus, apparently, is only virusspecific strain of the messenger RNA and translated into three viral structural protein, i.e., C, M and E, as well as in two major non-structural protein, NV4 and NV5 and complex number of smaller non-structural proteins. It is known that the major neutralizing epitopes for flaviviruses in the envelope E protein (Roehrig, 1986). Corresponding E-gene of hepatitis C virus and polypeptide encoding plot can be predicted based on homology with flaviviruses. Thus, the vaccine may consist of recombinant polypeptides containing epitope E virus hepatitis C. These polypeptides can be produced in bacteria, yeast or mammalian cells, or alternatively can be isolated from viral preparity antibodies to the virus of hepatitis C. Thus, polypeptides containing epitope E, C and M, can also be used separately or in combination in the vaccines of hepatitis C virus

In addition to the above, it was shown that immunization of non-structural protein 1 (NS1) leads to protection against yellow fever (Schlesinger et al. , 1986). This is true even if the immunization does not lead to the formation of the neutralized antibody. Thus, especially with this protein seems vysokomotivirovan among flaviviruses, it's better than the NS1 of hepatitis C virus protects against infection with hepatitis C. in Addition, it also shows that unstructured proteins can protect against viral pathogenicity, even if they do not affect the productivity of neutralizing antibodies.

In view of the aforesaid polyvalent vaccine against hepatitis C virus can consist of one or more structural proteins and/or one or more nonstructural proteins. These vaccines can contain, for example, recombinant polypeptides of hepatitis C virus and/or polypeptides isolated from virions. In addition you can use inactivated hepatitis C virus in vaccines; inactivation can be carried out upon receipt of virusler, the treatment with organic solvents or detergents, or by treatment with formalin. In addition, vaccines can be derived from attenuated strains of hepatitis C virus Receiving attenuated strains of hepatitis C virus are described below.

It is known that some proteins flaviviruses contain vysokokontsentrirovannye areas, i.e., it is expected some immunological cross-reactivity between hepatitis C virus and other flaviviruses. It is possible that shared epitopes between flaviviruses and hepatitis C virus will cause the formation of protective antibodies against one or more disorders, due to these pathogenic agents. Thus, it is possible to design multipurpose vaccines based on this knowledge.

Getting the vaccine containing the immunogenic polypeptide(s) as active ingredients, known to specialists in this field. Typically, such vaccines are prepared in the form of preparations for injection or in the form of liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The drug may be emulsified or protein encapsulated in liposomes. Active imimimi with the active ingredient. Suitable fillers are, for example, water, saline, dextrose, glycerol, ethanol and the like and combinations thereof. In addition, if necessary, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying means, the pH of the buffer means and/or additives that increase the efficacy of the vaccine. Examples of adjuvants which may be effective include, but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L - threonyl-D-isoglutamine, N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetyl-muramyl-L - alanyl-D-isoglutamine-L-alanine-2-(1'-2' -dipalmitoyl-sn - glycero-3 - hydroxyrisperidone)-ethylamine (CGP 19835A, hereinafter referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, dimycolate trehalose and cell membrane skeleton (MPL + TDM + CWS) in a 2% emulsion of squalene in tween 80. The effectiveness of adjuvant can be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an antigenic sequence of the hepatitis C virus, obtained from administration of this polypeptide in vaccines which are also included different is induced, or intramuscularly. Additional forms of medical preparations suitable for administration by other routes include suppositories and, in some cases - forms for administration orally. Suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories can be obtained from mixtures containing the active ingredient in the range from 0.5 to 10%, preferably 1-2%. Form preparations for oral administration include such commonly used excipients, such as mannitol pharmaceutical purity, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate and the like. These compositions give the form of solutions, suspensions, tablets, pills, capsules, forms with prolonged release of the active ingredient or powders and contain 10-95% of active ingredient, preferably 25-70%.

Proteins can be prepared as vaccines in neutral form or in salt form. Pharmaceutically acceptable salts include the acid additive salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, chloride-hydrogen or phosphoric acids, or such authority is passed carboxyl groups, can also be derived from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

II.F. Dosing and administration of vaccines

The vaccine was introduced in a way compatible with the dosage forms of drugs, and in such quantities that would be diagnostically and/or therapeutically effective. The amount of injected vaccine, which is typically in the range of 5 to 250 micrograms of antigen per dose, depends on the subject to be treated, the functional activity of the immune system of a subject to synthesize antibodies, and the degree of desired protection. The exact amount of the active ingredient required for introduction may depend on the opinions of a treating physician and can be strictly individual for each subject.

The vaccine may be prescribed for administration in a single dose, or preferably in a multiple dose. When assigning multiple-dose primary course of vaccination may conclude 1-10 separate doses, followed by other doses in a known interval of time required for maintenance and/or the eskers(a) - after a few months. The dosage will also be, at least partially, dependent on the opinions of a treating physician.

In addition, the vaccine containing the immunogenic antigens (antigen), hepatitis C virus, can be introduced in combination with other immune-regulating means such as immunoglobulin.

II.G. Antibodies against epitopes of hepatitis C virus

Immunogenic polypeptides, obtained as described above was used to produce antibodies, both polyclonal and monoclonal. If you need a polyclonal antibody, a selected mammal (i.e., mouse, rabbit, goat, horse and so on) were immunized immunogenic peptide bearing the epitope(s) of hepatitis C virus Serum immunized animal is collected and processed by the known methods. If serum containing polyclonal antibodies to an epitope of hepatitis C virus, contains antibodies to other antigens, the polyclonal antibodies can be purified

immunoaffinity chromatography. Methods of producing and processing polyclonal antisera known in this field, see, for example, Mayer and Walker 1987

Alternative polyclonal antibodies can be isolated from mammals is of atita C serum from uninfected individual, based on affinity chromatography and the use of the polypeptide fusion peroxide dismutase or polypeptide that is encoded in the circular DNA of clone 5-1-1 presented in section V. E.

Monoclonal antibodies directed against epitopes of the hepatitis C virus can also be easily obtained by a person skilled in the art. The General methodology of obtaining monoclonal antibodies hybridization are well known. Vital cell line producing antibodies can be generated by cell fusion, and also other methods, such as direct transformation of B lymphocytes oncogenic DNA, or transfection with a virus of Epstein-Barr. Cm. also M. Schreier et al., 1980; Hammerling et al., 1981; Kennett et al. 1980 ; see also U.S. patents NN 4341761, 4399121, 4427783, 4444887, 4466917, 4472500, 4491632 and 4493890. The list of monoclonal antibodies produced against epitopes of the hepatitis C virus can be tested for various properties, i.e., for isotype, affinity for the epitope, and so forth.

Antibodies, both monoclonal and polyclonal, which are directed against epitopes of the hepatitis C virus, especially useful in the diagnosis and neutralized antibodies useful in passive immunotherapy. Monoclonal antibodies, in particular, can be floor is ins who are the "internal image" of the antigen of the infectious agent from which the desired protection. See, for example, Nisonoff A. et al., 1981, and Dreesman et al., 1985

Metaly generating antihistimine antibodies known in the field. See, for example, Grzych (1985), MacNamara et al. (1984) and Uytdehaag et al. (1985). These antihistimine antibodies may also be useful in the treatment of hepatitis neither A nor B, but also to determine immunogenic sites of antigens of hepatitis C.

II.H. Diagnostic oligonucleotide samples and kits

Using disclosed in the application of a dedicated circular DNA of hepatitis C virus based, including sections of circular DNA, shown in Fig. 1-32, it is possible to obtain oligomers of approximately 8 nucleotides or more, which form hybrids with the genome of hepatitis C virus and are useful for the identification of the viral agent(s), further characterization of the viral genome(s), as well as in the determination of the virus(s) in diseased individuals. Samples on polynucleotide (natural or derived) have a length that allows the determination of a unique viral sequences by hybridization. While 6-8 nucleotides may be suitable for the length, sequence 10-12 of the nucleus is the century Preferably, these sequences were obtained from areas in which there is no heterogeneity. These samples can be obtained using conventional methods, including methods for automated synthesis of oligonucleotides. Among the useful samples, for example, can be called a clone 5-1-1 and additional clones disclosed in the description, and various oligomers, useful in testing pools circular DNA, is presented below. A complement to any unique portion of the genome of hepatitis C virus will be satisfactory. For use as samples desirable full complementarity, although it may be optional, because the length of the fragment increases.

To use these samples as a diagnostic process the sample that is to be analyzed, such as blood or serum, if necessary, for extracting the contained nucleic acids. Obtained from a sample nucleic acid can be subjected to gelelectrophoresis or another method of separation by size; alternatively, the sample nucleic acid may be radiografia without separation by size. Then sample mark. Suitable labels, and methods of labeled samples is known in this field and VCLT samples and chemoluminescence samples. Nucleic acid extracted from a sample, the process then labeled probes in hybridization conditions with appropriate precautions. Samples can be obtained completely complementary to the genome of hepatitis C. Therefore, comply with the special conditions of stringency, to prevent false findings. However, strict conditions must be met only if the samples are complementary to parts of the viral genome that have lost their heterogeneity. The stringency conditions of hybridization is determined by several factors during the hybridization and washing procedure, including temperature, ionic strength, length of time and the concentration of formamide. These factors are described, for example, Maniatis, T., (1982).

Typically, you should expect that the sequence of the genome of hepatitis C virus will be present in the serum of infected individuals at relatively low levels, i.e., about 102-103sequences in ml This level may require the use of methods of amplification with hybridization studies. Such methods are known in this field. For example, the "Bio-Bridge" Enzo biochemical Corporation uses end deoxynucleotidyl Shuttle service is associated to the nucleotide sequence of the target, and then in a modified Biotin poly-A sequence. Application PCT N 84 (03520) and the application for the European patent N 124221 describe research on DNA hybridization, in which: (1) analyte renaturierung breakdown ednonachalie DNA, which is complementary to the labeled enzyme to the oligonucleotide; and (2) received tailed duplex's hybrid in enzyme labeled oligonucleotide. The application for the European patent N 204510 describes research on DNA hybridization, in which the analyzed DNA in contact with the sample, having a tail, such as a poly-dT tail, increased thread which has a sequence, which it hybridises to the tail of the sample, such as a sequence of poly-A, and which is able to bind multiple labeled threads. A particularly desirable technique may first involve an increase of the target sequences of hepatitis C virus in the sera of about 10,000 times, i.e. up to about 106sequences per milliliter. This can be accomplished, for example, according to the method of Saiki et al., (1986). Increased sequence(s) can then be determined using hybridization analysis, which is described in the continuing application U.S. patent N 2300-0171, registered October 15, 1987 in the name of the successors to , to determine the sequence at the level of 106/ml, using polymers of nucleic acids that are associated with the analyzed ednonachalie nucleic acid and which also bind many admonisheth labeled oligonucleotides. The analysis phase separation of a suitable solution, which can be used with a labeled polynucleotide probes, and methods of sampling are described in the publication of European patent N 225807, published on 16 June 1987, owned by the assignees of the present application, and which are listed in the bibliographic reference.

Samples can be Packed into a diagnostic kit. Diagnostic kits include DNA samples, which can be too efficient; alternative DNA samples may not have labels and ingredients for the introduction of labels can be included in the kit. The kit may contain securely packaged reagents and materials needed for the particular hybridization, such standards and instructions for conducting the test.

II.I. Immunological analysis and diagnostic kits

Polypeptides that interact immunologically with antibodies serum containing hepatitis C virus, for example, polypeptides derived from the clones described in section IV.A, or encoded the C in these polypeptides (see, for example, section IV.E) are useful for conducting immunoassay for detection of antibodies against hepatitis C virus, or the presence of the virus and/or viral antigens in biological samples, such as samples of blood or serum. Implementation immunological analysis has to deal with variations and their diversity is known to specialists in this field. For example, in immunological analysis, you can use one viral antigen, such as a polypeptide, from any of the clones containing circular DNA virus hepatitis C, described in section IV.A, or from composite circular DNA obtained from circular DNA in these clones, or from the genome of the hepatitis C virus, which received the circular DNA of these clones; alternative in immunological analysis, you can use a combination of viral antigens derived from these sources. You can use, for example, a monoclonal antibody directed against a viral antigen, monoclonal antibodies, directed against different viral antigens, polyclonal antibodies directed against the same viral antigen or polyclonal antibodies directed against various viral and type sandwich. The analyses can also be used, for example, a rigid substrate, or may be using thus. Most analyses include the use of labeled antibodies or polypeptides; a label can be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Analyses in which amplify the signals from the sample, also known and examples of such analyses is the use of Biotin and avidin, and immunological assays with labeled enzymes and intermediate tests, such as ELISA analysis.

The model flavivirus for hepatitis C virus allows predictions about the likely diagnostic localization of epitopes for structural proteins of Periana. Plots C, pre-m, M and E probably all contain epitopes considerable potential for detection of viral antigens and in particular for the diagnosis. Similarly, localization of non-structural proteins, it is expected that contain important diagnostic epitopes (i.e., 5 NS, encoding the estimated polymerase, and NS 1, encoding the estimated complimentative antigen). Recombinant polypeptides or viral polypeptides that include epitopes from these specifications the infected patients.

In addition, antibodies directed against proteins E and/or M can be used in immunological assays to determine viral antigens in patients with hepatitis C virus, which causes hepatitis b neither A nor B, and infections of the blood donors. In addition, these antibodies can be extremely useful in identifying donors and patients in the acute phase.

Kits useful for immunoassay and contain appropriate labeled reagents, and equipped with necessary materials, including packaging polypeptides according to the invention, containing epitopes of hepatitis C virus, or analysis, directed against epitopes of hepatitis C virus in suitable containers, along with other reagents required for analysis, as well as a number of instructions for carrying out analysis.

II. J. Further characterization of the genome of hepatitis C virus, virions and viral agents, using samples obtained from circular DNA to viral genome

Sequence information circular DNA of hepatitis C virus in the clones described in section IV.A and shown in Fig. 1-32, inclusive, may be used to obtain further information about the sequence of the genome of hepatitis C, and d is outermost, including the nature of the genome, the structure of the viral particles and the nature of antigens, of which it is composed. This information, in turn, can lead to additional polynucleotide samples, polypeptides derived from the genome of the hepatitis C virus, and antibodies directed against epitopes of hepatitis C virus, which will be useful for the diagnosis and/or treatment of hepatitis neither A nor B is caused by hepatitis C virus

The sequence information circular DNA in the above-mentioned clones useful for the preparation of samples for the allocation of additional sequences circular DNA obtained from the as yet unidentified regions of the genome(s) of the hepatitis C virus, which received the circular DNA clones, in section IV. A. for Example, labeled sample containing a sequence of about 8 or more nucleotides, and preferably 20 or more nucleotides, which are obtained from areas adjacent to the 5'-endings or 3'-end family of sequences circular DNA virus hepatitis C, shown in Fig. 1, 3, 6, 9, 14 and 32, can be used to identify overlapping sequences circular DNA from pools of circular DNA virus of hepatitis C. These sequences that overlap the circular DNA is that of circular DNA in the above-mentioned clones does not occur, can be used for the synthesis of samples to identify other overlapping segments, which do not necessarily overlap the circular DNA in the clones described in section IV.A. Despite the fact that the genome of the hepatitis C virus is divided into segments and common sequences missing segments, it is possible to trace the complete viral genome(s) using the method of allocation of overlapping circular DNA obtained from the viral genome(s). Although unlikely, if the genome is segmented genome, which lacks a common sequence, but the sequence of the genome can be identified by serological screening of pools of circular DNA virus hepatitis C lambda gt11, as used for the selection of clone 5-1-1, identify sequences in isolates circular DNA and use a dedicated circular DNA to highlight overlapping fragments using the method described for selecting and determining the sequences of the clones are given in section IV.A. An alternative characterization of the genomic segments may come from the viral genome(s) isolated from purified virus particles of hepatitis C. Methods of cleaning particles of the hepatitis C virus and defining them in the cleaning process shown in he process which can be used shown in the example of section IV.A.1. Selected genomic segments can then be cloned and determined their sequence. Thus using the information here, you can spend the cloning and sequencing of the genome(s) of the hepatitis C virus, regardless of its nature.

Methods of pooling circular DNA known in the field and discussed below and is discussed above; the method of constructing pools circular DNA of hepatitis C virus in lambda gt11 described below in section IV.A. However, the pools circular DNA, useful for sampling nucleic acids, can also be established in other vectors known in this field, for example a lambda gt10 (Huynk et al., 1985). Circular DNA derived from hepatitis C virus, certain samples obtained from circular DNA in Fig. 1-32 and from samples synthesized from polynucleotides derived from these circular DNA can be isolated from the clone by fermentation of a selected polynucleotide appropriate limiting enzyme(s) and determined its sequence. See, for example, section IV. A. 3 and section IV.A.4, the methods used for isolation and sequence determination of circular DNA virus GE is installed for isolation and sequence determination of circular DNA virus hepatitis C, which overrides the one in the clone 81, and section IV.A.8 and IV.A.9 - to highlight and define the sequence in clone that overlaps another clone (clone 36), which overlaps clone 81.

Sequence information obtained from these overlapping circular DNA virus hepatitis C are useful for identifying regions of homology and heterogeneity in viral genome(s), which may indicate the presence of different strains of the genome and/or populations of defective particles. It is also useful for making hybridization assays to detect the hepatitis C virus or antigens of the hepatitis C virus, or nucleic acid of hepatitis C virus in biological samples, and in the process of selection of hepatitis C virus (described below, using the methods described in section II.G). In addition, overlapping circular DNA can be used to create expression vectors for polypeptides derived from the genome(s) of the hepatitis C virus, which also encode polypeptides that are encoded in clone 5-1-1, 36, 81, 91 and 1-2 in the other clones described in section IV.A. How to create these polypeptides containing epitopes of hepatitis C virus, and antibodies directed against epitopes of hepatitis C virus contained therein, and is of linesta circular DNA virus hepatitis neither A, neither B, contained in the clones 5-1-1, 32, 35, 36, 1-2, 81 and 91 discussed above and below.

Encoded in the family of circular DNA sequence contained in the clones 5-1-1, 32, 35, 36, 81, 91, 1-2 and other clones described in section IV.A are the antigen(s) that contain epitopes that are apparently unique to the hepatitis C virus, i.e., antibodies directed against these antigens are absent in individuals infected with hepatitis A virus or hepatitis B virus, and in individuals not infected with hepatitis C (see serological data presented in section IV.B). In addition, the comparison of sequences of these circular DNA sequences of hepatitis A virus, hepatitis B virus, hepatitis D and genomic sequences in gene Bank shows that there is little homology between these circular DNA and nucleotide sequences of those sources. Thus, antibodies directed against antigens encoded in the circular DNA of these clones can be used to identify virus particles BB-neither A nor B, isolated from infected individuals. B addition they are also useful for isolation of the causative agent(s) of hepatitis b neither A nor B.

H the culture cells by any known method in this field, including, for example, methods based on the size distribution, such as sedimentation, or elimination methods, or methods based on density, such as ultracentrifugation in density gradients, or precipitation agent such as polyethylene glycol, or chromatography on various materials, such as anyone - or cation-exchange materials that bind due to hydrophobicity, and affinity of the colony. In the process of allocating the presence of the hepatitis C virus can be determined by hybridization analysis of the extracted genome, using samples obtained from circular DNA virus hepatitis C described above, or immunological analysis (see section II.I) using as samples of antibodies directed against antigens of the hepatitis C virus encoded in a family of ring sequences of DNA, shown in Fig. 1-32, and directed against antigens of the hepatitis C virus encoded in overlapping sequences circular DNA virus hepatitis C described above. Antibodies may be monoclonal or polyclonal and may be desirable purification of antibodies prior to their use in immunological analysis. The process of purification of polyclonal antibodies, which can be used for antibody, directed against other antigens of hepatitis C.

Antibodies directed against antigens of the hepatitis C virus encoded in the family of circular DNA, shown in Fig. 1-32, as well as antigen encoded in an overlapping circular DNA virus hepatitis C, which is fixed on a solid substrate, useful for selection of hepatitis C virus in immunoaffinity chromatography. Methods immunoaffinity chromatography are known in this field, including methods of fixation of antibodies on solid substrates, so that they retain their selective immunological activity; methods may involve the adsorption of the antibody to the substrate (see , for example, Kurstak in Enzyme Immunodiagnosis, pages 31-37), as well as methods in which antibodies are covalently bound to the substrate. In General, methods similar to those used for covalent binding of the antibodies to a solid substrate, which in General is described in section II.C; however, space group can be included in the bifunctional linking agents in order to localize the binding of antigen remained accessible for antibodies.

In the process of cleaning the presence of the hepatitis C virus can be determined and/or confirmation is tion sequences circular DNA virus hepatitis C, presented on Fig. 1-32, and sequences overlapping circular DNA virus hepatitis C described above. In this case, the fraction is treated under conditions that cause the gap viral particles, for example, detergents in the presence of chelat forming means, and the presence of viral nucleic acid is determined by the hybridization method described in section II.H. Further confirmation that the selected particles are agents that cause hepatitis C, can be obtained by infection of chimpanzees selected viral particles with the subsequent definition of what are the symptoms of hepatitis neither A nor B the result of infection.

Viral particles purified preparation can be investigated further. Purified genomic nucleic acid. Based on its sensitivity to the ribonuclease, and not the desoksiribonukleaza I, it becomes obvious that the virus comprises a genome RNA. Cm. the example in section IV.C.2 below. The number of threads and cyclization or necklace can be defined known in the field of methods, including, for example, the observation under an electron microscope, migration in dense gradients and sediment characteristics. Based on hybridi the tsya, that hepatitis C may include a positive filamentous genome RNA (see section IV.H.1). Such methods are described, for example, Methods in Enzymology. B addition, the purified nucleic acid can be blocked and determined its sequence of known methods, including reverse transcription, because the material of the genome is RNA. Cm. for example, Maniatis (1982) and Glover (1985). The use of nucleic acid derived from viral particles, allows you to monitor the whole genome is segmented or not.

The study of the homology of the polypeptide encoded in continuous openly readable structure of the United clones 14i with at 39c, inclusive (see Fig. 26), indicates that the polypeptide of hepatitis C virus contains regions of homology with the corresponding protein in canned parts of flaviviruses. The example described in section IV.H.3. This discovery has many important ramifications. First, this evidence, combined with the results showing that the hepatitis C virus contains a positive filamentous genome, the size of which is about 10,000 nucleotides, consistent with the assumption that the hepatitis C virus is flaviviruses or flaviopolis virus. In General, the virions flavivirus and it is 1988). Thus, the structural genes encoding polypeptides C, pre-M/M and E can be localized in the 5'-end of the genome at the top in the direction of the clone 14i. In addition, the use of comparisons with other flaviviruses can predict the exact localization sequences encoding these proteins.

The selection of sequences up from the sequences in the clone 14i may be performed in various ways, which, having given in the description of the information will be obvious to the person skilled in the art. For example, the "wandering" of the genome can be used to highlight other sequences, which is the 5'-end to the sequences in the clone 14i, but which overlap this clone; this in turn leads to the isolation of additional sequences. This method is extensively described in section IV below.A. For example, it is well known that flavivirus have conserved epitopes and lots of canned sequences of nucleic acids. Polynucleotide containing conserved sequences, can be used as samples that bind the genome of hepatitis C virus, thus allowing its selection. In addition these scanservice C, presented on Fig. 22, can be used to design the nucleating systems that increase genome sequences up from the sequences in the clone 14i, using the growth response polymerase chain. Such an example is described below.

The structure of the hepatitis C virus can also be determined and the components selected. The morphology and dimensions can be determined, for example, by electron microscopy. Identification and localization of specific viral polypeptide antigens, such as coating or membrane antigens or internal antigens, such as proteins that bind nucleic acids, the core antigens and polynucleotide polymerase(s) can be determined, for example, of whether the antigens as major or minor components of the virus, as well as the use of antibodies directed against specific antigens encoded in the selected circular DNA samples. This information is useful for making vaccines; for example, it may be preferable to enable the foreign antigen in the preparation of the vaccine. Multivalent vaccines can be formed, for example, of the polypeptide derived from the genome encoding the structural protein, for example, b is CLASS="ptx2">

II. K. System cell cultures and systems of animal models for replication of hepatitis C virus

The assumption that the hepatitis C virus is flaviviruses or flaviopolis virus, also offers information about the methods of cultivation of the virus of hepatitis C. the Term "flaviopolis" means that the virus shows a significant number of homology to known conserved areas flaviviruses and that a large part of the genome is one openly readable structure. Metaly growing flaviviruses known to specialists in this field (see, for example, reviews Brinton (1986) and Stollar, V. (1980)). In General, suitable cells or cell culture for the cultivation of the virus of hepatitis C can include cells that support the replication of flaviviruses, such as the following: cell line of monkey kidney (i.e. MK2, VERO) cell line kidney pig (i.e. PS), a line of renal cells of a young hamster (i.e., KSS), line mikrofalowe cells Moray eels (i.e., P388D1, MK1, Mm1), line mikrofalowe human cells (i.e., U-937), peripheral blood leukocytes of man, stuck a human monocytes, hepatocytes and cell lines hepatocytes (i.e., HUH7, HEPG2), embryos and germ cells (i.e. fibroblasts of chicken embryos or cells derived from invertebrates, PR is the turn of cells mosquitoes (i.e., A. Albopictus, Aedes aegypti, Cutex tritaeniorhynchus) or cell line ticks (i.e., RML-14, Dermacentor rumapertus).

Perhaps that will be grown in primary hepatocytes and then infected with hepatitis C; or alternative culture of hepatocytes can be obtained from the liver of infected individuals (i.e., human or chimpanzee). In the latter case, it would be an example of cells that are infected in vivo and intertwined in vitro. In addition can be used various methods of recovery, in order to obtain a cell line derived from cultures of hepatocytes. For example, primary cultures of liver (before and after enrichment of the population) can be merged into a variety of cells to maintain stability. For example, culture can also be infected and transformed with a virus or infected alien transformed genes to obtain permanent and semi-permanent cell line. In addition, for example, cells in cultures of the liver can be fused to the recovered cell lines (i.e., HepG2). Methods merge cells known in the field and include, for example, using the merge agents such as polyethylene glycol, virus Sendan and the virus of Epstein-Barr.

As discussed above, hepatitis C is the t to be implemented methods, known in this area the infection of cells by flaviviruses. These methods include, for example, incubation of cells with viral drugs under conditions that allow virus penetration into the cell. In addition it is possible to obtain viral production by infected cells viral polynucleotide. It is known that togavirus and flavivirus as RNA-containing viruses that are infectious in a variety of vertebrate cell lines (Pfefferkorn and Shapiro, 1974) and in cell lines mosquitoes (Peleg, 1969). Methods infection of tissue culture cells by RNA duplexes, positive filamentous RNA and circular DNA and other DNA) is known in this field and include, for example, methods that use electroporation and the precipitation of DEAE-dextran or calcium phosphate. A rich source of the RNA of the hepatitis C virus can be obtained by carrying out in vitro transcription circular DNA of hepatitis C virus on the complete genome. Transfection of this material or cloned circular DNA of the hepatitis C virus will manifest itself in viral replication and reproduction of the virus in vitro.

In addition to the cultured cells can be used in the system of animal models for viral replication: animal systems where known flavivirus familiar specialcoll chimp and also marmosets and suckling mice.

II.L. Check (screening) antiviral agents for hepatitis C

The presence of cell culture systems and animal models for hepatitis C virus also makes it possible to check the anti-virus agents, which ingibiruet replication of hepatitis C virus, and particularly those agents which mainly contribute to cell growth and increases in their numbers, inhibit at the same time viral replication. These screening methods known to experts in this field. In General, antiviral agents was tested at various concentrations for their action to prevent viral replication in cultures of cells that support viral replication, and then on the inhibition of infectivity or viral pathogenicity (and at the lowest level of toxicity) in the model system of the animal.

Methods and compositions are provided for in the description to identify antigens of the hepatitis C virus and polynucleotides of hepatitis C virus, useful for testing antiviral agents because they represent an alternative and generally more sensitive tools to detect the boiling agent of viral replication than the sockets or test Inga is to quantify viral nucleic acid, produced in cell culture. This can be accomplished, for example, hybridization or competitive hybridization of infected nucleic acids of the cells with labeled polynucleotides of hepatitis C virus samples. For example, antibodies directed against the hepatitis C virus, can be used to identify and quantify antigen(s) of hepatitis C virus in cell culture using an immunological assay, described above. In addition, since it may be desirable to quantify the antigen of the hepatitis C virus in the infected cell cultures competitive analysis, the polypeptides encoded in the circular DNA of hepatitis C virus, as described in the application, helpful for these competitive analyses. In General, recombinant polypeptides of hepatitis C virus obtained from circular DNA virus hepatitis C should be marked and the inhibition of binding of these labeled polypeptides with a polypeptide of hepatitis C virus by antigen produced in the system of culture cells, can be registered. In addition, these techniques are especially useful in cases where the hepatitis C virus may be capable of replication in a cell line without causing their death.

II.M. Poluchenie and/or systems model animals, you can allocate the weakened line of hepatitis C. These lines may be suitable for vaccines or for the selection of viral antigens. Weakened lines can be selected after multiple passages in cell culture and/or animal model. The definition of a weakened strain in infected cells or the individual is carried out by methods known in this field, and may include, for example, using antibodies to one or more epitopes encoded in the hepatitis C virus in the sample, or the use of polynucleotide containing a sequence of hepatitis C virus of at least 8 nucleotides, as a probe. Alternative or in addition attenuated strain can be obtained using the information of the genome of hepatitis C virus given in the description, and the use of recombinant methods. In General you can try to destroy the region of the genome that encodes, for example, a polypeptide related to pathogenicity, but which allows the replication of the virus. In addition, the structure of the genome should allow the expression of the genome, leading to the formation of neutralizing antibodies to hepatitis C. This modified gene can then be used to transform cells, allowing rap is s hepatitis C virus is useful not only for the purposes associated with the production of vaccines, but also as a source of commercial production of viral antigens, because the processing of these viruses will require less stringent protective measures for users involved in getting viruses and/or viral products.

III. Common methods

Common methods used for the extraction of the genome of the virus, obtaining and testing a pool of circular DNA, determining the sequences of the clones, constructing expression vectors, transforming cells, conducting radioimmunological analysis and enzyme linked immunosorbent assay for growing cells in culture, and the like known in the field and lab manuals are available describing these methods. However, for General guidance the following are some sources currently available for carrying out such methods, and materials useful in their implementation.

III.A. The hosts, and control sequences in the expression

Prokaryotic and eukaryotic cells-the hosts can be used for expression of a desired coding sequence, if you use the appropriate control sequence compatible with a specific host. Clearitol include promoters, optionally containing part of the gene-operator, and localization of the binding of ribosomes. The transfer vectors compatible with prokaryotic hosts usually receive, for example, pBR322, a plasmid containing operons, which imparts resistance to ampicillin and tetracycline, and various pUC vectors, which also contain sequences that convey markers of antibiotic resistance. These markers can be used to obtain transformants selection. Commonly used prokaryotic control sequences include betalactamases (penicillinase) and lactose promoter (Chang et al., 1977 ), the tryptophan promoter (trp) (Goeddel et al., 1980) and the lambda-derived PLthe promoter and localization of binding of ribosome N gene (Shimatake et al. , 1981) and the hybrid tac (promotor De Boer et al., 1983) obtained from the sequence of the trp and lac UV5 promoters. Previous systems are particularly compatible with E. coli; if necessary, it can be used other prokaryotic hosts, such as strains of Bacillus or Pseudomonas with the appropriate control sequences.

Eukaryotic hosts include yeast and mammalian cells in culture. Saccharomyces cerevisiae and Saccharomyces carlsbergensis n the vectors are markers, which allow for the selection of successful transformants transfer prototrophic autotrophy mutants or resistance to heavy metals strains wild type. Compatible yeast vectors can apply dvuhstronnih source replication (Broach et al., 1983 ), the combination of CEN3 and ARS1 or other means to ensure replication, such as sequences that lead to the implementation of the corresponding fragment in the genome of the host cell. Control sequences for yeast vectors are known in this area and include the promoters for synthesis of glycolytic enzymes (Hess et al., (1968); Holland et al. (1978)), including the promoter for 3-phosphoglycerate (Hitzeman, 1980). May also include stopping the synthesis of agents (the terminator), such as derived from analazing gene (Holland, 1981). Especially useful control systems are systems that contain glyceraldehyde-3 - phosphatedehydrogenase promoter (GAPDH) or alcohol dehydrogenase regulatory promoter, terminators, also derived from glyceraldehyde-3-phosphate dehydrogenase and, if you need to call secretion, then the parent sequence of the yeast alpha-factor. In addition, the sites of transcriptional regulation United in the body of the wild type. These systems are described in detail in European patent N 120551, published October 3, 1984; European patent N 116201 limited edition, published August 22, 1984, and in the European patent N 164556, published on 18 December 1985; all patents owned by the present applicant and described in the bibliographic reference.

Lines of mammalian cells that are useful as hosts for expression are known in this area and include many lively cell line, obtained from the collection of type cultures of America (ATS), for example HeLa cells, cells of the Chinese hamster ovary (CHO), kidney cells baby hamster (KSS) and a number of other cell lines. Suitable promoters are well known in the field and include viral promoters, such as promoters from simian vacuolating virus (OB 40), sarcoma virus rose (HRV), adenovirus and virus bovine papilloma (EBP). Mammalian cells may also need to target sequences and poly A additional sequences may also be included reinforcing sequences that enhance expression, and consistency, which cause the increase in the number of genes may also be desirable. These sequences are known in the field. Vesti, to ensure the integrity of the respective sequences encoding epitopes of the virus neither A nor B in the genome of the host.

III.B. Transformation

The transformation can be carried out by any known method of introducing polynucleotides into the host cell, including, for example, packaging polynucleotide in the virus and transfer to the cell of the host genetic material of a virus or a direct uptake polynucleotide. The process of transformation depend on the host to be transformed. For example, transformation of host cells E. coli by lambda gt11, containing sequence of the hepatitis BB-neither A nor B, discussed in this section below. Bacterial transformation can be by direct absorption, usually involving the processing of calcium chloride or rubidium chloride (Cohen (1972); Maniatis (1982)). Transformation of yeast direct absorption can be carried out using metol Hinnen et al., 1978 Transformation of mammalian cells by direct uptake may be performed using the method of deposition of calcium phosphate method (Graham and Van der Eb, 1978) or various known modifications to it.

III.With. Construction of vectors

When designing a century the appropriate limiting enzymes in conditions which is usually determined by the manufacturer of these commercially available enzymes. In General, about 1 microgram of plasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 microliter buffer solution by incubation for 1-2 hours at 37oC. After incubation with limiting enzyme protein is removed by extraction with a mixture of phenol-chloroform and DNA allocate precipitation with ethanol. Cleaved fragments can be separated using polyacrylamide or by electrophoresis on Agronova the gel according to the General methods described in Methods in Enzymology (1980), 65:499-560.

The cleavage fragments with sharp ends can become with ragged ends, using polymerase 1 in DNA of E. coli (Maples) in the presence of an appropriate deoxynucleotides, present in the mixture. Can be used as the processing of S1 nuclease, resulting in hydrolysis of any parts ednonachalie DNA.

The ligation can be performed using standard buffer and standard temperature conditions using DNA ligase T4 and triphosphate and less ligase than bonds with ragged edges. If fragments of the vector is used as part ligation mixture, the fragment of the vector often obakin way prevent re-ligation of the vector; alternative resorption limiting enzyme unwanted

fragments can be used to prevent ligation.

Ligation mixture is converted into a suitable cloning hosts, such as E. coli, and effective transformants are selected, for example, resistance to antibiotics, and explore the true structure.

III.D. The construction of the desired DNA sequences

Synthetic oligonucleotides can be obtained using the automated synthesizer, as described (1984). If you wish, synthetic threads can be labeled with phosphorus-32 in the processing of polynucleotide kinase in the presence labeled with phosphorus-32 ATP, using standard reaction conditions.

DNA sequences, including those isolated from pools of circular DNA can be modified by methods including, for example, locally directed mutagenesis, as described by Zoller (1982). In short, subject to modification of the DNA packaged in the phage, as admonitory sequence, and pay in dantcathy DNA polymerase DNA, using as a seed crystal synthetic oligonucleotide complementary to part of the DNA, which must be modifcata DNA is transformed into a phage, support bacteria-host. Cultures of the transformed bacteria, which contain a replication of each thread phage, was placed in a Cup with agar medium to obtain a spot. Theoretically, 50% of new spots contain the phage having the mutated sequence, and the remaining 50% have the original sequence. The replicate spots hybridizer in labeled plastic sample at temperatures and conditions that allow hybridization with the correct thread, but not with the unmodified sequence. Sequences that were identified by hybridization, were identified and formed the clones.

III.E. Hybridization with a break

Pools of DNA can be tested according to the method of Grunstein and Hogness (1975). Briefly, in this method, the DNA to be tested, fixed on nitrocellulose filters was denaturiruet and previously was hybridisable with buffer containing 0-50% formamide, to 0.75 mole of sodium chloride, 75 mm sodium nitrate, 0.02 percent (weight by volume) of each: of bovine serum albumin, polyvinylpyrrolidone and Ficoll sodium (pH 6.5), 0.1% sodium dodecyl sulfate, and 100 micrograms per milliliter of media denatured DNA. The percentage of formamide in the buffer, and the time and temperature conditions the STI. Oligomeric samples that require for more mild conditions of stringency, typically use a low percentage of formamide, require a lower temperature and longer time of hybridization. Samples containing more than 30 or 40 nucleotides such as samples obtained from circular DNA or genomic sequences, usually require higher temperatures, i.e. about 40-42oC and high interest rates, i.e., 50% formamide. During the pre-hybridization buffer was added labeled with phosphorus-32 5'-oligonucleotide sample, and the filters were incubated in this mixture, under conditions of hybridization. After washing the treated filters were subjected to autoradiography to determine the localization of the hybridized sample DNA in the appropriate locations on the original agar plates was used as the source of the desired DNA.

III.F. Design confirmation and sequencing

For traditional constructions of vectors ligation mixture transformed into E. coli strain HB101 or other suitable host and efficient transformants selected by antibiotic resistance or other markers. Plasmids from transformants were then prepared by methods generally a constraint definition of enzyme activity and/or sequencing. Sequencing can be performed by dimethoxymethane Sanger et al. (1977), as further described by Messing et al. (1981), or by the method of Maxam et al. (1980). The problems associated with compression of the bands observed sometimes in areas that are rich in GC, overcome by the use of T-deazaguanosine according to Barr and co-authors (1986).

III.G. ELISA analysis with the enzyme label

Enzyme-linked immunosorbent assay with enzyme label (ELISA) can be used to measure the concentrations of either antigen or antibody. This method depends on the confluence of the enzyme with either antigen or antibody and uses the binding activity of the enzyme as a quantitative marker. To measure antibodies, known antigen is fixed on the solid phase (i.e., micro-plastic or plastic Cup), incubated with dilutions of the test serum, washed, incubated with immunoglobulin, enzyme labeled, and again washed. Suitable enzymes for the introduction of labels known in the field and include, for example, the peroxidase of lozhechnitsy. The enzyme activity associated with the solid phase, measured by the addition of a specific substrate and determine the formation of the product or use of the colorimetric substrate m is smiriti antigen, known specific antibody fixed to a solid phase and add the test material containing the antigen; after incubating the solid phase is washed and add the second enzyme labeled antibody. After wash add substrate and the enzyme activity determined colorimetrically and refers to the concentration of antigen.

IV. Examples

The following examples of implementation of the present invention are purely illustrative and do not limit the scope of the invention. In the light of the present description, numerous implementation in the scope of the invention will be obvious to an ordinary person skilled in the field. Presents procedures, for example, in section IV.A can be optionally repeated, because the methods available for constructing the nucleotide sequences based on the information provided by the invention. The expression described in example E. coli, however, and other systems that are described more fully in section III.A available. Can be obtained for more epitopes derived from genomic structure and used for the formation of antibodies, as described below.

IV. A. Obtaining, allocating and determining the sequences of circular DNA virus hepatitis C-6dilution of this deposited serum, administered intravenously, caused by the hepatitis b neither A nor B the other chimpanzees, her infectious dose for chimpanzees was at least 106/ml, i.e., had a high infectious viral titer.

The pool is circular DNA from pooled plasma with high titers generated in the following way. First was isolated virus particles from plasma: 90 ml aliquot of sample was diluted in 310 ml of a solution containing 50 mm Tris-HCl, pH 8.0, 1 mm ethylenediaminetetraacetic acid, 100 mm sodium chloride. Debris was removed by centrifugation for 20 minutes at 15000 g and 20oC. Viral particles in the obtained supernatant layer obrawalis. To release the viral genome, particle tore suspendirovanie balls in 15 ml of a solution containing 1% sodium dodecyl sulfate, 10 mm ethylenediaminetetraacetic acid, 10 mm ethylenediaminetetraacetic acid, 10 mm Tris-HCl buffer, pH 7.5, containing 2 mg/ml proteinase K, followed by incubation at 45oC for 90 minutes. Nucleic acids were isolated by addition of 0.8 micrograms MS2 1H bacteriophage as a carrier and the mixture was extracted with 4 times with a mixture of phenol-chloroform in the ratio of 1:1 (phenol saturated with 0.5 M Tris-HCl, pH 7.5; 0.1% (by volume) beta-mercaptoethanol, 0.1 percent (weight by volume) oxygenology, followed by extraction with chloroform twice. The aqueous phase was concentrated with 1-butanol before precipitation with 2.5 volumes of absolute ethanol at -20oC and left overnight. Nucleic acid was isolated by centrifugation in a Beckman rotor SW41 at 40,000 rpm for 90 minutes at 4oC and dissolved in water which has been treated with a 0.05% (by volume) diethylpyrocarbonate and autoclaved.

Nucleic acid obtained in the previous operation (< 2 micrograms) was denaturiruet 17.5 mm CH3HgOH; circular DNA was synthesized using the denatured Nokia Huynh (1985 ), except that random seed substituted oligo(dT) 12-18 in the process of synthesis of the first filament of circular DNA reverse transcriptase (Taylor et al., 1976). Received dontcha DNA was fractionated by size on a column of sepharose CL-4B; suirvey material approximately with the average size of 400, 300, 200 and 100 base pairs were deposited in pools circular DNA 1, 2, 3 and 4 respectively. Pool lambda gt11 circular DNA was generated from a pool of N 3 circular DNA.

The pool is circular DNA lambda gt11 generated from a pool of N 3, researched epitopes that could specifically bind to a serum of a patient who has previously undergone hepatitis neither A nor B. Was investigated about 106phages with sera of patients using methods Huynh et al. (1985), except that the associated human antibody was determined by human immunoglobulin in anticigarette sheep, labeled with radioactive iodine-125.

Identified five positive phages that have been cleared. Then five positive phages were tested for specificity of binding to serum 8 different persons already infected with the causative agent of hepatitis neither A nor B, using the same method. Four phage encodes the polypeptide, which inter the initial screening pool of phages. Fifth phage encodes a polypeptide that reacted immunologically with 5 out of 8 tested sera. In addition, this polypeptide was not neuronova immunologically with serum from 7 normal blood donors. So it seems that the clone 5-1-1 encodes a polypeptide that is specifically recognized by immune sera of patients with hepatitis neither A nor B.

IV. A. 2. The sequence of the circular DNA of hepatitis C virus in recombinant phage 5-1-1 and of the polypeptide encoded in the sequence

Circular DNA in recombinant phage 5-1-1 was subjected to determination of the sequences according to the method of Sanger et al. (1977). In essence circular DNA was fragmented using EcoRI, isolated by fractionation by size using gel electrophoresis. Fragments of EcoRI restriction were subcloned into M13 vectors mp18 and mpl9 (Messing, 1983) and studied their sequence using the method of termination of the double detoxicate by the method of Sanger et al. (1977). The resulting sequence is shown in Fig. 1.

The polypeptide encoded in Fig. 1, i.e., encoded in the circular DNA of the hepatitis C virus is in the same structure as that of the N-terminal part of the molecule beta-galactosidase, cat is pitop(s), specifically recognized by sera of patients with humans and chimpanzees, with infections of hepatitis neither A nor B.

IV.A.3. The selection of the overlap of the genetic code circular DNA of hepatitis C virus with circular DNA in clone 5-1-1

The overlap of the circular DNA of hepatitis C virus with circular DNA in clone 5-1-1 was obtained by the study of the same pool of lambda gt11, created as described in section IV.A.1, with synthetic polynucleotides obtained from a sequence of circular DNA virus hepatitis C in clone 5-1-1, as shown in Fig. 1. The sequence of polynucleotide used for screening was:

5'-TCC CTT GCT CGA TGT ACG GTA AGT GCT GAG AGC ACT CTT CCA TCT CAT CGA CTC ACT GGT AGA GGA CTT CCC TGT CAG GT-3'.

Pool lambda gt11 was investigated in this breakdown, using the method described by Huynh (1985). About one in 50,000 clones hybridizes with the sample. Three clone, hybridizers with a synthetic sample were numbered 81, 1-2, and 91.

IV. A. 4. The nucleotide sequence in overlapping circular DNA virus hepatitis C circular DNA in clone 5-1-1

The sequence of nucleotides in the three clones: 81, 1-2, 91 defined, essentially, as in section IV.A.2. The sequence of these clones relative to posledovatelno is certain epitope of hepatitis C virus, and where homologues in nucleotide sequences are indicated by vertical lines between the sequences.

The sequence of the cloned circular DNA of hepatitis C virus of vysokoekonomichny in the overlapped areas (see Fig. 2). However, there are differences in the two plots. Nucleotide 67 clone 1-2 is a thymidine, whereas the other three clones contain sitively residue in this position. It should be noted that the same amino acid is encoded, if this situation is either C or t

The second difference is that clone 5-1-1 contains 28 base pairs that are not present in the other three clones. These pairs of bases occupy the beginning of a sequence of circular DNA in 5-1-1 and denoted by lowercase letters. Based on radioimmunoassay data analysis, which is discussed below in section IV.D, we can assume that the epitope of the hepatitis C virus can be encoded in this 28 p. O. (base pairs) of land.

No 28 base pairs in the clones 31, 1-2, 91, present in clone 5-1-1, may indicate that the circular DNA in these three clones obtained from the defective genomes; alternative 28 p. O. plot may be a limit artifact in clone 5-1-1.

Posledovatelno were not found in the other circular DNA because circular DNA that overlaps these areas hitherto not yet allocated.

Composite sequence of circular DNA virus hepatitis C received from overlapping DNA clones 5-1-1, 81, 1-2, 91, shown in Fig. 3. However, this figure unique 28 base pairs clone 5-1-1 skipped. The figure shows the sequence of the polypeptide encoded in an openly readable structure of composite circular DNA virus of hepatitis C.

IV. A. 5. The allocation of overlapping circular DNA of hepatitis C virus with circular DNA in clone 81

The selection of ring sequences of the DNA of the hepatitis C virus, overlapping circular DNA in clone 81 in the upper part, was carried out as follows. Lambda gt11 pool of circular DNA, obtained as described in section IV. A. 1 was checked by hybridization with synthetic polynucleotide breakdown homologous to the 5'end sequence of clone 81. The sequence of clone 81 shown in Fig. 4. This sequence synthetic polynucleotide used for screening were:

5' CTG TCA GGT ATG ATT GCC CGC TTC CCG GAG 3'.

Methods selection was basically in line described by Huynh (1985), except that Phil>C for 30 minutes each wash. About one in 50,000 clones formed a hybrid with the breakdown. Positive recombinant phage containing circular DNA, giving the hybrid sequence, was isolated and purified. This phage was numbered as the clone 36.

Descending sequence of circular DNA that overlaps the ending carboxyla sequence of circular DNA in clone 81, were selected using a method similar to that described for the selection of the ascending sequence of circular DNA, except that the sample synthetic oligonucleotide received homologous to the 3'end of the sequence in clone 81. Sequence synthetic polynucleotide used for selection of ring sequences of DNA, was the following:

5' TTT GGC TAG TGG TTA GTG GGC TGG TGA CAG 3'.

Positive recombinant phage containing circular DNA, forming a hybrid with this last sequence, was isolated, purified and got the numbering of the clone 38.

IV. A. 6. The nucleotide sequence of the circular DNA of hepatitis C virus in clone 36

The nucleotide sequence of circular DNA in clone 36 was determined essentially by the method, opisannoe 81 and the polypeptide, coded open-readable structure shown in Fig. 5.

Openly readable structure in clone 36 is in the same area as the antigen of the hepatitis C virus encoded in clone 81. Thus in combination openly readable patterns in clones 36 and 81 encode a polypeptide that is part of a major antigen of hepatitis C. the Sequence of this supposed polypeptide of hepatitis C virus and its sequence encoding dunatoi DNA, which is obtained from a combination of openly readable structures circular DNA of hepatitis C virus clones 36 and 81, shown in Fig. 6.

IV.A.7. The sequence of nucleotides circular DNA virus hepatitis C in clone 32

The nucleotide sequence of circular DNA in clone 32 was determined basically as described in section IV.A.2 for the sequence of clone 5-1-1. Sequence data indicate that the circular DNA in recombinant phage clone 32 obtained from two different sources. One piece of circular DNA included 418 nucleotides derived from the genome of hepatitis C virus; another fragment contained 172 nucleotides derived from the genome of the bacteriophage MS2, which was used as a carrier in prematurity sequence of the genome of hepatitis C virus, presented on Fig. 7. A sequence region that overlaps that of clone 81, and the polypeptide encoded openly readable structure, also shown in this figure. This sequence contains one continuous openly readable structure, which is in the same translational structure as the antigen of the hepatitis C virus encoded by clone 81.

IV.A.8. Selection of overlapping circular DNA of hepatitis C virus with circular DNA in clone 36

Allocation of the upstream sequences of circular DNA virus hepatitis C that overlaps the sequence of circular DNA in clone 36, was carried out as described in section IV.A.5, for a sequence that overlaps the circular DNA of clone 81, except that the synthetic polynucleotide was based on the 5'-site of clone 36. Sequence synthetic polynucleotide used for screening was:

5' AAG CCA CCG TGT GCG CTA GGG CTC AAG CCC 3'.

About one in 50,000 clones occurred hybridization with the sample. Selected, purified clone of recombinant phage containing circular DNA, which was hybridisable with this sequence was named clone 35.

IV.A.9. The nucleotide sequence of the ring is essentially so, as described in section IV.A.2. The sequence, its area of overlap with the sequence of circular DNA in clone 36 and the expected polypeptide encoded in clone 36, clone 81, and the clone 32. In Fig. 9 shows the sequence of the longest continuous open reading patterns, which extends through the clones 35, 36, 81 and 32, together with the putative polypeptide encoded therein. This combined sequence was confirmed by using other independent clones circular DNA obtained from the same pool of circular DNA lambda gt11.

IV.A.10. The selection of overlapping circular DNA of hepatitis C virus with circular DNA in clone 35

Allocation of the upstream sequences of circular DNA virus hepatitis C that overlap the sequence of circular DNA in clone 35 was carried out as described in section IV.A.8, for sequences that overlap the circular DNA of clone 36, except that the synthetic polynucleotide was based on the 5'-site of clone 35. Sequence synthetic polynucleotide used for screening was the following:

5' CAG GAT GCT GTC TCC CGC ACT CAA CGT 3'.

About one in 50,000 clones were hybridisable with breakdown. Selected, cleaned the , was named clone 37b.

IV.A.11. The nucleotide sequence of hepatitis C virus in the clone 37b

The nucleotide sequence of circular DNA in the clone 37b was determined basically as described in section IV.A.2. The sequence plot of its overlap with the sequence of circular DNA in clone 35 and the expected polypeptide encoded therein, shown in Fig. 10.

5'-terminal nucleotide of the clone 35 is T (thymine), while the corresponding nucleotide in the clone 37b is adenine (A). The circular DNA of the other three independent clones, which were isolated during the procedure of selection of clone 37b, described in section IV.A.10 were subjected to determination of the sequences. The circular DNA of these clones also contained an adenine at this position. Thus, the terminal 5' thymine in clone 35 may be an artifact of the cloning process. It is known that the artifacts often appear in the 5'-ends of the molecules circular DNA.

Clone 37b obviously contains one continuous openly readable structure that encodes the polypeptide, in the continuation of the polypeptide encoded in an openly readable structure, which extends through the overlapping clones 35, 36, 81 and 32.

IV. A. 12 is of sledovatelnot circular DNA of hepatitis C virus to clone 32 was carried out as follows. First allocated clone of cla using synthetic hybridization sample, which was based on the nucleotide sequence of the circular DNA of hepatitis C virus and the sequence in clone 32. Method of allocation does not differ materially from those described in section IV. A. 5, except that the synthetic sample was as follows:

5' AGT GCA GTG GAT GAA CCG GCT GAT AGC CTT 3'.

Using the nucleotide sequence from clone cla, synthesized a different nucleotide sequence:

5' TCC TGA GGC GAC TGC ACC AGT GGA TAA GCT 3'.

Selection from the pool of lambda gt11 using obtained from clone cla sequence as the samples were given approximately one clone 50,000-positive colonies. Selected purified clone, which formed a hybrid with this breakdown, was named clone 33b.

IV.A.13. The nucleotide sequence of circular DNA in the clone 33b

The nucleotide sequence of the clone 33b was determined essentially as described in section IV.A.2. The sequence plot of its overlap with the sequence of circular DNA in clone 32 and the intended polypeptide encoded therein, shown in Fig. 11.

Clone 33b obviously contains one continuous open, 6, 81 and 32. The polypeptide encoded in clone 33b, is in the same translational structure as that of the polypeptide encoded in the longest open reading these overlapping clones.

IV.A.14. The allocation of overlapping circular DNA of hepatitis C virus with circular DNA clone 37b and circular DNA clone 33b

To highlight the circular DNA virus hepatitis C that overlap the circular DNA clones 37b and 33b, there were used the following synthetic samples obtained from circular DNA in the clones that were used to test pool lambda gt11 using essentially the method described in section IV.A.3. The used samples were:

5' CAG GAT GCT GTC TCC CGC ACT CAA CGT C 3'

and

5' TCC TGA GGC GAC TGC ACC AGT GGA TAA GCT 3',

to identify colonies containing the sequence of circular DNA virus hepatitis C that overlap the sequence of circular DNA in the clone 37b and 33b, respectively. Approximately one out of 50,000 colonies was determined on each trial. The clone containing circular DNA, the ascending and overlapping circular DNA clone 37b was named clone 40b. The clone that contained the descending circular DNA that overlaps the circular DNA clone 33b, was named clone 25s.

the Idov in the circular DNA clone 40b and clone 25c determined essentially so, as described in section IV.A.2. Sequence 40b and 25c, their areas of overlap with the circular DNA clones 37b and 33b and putative polypeptides encoded in them, is shown in Fig. 12 (clone 40b), and Fig. 13 (clone 25c).

5'-terminal nucleotide of the clone 40b is C. However, the circular DNA from five other independent clones, which were selected in the selection process clone 40b described in section IV.A.14, were also subjected to tests for determining sequences. Circular DNA from these clones also contained a nucleotide T at this position. Thus, G can represent a cloning artifact (see the discussion in section IV.A.11).

5'-terminating codon clone 25c is ACT, but the sequence of this area in the clone cla (sequence not shown) and clone 33b is TCA. This difference may also be an artifact of cloning, as you can imagine such 28 extra 5'-terminal nucleotides in clone 5-1-1.

Clones 40b and 25c each obviously contain openly readable structure, which is the length of the continuous open reading patterns in the clones' previously defined sequences. The nucleotide sequence revealed readable structure of the peptide, encoded in it, is shown in Fig. 14. In this figure the potential artifacts are excluded from the sequence and instead shows the corresponding sequence in nikocevic 5'-sites of multiple overlapping clones.

IV. A. 16. The production of composite circular DNA from circular DNA clones 36, 81 and 32

Composite circular DNA virus hepatitis C, C100, was created as follows. First, cut out circular DNA from clones 36, 81 and 32 with EcoRI. The EcoRI fragment of circular DNA from each clone individually cloned in the localization EcoRI vector pGEM3-blue (Promega Biotec). The obtained recombinant vectors that contain circular DNA from clones 36, 81 and 32, were named as pGEM3-blue/36, pGEM3-blue/81 and pGEM3-blue/32, respectively. Suitably oriented recombinant pGEM3-blue/81 was spaziali NaeI and NarI, and large (2850 p. O.) fragment was purified and Legerova small (570 p. O.) purified mixture of NaeI-NarI limiting fragment from the vector pGEM3-blue/36. This composite circular DNA from clones 36 and 81 was used to generate another pGEM3-blue vector containing continuous openly readable structure of hepatitis C virus contained in an overlapping circular DNA in these clones. Hollow PLA is Wali small (580 p. O.) a fragment of the PvuII-EcoRI, selected from the respectively oriented plasmid pGEM3-blue/32? and composite circular DNA from clones 36, 81, and 32 was Legerova an elongated in-line EcoRI vector pSODcf1, which is described in section IV.B.1 and which is used for the expression of clone 5-1-1 in bacteria. Recombinants containing 1270 p. O. EcoRI fragment of the composite DNA of hepatitis C virus (C100) were selected and circular DNA from the plasmids was grown with EcoRI and purified.

IV.A.17. The selection of the nucleotide sequences of circular DNA virus hepatitis C in clones 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c

Circular DNA virus hepatitis C in clones 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c were isolated by the methods of allocating fragments overlapping circular DNA from a pool of circular DNA virus hepatitis C lambda gt11, described in section IV. A. 1. The method used was essentially been the same as those described in section IV.A.3, except that the used samples were obtained from the nucleotide sequence of the last selected clones from the 5'- and 3'-endings United sequences of hepatitis C. the Frequency of clones, hybridizers with samples described below and was approximately one clone 50,000 in each case.

Of a nucleotide sequence number of the Le IV.A.2, except that circular DNA isolated from clone 5-1-1, replaced with circular DNA cut from these phages.

Clone 33c was isolated using the hybridization probe based on the sequence of nucleotides in clone 40b. The nucleotide sequence of clone 40b shown in Fig. 12. The nucleotide sequence of samples used for isolation of clone 33c, was the following:

5' ATC ACG ACC GGG GTG ACA ACA ATT ACC ACT 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 33c, and the overlap with the sequence in clone 40b shown in Fig. 15, which also includes the sequence of amino acids encoded therein.

Clone 8h was isolated using probes based on the sequence of nucleotides in clone 33c. The nucleotide sequence of the sample was:

5' AGA GAC AAC CAT GAG GTC CCC GGT GTT C 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 8h, overlapped with the sequence in clone 33c, and the amino acids encoded therein, shown in Fig. 16.

Clone 7e was isolated using probes based on the sequence of nucleotides in clone 8h. The nucleotide sequence of the sample was:

5' TCG GAC CTT TAC CTG GTC ACG of AGG, encoded in it, is shown in Fig. 17.

Clone 14C was highlighted with a breakdown based on the sequence of nucleotides in clone 25c. The sequence of clone 25C shown in Fig. 13. The sample for the selection of clone 14C had the sequence:

5' ACC TTC CCC ATT AAT GCC TAC ACC ACG GGC 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 14c, its overlap with the sequence in clone 25c and encoded in its amino acid shown in Fig. 18.

Clone 8f was isolated using probes based on the sequence of nucleotides in clone 14C. The nucleotide sequence of the sample was:

5' TCC ATC TCT CAA GGC AAC TTG CAC CGC TAA 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 8f, its overlap with the sequence in clone 14C and amino acids encoded therein, shown in Fig. 19.

Clone 33f was isolated using probes based on the sequences present in the clone 8f. The nucleotide sequence of the sample was:

5' TCC ATG GCT GTC CGC TTC CAC CTC CAA AGT 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 33f, its overlap with the sequence in clone 8f and the amino acids encoded therein, shown in Fig. 20.


5' GCG ACA ATA CGA CAA CAT CCT CTG AGC CCG 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 33g, its overlap with the sequence in clone 33f and encoded in its sequence of amino acids shown in Fig. 21.

Clone 7f was isolated using a probe based on the sequence of nucleotides in clone 7e. The nucleotide sequence of the sample was:

5' AGC AGA CAA GGG GCC TCC TAG GGT GCA TAA T 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 7f, its overlap with clone 7e and the amino acids encoded therein, shown in Fig. 22.

Clone 11b was isolated using probes based on the sequence of clone 7f. The nucleotide sequence of the sample was:

5' CAC CTA TGT TTA TAA CCA TCT CAC TCC TCC 3'.

The sequence of the circular DNA of hepatitis C virus in clone 11b, its overlap with clone 7f and the amino acids encoded therein, shown in Fig. 23.

Clone 14i was isolated using probes based on the sequence of nucleotides in clone 11b. The nucleotide sequence in the sample was:

5' CTC TGT CAC CAT ATT ACA AGC GCT ATA TCA 3'.

The sequence of circular DNA virus hepatitis C in selected using samples based on the sequence of nucleotides in clone 33g. The nucleotide sequence of the sample was:

5' CTC GTT GCT ACG TCA CCA CAA TTT GGT GTA 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 39c, its overlap with clone 33g and encoded in its amino acid shown in Fig. 25.

IV.A.18. The sequence of composite circular DNA virus hepatitis C received from the selected clones containing circular DNA virus hepatitis C

Sequence of circular DNA virus hepatitis C, described above, was built in line, to create a series of composite circular DNA virus of hepatitis C. the Selected clones arranged in a line in the direction 5' to 3', were: 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g and 39c.

The sequence of composite circular DNA virus hepatitis C received from the selected clones, and is encoded in its amino acid shown in Fig. 26.

When creating a composite sequence were taken into account the following sequence heterogeneity. Clone 33c contains circular DNA virus hepatitis C and 800 base pairs, which overlaps the circular DNA clones 40b and 37c. In the clone 33c, and also in five other what nucleotide is alanine. The difference sequences creates obvious heterogeneity in the amino acids encoded in them, which will either Cys or Tyr for guanine or adenine, respectively. This heterogeneity may have important ramifications from the point of view of protein synthesis.

Nucleotide residue No. 2 in the clone 8h circular DNA virus hepatitis C is a thymine. However, as shown below, the corresponding residue in the clone 7e is adenine; moreover, adenine at this position are also found in three other isolated overlapping clones. Thus, timiney balance in the clone 8h may be a cloning artifact. Therefore in Fig. 26 the residue in this position is designated as A (adenine).

3'-terminal nucleotide in the clone 8f circular DNA virus hepatitis C is a guanine. However, the corresponding residue in the clone 33f and the other two overlapping clones is thymine (T). Therefore in Fig. 26 the residue in this position is designated as T.

the 3'termination sequence in clone 33f circular DNA virus hepatitis C is TTGC. However, the corresponding sequence in the clone 33g and the other two overlapping clones is ATTS. Therefore in Fig. 26, a corresponding portion of odnako in clone 33f and the other two overlapping clones corresponding residue is A. Therefore in Fig. 26 corresponding to the area designated as A.

the 3'termination codon clone 14i is AA, while the corresponding dinucleotide in clone 11b and three other clones is TA. Hence, in Fig. 26 specify the remainder of the TA.

The separation of other heterogeneous sequences discussed above.

Testing of composite circular DNA of hepatitis C virus show that it contains one large open-readable structure. This suggests that the viral genome is translated into a large polypeptide that is processed concomitantly or sequentially with the broadcast.

IV.A.19. The selection of the nucleotide sequences of circular DNA virus hepatitis C in clones 12f, 35f, 19g, 26g and 15e

Circular DNA virus hepatitis C in clones 12f, 35f, 19g, 26g and 15e were isolated essentially by the method described in section IV.A.17, except that he used the following sample. The frequency of clones that were hybridisierung the sample was approximately one clone from 50,000 in each case. Nucleotide sequence of the circular DNA of these clones was determined mainly by the method described in section IV.A.2, except that the circular DNA isolated from clone 5-1-1, replacing the circular DNA virus hepatitis C in Fig. 26, was performed using a hybridization sample, based on the sequence of nucleotides in clone 14i. The nucleotide sequence of the sample was:

5' TGC TTG TGG ATG ATG CTA CTC ATA TCC CAA 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 12f, its overlap with clone 14i, and the amino acids encoded therein, shown in Fig. 27.

The selection of clone 35f, which contains circular DNA falling to a circular DNA virus hepatitis C in Fig. 26, was performed using hybridization samples based on the sequence of nucleotides in clone 39c. The nucleotide sequence of the sample was:

5' AGC AGC GGC GTC AAA AGT GAA GGC TAA CTT 3'.

The sequence of clone 35f, its overlap with the sequence in clone 39c and the amino acids encoded therein, are shown in Fig. 28.

The selection of clone 19g was performed using a hybridization sample, based on the 3'sequence of clone 35f. The nucleotide sequence of the sample was:

5' TTC TCG TAT GAT ACC CGC TGC TTT GAC TCC 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 19g, its overlap with the sequence in clone 35f and the amino acids encoded therein, shown in Fig. 29.

The separation of the Nucleotide sequence of the sample was:

5' TGT GTG GCG ACG ACT TAG TCG TTA TCT GTG 3'.

The sequence of the circular DNA of hepatitis C virus clone 26g, its overlap with the sequence in clone 19g and the amino acids encoded therein, shown in Fig. 30.

Clone 15e was isolated using a hybridization sample, based on the 3'sequence of clone 26g. The nucleotide sequence of the sample was:

5' CAC ACT CCA GTC AAT TCC TGG CTA GGC AAC 3'.

The sequence of the circular DNA of hepatitis C virus in the clone 15e, its overlap with the sequence in clone 26g and the amino acids encoded therein, shown in Fig. 31.

The clones described in this section are deposited in the American collection of cell cultures under the name and under the conditions described in section II.A, and registered under the numbers presented in the table. B.

Sequence of circular DNA virus hepatitis C in selected clones described above and arranged in a line to create a sequence of composite circular DNA virus of hepatitis C. the Selected clones arranged in a line in the direction 5'-3' are: 12f, 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g, 39c, 35f, 19g, 26g and 15e.

The sequence of composite circular DNA virus hepatitis is. . 20. An alternative method of allocation sequences circular DNA ascending sequence of circular DNA virus hepatitis C in clone 12f

Based mostly on the 5'sequences of hepatitis C in Fig. 32 received from the circular DNA of hepatitis C virus in the clone 12f, synthesized small synthetic oligonucleotide priming reverse transcriptase and used them to bind with the corresponding sequence in the genomic RNA of hepatitis C virus to initiate reverse transcription of ascending sequences. Initiating a sequence closely related known 5'-end terminal sequence of clone 12f, but much lower to allow for the construction of sequences of samples in an upward direction to the sequences of the seed. Used known methods of seed and clone. The resulting pools of circular DNA was investigated by sequences of ascending to the originating locations (as inferred from the explanation of the sequence in clone 12f). The genomic RNA of hepatitis C virus received either from plasma or samples from the liver of chimpanzees with hepatitis neither A nor B, or from similar samples of a person with hepatitis neither A nor B.


To allocate additional 5'-terminal sequence of the RNA genome of the hepatitis C virus, the product of circular DNA the first stage of reverse transcription, which doubled the RNA matrix, forms a ridge with oligochitosan. This is achieved by incubation of the product from end terminal transferase in the presence of the PAGE. A second round of synthesis circular DNA, which gives the complement of the first thread of circular DNA by using oligohaline as a seed for the reaction of reverse transcriptase. Sources of genomic RNA of hepatitis C virus described in section IV.A.20. Methods of forming processes with end terminal transferase and for the reaction of reverse transcriptase described by Maniatis et al. (1982). Then the products of circular DNA was analyzed and determined their sequence.

IV. A. 22. Alternative methods of cultivation processes for the selection of sequences from the 3'-terminal region of the genome of hepatitis C virus

This method is based on the previously used methods cloning circular DNA RNA containing flavivirus. In this method, RNA is placed in conditions of denaturirovannyj to remove secondary structures at the 3'termination codon, and then grow process with poly-A-polymerase, COI is aliroot, using oligo dT as a seed. Then synthesize the second strand circular DNA and clone products, filter out and determine the sequence.

IV. A. 23. Pooling lambda gt11 circular DNA of hepatitis C virus containing large inserts of circular DNA

The method used to create and test pools lambda gt11 essentially the same as described in section IV.A.1, except that the pool was created from a larger circular DNA, buervenich from column sepharose CL-4B.

IV. A. 24. Pooling circular DNA of hepatitis C virus using synthetic oligomers as the initiators of the reaction

New pools circular DNA of the hepatitis C virus was created from RNA obtained from pooled plasma of infectious chimpanzee, described in section IV.A.1, and the fraction of poly-A+RNA obtained from the liver of an infected animal. Circular DNA was designed basically as described after Gubler and Hoffman (1983), except that the seed for the synthesis of the first filament of circular DNA served two synthetic oligomer, based on the above-described sequence of the genome of hepatitis C virus Seed based on the sequence in clone 11b and 7e, were respectively as follows:

IV. B. the expression of the polypeptides encoded in the circular DNA of hepatitis C virus, and identification of reproduced products as induced antigens of the hepatitis C virus

IV.B.1. The expression of the polypeptide encoded in clone 5-1-1

The polypeptide encoded in clone 5-1-1 (see section IV.A.2 above), was reproduced as a polypeptide fusion with peroxydisulfate. This was done by sublimemovies circular DNA clone 5-1-1, insert in the expression vector pSODcfl (Steimer et al., 1986), as follows.

First, DNA extracted from the vector pSODcfl was treated with BamHI and EcoRI and the following merger was digirolamo linear DNA, created the limiting enzymes:

5' GAT CCT GGA ATT CTG ATA A-3',

3' GA CCT TAA GAC TAT TTT AA 5'.

After cloning, the plasmid containing the insert were isolated.

The plasmid containing the insert was limited EcoRI. Insert the circular DNA of hepatitis C virus in clone 5-1-1 was cut EcoRI and legirovannye in this enzyme EcoRI, arranged in the line of plasmid DNA. The mixture of DNA used for transformation dryto readable patterns, it is shown in Fig. 1. They were identified restrictive mapping and determination of the nucleotide sequence.

Recombinant bacteria of the same clone were induced for expression of the polypeptide peroxydisulfate-neither A nor B5-1-1growing bacteria in the presence of IPTG.

IV.B.2. The expression of the polypeptide encoded in clone 81

The circular DNA of hepatitis C virus contained in clone 81 was reproduced as a polypeptide merge peroxydisulfate-neither A nor B81. Method of obtaining vector encoding the polypeptide of the merger, was similar to the method used to create a vector encoding peroxydisulfate-neither A nor B5-1-1except that the source of circular DNA virus hepatitis C was the clone 81, which was isolated as described in section IV.A.3, and for which the sequence of the circular DNA was determined as described in section IV. A. 4. The nucleotide sequence of circular DNA virus hepatitis C in clone 81, and the estimated amino acid sequence of the polypeptide encoded therein, shown in Fig. 4.

Insert the circular DNA of hepatitis C virus in clone 81 was cut by EcoRI and Legerova in pSODcf1, kotoula to transform E. coli strain D1210. Recombinants with the circular DNA of hepatitis C virus clone 81 in the correct orientation for expression openly readable structure shown in Fig. 4, were identified limiting mapping and determination of nucleotide sequences.

Recombinant bacteria of the same clone were induced for expression of the polypeptide peroxydisulfate-neither A nor B81growing bacteria in the presence of IPTG.

IV.B.3. Identifikacija polypeptide, encoded by the clone 5-1-1 as antigen associated with hepatitis C and hepatitis b neither A nor B

The polypeptide encoded in the circular DNA of hepatitis C virus clone 5-1-1, was identified as an antigen associated with hepatitis neither A nor B by demonstrating that serum chimpanzees and humans infected with hepatitis neither A nor B, immunologically reacted with the polypeptide merge peroxydisulfate-neither A nor B5-1-1that contained peroxydisulfate in its N end and structural 5-1-1 antigen in its C-terminal codon. This was carried out by "Western" Platinium (Towbin et al., 1979), as follows.

Recombinant bacterial strain, transformed with the expression vector coding for the polypeptide peroxydisulfate-neither A nor B5-1-1

Characteristics of the chimpanzee sera used for Western spots, and the results are shown on pictures autoradiographically strips shown in Fig. 33. Nitrocellulose strips containing polypeptides were incubated with sera obtained from chimpanzees in different periods of time during acute infection with hepatitis A or B (line Hutchinson) (lines 1-16), infection with hepatitis A (lines 17-24 and 26-33) and infection of hepatitis B (lines 34-44). Line 25 and 45 show the positive controls, in which the immune spots were incubated with patient serum, used to identify recombinant clone 5-1-1 in the initial screening pool circular DNA lambda gt11 (see section IV.A.1).

Band, see what iania with peroxydisulfate. These antibodies do not show binding only with peroxydisulfate, because it was also included as a negative control in these samples appeared as a band migrating faster than the polypeptide merge peroxydisulfate-neither A nor B5-1-1.

Rows 1-16 in Fig. 33 shows the binding of antibodies in the serum samples of four chimpanzees: sera were obtained just before infection with hepatitis neither A nor B and sequentially during acute infection. As can be seen in the figure, while antibodies that react immunologically to a polypeptide peroxydisulfate-neither A nor B5-1-1was absent in the serum samples obtained before the introduction of the inoculum virus infection hepatitis C and in the early acute phase of infection, all four animals ultimately induced circulating antibodies to this polypeptide on the last part of the acute phase or later during the acute phase. Additional bands observed in immunological stains in the case of chimpanzees number 3 and 4 were due to background binding to proteins of the bacterial host.

In contrast to the results obtained with serum from a chimpanzee infected with hepatitis neither A nor B, an education is m hepatitis A, or three chimpanzees infected with hepatitis B. the Only binding in these cases was the background binding proteins of the bacterial host, which is also infected with the hepatitis C virus samples.

Characteristics of the human sera used for "Western" spots, and the results are shown on pictures autoradiographically bands shown in Fig. 34. Nitrocellulose strips containing polypeptides were incubated with serum obtained from persons in different periods of infection with hepatitis neither A nor B (lines 1-21), hepatitis A virus (lines 33-40) and hepatitis B virus (lines 41-49). Line 25 and 50 indicate positive controls, in which immunological stains were incubated with patient serum used for the initial selection in the pool of lambda gt11 described above. Lines 22-24 and 26-32 show "uninfected controls, in which serum obtained from normal blood donors.

As can be seen in Fig. 34, serum from nine patients with hepatitis neither A nor B, including serum, used for screening the pool of lambda gt11, contained antibodies to part neither A nor B5-1-1polypeptide fusion. Sera of three patients with Her in the future in these patients. It is also possible that the lack of response stems from differences pathogen - virus neither A nor B is responsible for the induction of disease in individuals who had taken nariakira serum.

In Fig. 34 it is also shown that the serum of many patients infected with hepatitis A virus and hepatitis B virus, do not contain antibodies against either A or B5-1-1and that these antibodies are present in sera from normal controls. Although one patient with hepatitis A virus (line 36) obviously contains antibodies against either A or B5-1-1, it is possible that this patient was first infected with the hepatitis C virus, because the prevalence of diseases hepatitis neither A nor B is very high and because it is often asymptomatic.

These serological studies show that circular DNA in clone 5-1-1 encodes epitopes that are recognized by specific sera sick people and animals infected with BB-neither A nor B. In addition, circular DNA does not seem clearly derived from the genome of primates. Hybridization probe prepared from clone 5-1-1 or from clone 81, not it hybridises to the "South" spots of genomic DNA and control of human and chimpanzee from uninfected individuals in the conditions under which Autonomous DNA bovine serum.

IV. 4 b. The expression of the polypeptide encoded in the composite circular DNA virus hepatitis C in clones 36, 81 and 32

The polypeptide of hepatitis C virus, which is encoded in an openly readable structure stretching across the clones 36, 81 and 32, has been reproduced as a polypeptide fusion with peroxydisulfate. This was carried out by embedding a composite circular DNA, C100, in the expression vector and expression of the polypeptide in yeast.

A cassette for holding an expression containing composite C100 circular DNA obtained from clones 36, 81 and 32, designed by the embedding of the fragment 1270 p. O. EcoRI in localization EcoRI vector P33-56 (also called pS356), giving plasmid pS3-56C100. Design C100 described in section IV.A.16 above.

Vector S3-56, derived from pBP322, contains an expression cassette that contains the hybrid ADH2/GAPDH yeast promoter ascending to gene peroxydisulfate person and descending GAPDH termination of transcription. A similar cartridge that contains these controlling elements and the gene proxydemocracy, was described by Cousens et al. (1987) and continuing the application for the European patent N 196056, published October 1, 1986, owned by the present applicants. The cassette in the vector S3-56 great for what I lost, and the fact that gene peroxydisulfate is accompanied by a sequence of the adapter that contains the localization of EcoRI. The sequence of the adapter is:

5'-AAT TTG GGA ATT CCA TAA TGA G-3'

AC CCT TAA GGT ATT ACT CAG CT

Localization EcoRI allows integration of heterologous sequences that are being reproduced from the vector containing the cassette, give polypeptides, fused with peroxydisulfate through Oligopeptide the bundle containing the amino acid sequence:

-ans-leu-gly-ile-arg-

(-asparagine-leucine-glycine-isoleucine-arginine-).

The sample vector pS356 deposited on April 29, 1988 in the American collection of type cultures (ATSS). 12301 Parklawn Dr., Rockville, Maryland 20853 in terms Budapest/Treaty and was assigned a room 67683. The terms and conditions of access to the deposited vector are the same as for strains containing circular DNA virus hepatitis neither A nor B, defined in section II.A. The Deposit is meant for convenience only and is not required to implement the present invention in the presence of the present description. The deposited material are listed in the bibliographical note.

After recombinants containing the insert C100 circular DNA in a precise orientation were selected, expression cash is RIT cassette, were isolated and purified. This fragment was then integrated into the localization BamHI yeast vector raw.

Plasmid raw, the essential features of which are shown in Fig. 35 is a yeast Shuttle vector, which contains the full dvuhmetrovoy sequence replication (Broach, 1981) and the pBR322 sequence. It also contains the yeast URA3 gene derived from plasmid YEp24 (Botstein et al., 1979) and yeast LEU2dgene derived from plasmid pCl/1 (publication of the European patent N 116201 fine). Plasmid raw was synthesized by fermentation of plasmid YEp24 with EcoRI and delegacia vector to remove partial dvuhstronnih sequences. The resulting plasmid, YEP24deltaRI was ordered linearly by fermentation with ClaI and Legerova full dvuhmetrovoy the plasmid, which was ordered in line with ClaI. The obtained plasmid pCBou was then brozena with XbaI and the fragment of the vector 8605 p. O. was selected gel filtration. This dedicated XbaI fragment was Legerova fragment 4460 p. O. XbaI containing LEU gene2dextracted from pCl/1; orientation LEU2dgene in the same direction as the URA3 gene. Embedding expression implemented in a unique localization BamHI sequence pBR322, thus interrupting the gene for b the Aza-C100 expression cassette raws-3, was transformed into yeast strain: 308, as well as in other yeast strains. Cells were modified as described Hinnen et al. (1978), and grown on uretilecektir plates. Individual colonies were inoculated in latinalicious environment for growing. Culture stimulated the expression of the polypeptide peroxydisulfate-C100 (named C100-3) grown in YEP containing 1% glucose.

Strain: 308 belongs to the genotype MAT @ leu2, ura3 (del) DM15 (GAP/ADR1), integrated into the locus ADR1. In the strain JSC 308 overexpression of positive activator of gene product ADR1 leads to hyperreflexia (relative to control wild-type ADR1) and significantly higher outputs expressive heterologous proteins, if such proteins are synthesized through ADH2 UAS regulatory system. Construction of yeast strains: 308 described in the continuing application U.S. serial number (receipt verified N 2300-0229) registered at the same time, and which are listed in the bibliographic reference. Sample: 308 was deposited on may 5, 1988 in ATSS under the terms of the Budapest Treaty and received the number 20879. Storage time and conditions of access to the Deposit is the same as described in section II.A for strains containing calcev the 154 amino acids peroxydisulfate person when aminoterminal, the remainder of the five amino acids from synthetic adapter that contains the localization of EcoRI, the remains of 363 amino acids derived from circular DNA C100 and 5 carboxyterminal amino acids derived from the sequence of nucleotide MS2 connected to a ring sequence DNA in clone 32 (see section IV. A. 7). The putative amino acid sequence of carboxykinase this polypeptide, beginning at Polukhina Ala-residue peroxydisulfate shown in Fig. 36; also shown is the nucleotide sequence encoding this part of the polypeptide.

IV.B.5. Identification of the polypeptide encoded in the C100 as associated with hepatitis neither A nor B antigen

Polypeptide merge C100-3, obtained by expression from a plasmid rows-3 in yeast strain: 308, was characterized based on the size and the polypeptide encoded in the C100 was identified as associated with hepatitis neither A nor B antigen on its immunological reactivity with human sera, patient with chronic hepatitis neither A nor B.

Polypeptide C100-3, obtained by expression, as described in section IV.B.4, were analyzed as follows. Yeast cells of the strain JSC 308 were transformed yeast vector raw or plasma is these yeast cells in 1 ml culture (OD650nanometers 20) was converted into beads by centrifugation at 10000 rpm for one minute and then subjected to lysis energetic swirl (10 times in one minute) with 2 volumes of solution and one volume of glass beads (0,2 millimicrons diameter). The solution contained 50 mm Tris-HCl, pH 8.0, 1 mm ethylenediaminetetraacetic acid, 1 mm fluoride phenylmethylsulfonyl and micrograms/ml pentalina. Insoluble material in the lysate, which includes the polypeptide C100-3, collected by centrifugation (at 10,000 rpm for five minutes) and was dissolved by boiling for five minutes in sample buffer sodium dodecyl sulfate (see Laermmli, 1970). The number of polypeptides, equivalent to 0.3 ml stimulated yeast culture were subjected to electrophoresis through 10% polyacrylamide gel in the presence of sodium dodecyl sulfate according to the method of Laemmli (1970). Standard proteins were subjected to parallel electrophoresis gels. Gels containing polypeptides subjected to depression, or were stained with brilliant blue Coomassie, or were subjected to Western patrinia, as described in section IV.B.2, using the serum of a patient with chronic hepatitis A or B to determine the immunological reactivity of polypeptides, polsani brilliant blue Coomassie. Insoluble polypeptide(s) from strain JCS 308, transformed raw, and from two different colonies of strain JSC 308 transformed rows-3, shown on line 1 (raw) and on lines 2 and 3, respectively. Comparison of row 2 and row 3 from row 1 shows that stimulated the expression of the polypeptide corresponding to the molecular weight of 54000 Dalton from strain JSC 308 transformed rows-3, which is not stimulated in the strain JSC 308 transformed with plasmid raw. This polypeptide is indicated by the arrow.

In Fig. 37B presents the results of the "Western" spots insoluble polypeptides, obtained by expression in a transformed plasmid raw strain: 308 (line 1) or plasmid rows-3 (line 2). The polypeptides obtained by expression from raw were immunological directionspanel with the serum of a person infected with hepatitis neither A nor B. However, as shown by the arrow, the strain JSC 308 transformed with plasmid raws-3, synthesizes a polypeptide with a molecular weight of about 54000 daltons, which reacts immunologically with serum of a person infected with hepatitis neither A nor B. Other immunologically reactive polypeptides in line 2 can be decay products and/or aggregation this is the Polypeptide merge C100-3, containing peroxydisulfate at N-end, and structural polypeptide of hepatitis C virus C100 at the C-end, was purified by differential extraction of the insoluble fraction extracted yeast host cells, which was synthesized polypeptide.

Polypeptide merge C100-3 was synthesized in the strain, C308, transformed rows-3, as described in section IV.B.4. Yeast cells were then subjected to lysis, homogenization, insoluble material in the lysate was extracted at a pH of 12.0 and C100-3 in the remaining insoluble fraction was transferred into the dissolved state in a buffer containing sodium dodecyl sulphate.

Yeast lysate was prepared mainly by the method Nagahuma et al. (1984). Received a suspension of yeast cells containing 33% of cells suspended in a solution (buffer A), consisting of 20 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol and 1 mm fluoride phenylmethylsulfonyl. An aliquot of the suspension (15 ml) was mixed with an equal volume of glass spheres (diameter of 0.45-0.50 mm), zavarelli with a maximum speed supermicar (Lab-Line Instruments, Inc.) within eight minutes. The homogenate and glass beads were separated by glass beads three times washed with the same volume of buffer A, as originally Packed cells. the Nata at 7000 g for 15 minutes at 4oC, resuspending obtained beads in buffer A, equal to twice the original volume of Packed cells, and re-obtaining beads material by centrifugation at 700 g for 15 minutes. The washing process was repeated three times.

Insoluble material from the lysate was extracted at pH 12,0 as follows. Sediment suspended in buffer containing 0.5 M sodium chloride, 1 mm ethylenediaminetetraacetic acid, in which the volume of the suspension was equal to 1.8 times the original volume of Packed cells. Suspension supported pH by adding 0.2 volume of 0.4 M nutrifaster buffer, pH to 12.0. After stirring, the suspension was centrifuged at 7000 g for 15 minutes at 4oC and the supernatant layer was removed. The extraction was repeated twice. Extracted beads were washed with suspendirovanie them in 0.5 M sodium chloride, 1 mm ethylenediaminetetraacetic acid, using the amount of suspension is two volumes originally canned cells, followed by centrifugation at 7000 g for 15 minutes at 4oC.

Polypeptide C100-3 in the extracted sediment was transferred into a soluble state by treatment with sodium dodecyl sulfate. Balls suspended in obyavlenie in suspension. After stirring the suspension was centrifuged at 7000 g for 15 minutes at 4oC. the precipitate three times were extracted with sodium dodecyl sulfate. The obtained supernatant layers, which contained C100-3, were combined into the General Fund.

This procedure clears the C100-3 more than 10 times of the insoluble fraction of yeast homogenate and secretion of the polypeptide was more than 50%.

The purified preparation of the polypeptide of the fusion were analyzed by electrophoresis on a polyacrylamide gel according to Laemmli (1970). Based on the data analysis, the polypeptide had a purity of more than 80% and had an average molecular weight of about 54000 Dalton.

IV. C. Identification of RNA of infected individuals, which forms hybrids with the circular DNA virus hepatitis C

IV. C. 1. Identification of RNA in the liver of chimpanzees with hepatitis neither A nor B, which hybridize with circular DNA virus hepatitis C

RNA from the liver of chimpanzees infected with hepatitis neither A nor B, it is found that contains the RNA, forming hybrids with circular DNA contained in clone 81, when the North patenie as follows.

RNA was isolated from liver biopsy chimpanzees, from which was obtained a high titer plasma (see section IV.A.1), solpol-A+- and poly-A--faction. These fractions RNA subjected to electrophoresis on a formaldehyde-agarose gel (1% by weight by volume) and transferred to nitrocellulose. (Maniatis et al., 1982). Nitrocellulose filters were hybridisable with the circular DNA of hepatitis C virus from clone 81, provided with a radioactive label. (See in Fig. 4 the nucleotide sequence of the insert). To obtain a radioactive label, insert the circular DNA of the hepatitis C virus was isolated from clone 81, and were labeled with radioactive phosphorus-32 exact translation using DNA polymerase 1 (Maniatis et al., 1982). Hybridization was performed for 18 hours at 42oC in a solution containing 10% (weight by volume) of dextrin sulfate, 50% (weight, volume) deionized formamide, 750 mm NaCl, 75 mm sodium citrate, 20 mm acidic sodium phosphate, pH 6.5, 0.1% sodium dodecyl sulfate, 0,02% (weight/volume) bovine serum albumin, 0.02 percent (wt/vol) Ficoll-400, 0.02 percent (weight/volume) of polyvinylpyrrolidone, 100 micrograms/ml DNA salmon sperm that was hydrodynamically fragmentarily ultrasound and denaturiruet, and the sample is accurately labeled translated circular DNA activity 106registrations pulses per millimeter.

An autoradiograph of the investigated filters shown sleduushem volume: band 2 contains 30 micrograms of total RNA; lane 3 contains 30 micrograms of poly (A--RNA and lane 4 contains 20 micrograms of poly (A+-RNA. As shown in Fig. 38, the liver of chimpanzees, a patient with hepatitis neither A nor B contains a heterogeneous population of related molecules poly-A+-RNA, which it hybridises sample circular DNA virus hepatitis C and which, apparently, is approximately from 5,000 to 11,000 nucleotides in size. This RNA, which forms a hybrid with the circular DNA of the hepatitis C virus can be a virus genomes and/or specific transcripts of the viral genome.

The experiment described in section IV.C.2 below, is consistent with the assumption that the hepatitis C virus genome contains RNA.

IV.C.2. Identification of RNA derived from hepatitis C virus in serum inficirovannyh individuals

Nucleic acid was extracted from the particles separated from the high titer plasma chimpanzees, sick with hepatitis neither A nor B, as described in section IV. A. 1. Aliquots (equivalent to 1 ml of the original plasma) selected nucleic acid is suspended in 20 microliths 50 mm Hepes, pH 7.5, 1 mm ethylenediaminetetraacetic acid and 16 micrograms/ml yeast soluble RNA. Samples were denaturiruet by boiling for five minutes, followed ethylenediaminetetraacetic acid, 40 mm Hepes pH 7.5), or desoksiribonukleaza 1 (5 microlitres containing one unit of deoxyribonuclease/ 10 mm magnesium chloride, 25 mm Hepes, pH 7.5); control samples were incubated without enzyme. After incubation were added 230 microliters ice 2 x SSC containing 2 micrograms/ml of soluble yeast RNA, and the samples were filtered on nitrocellulose filter. Filters were hybridisable with a sample of circular DNA derived from clone 81, which was labeled with phosphorus-32, broadcast label. In Fig. 39 presents autoradiography filter. The hybridization signals were determined in treated desoksiribonukleaza samples and control samples (lanes 2 and 1, respectively), but was not detected in treated with ribonuclease sample (lane 3). Thus, since the treatment with ribonuclease 1 destroys nucleic acid selected from particles, and processing desoksiribonukleaza 1 does not cause such action, it strongly suggests that the genome of hepatitis C virus consists of RNA.

IV. C. 3. The definition of extended sequences of the nucleic acid of hepatitis C virus obtained from nucleic acids sequences of hepatitis C virus in the liver and plasma samples chimpanzees, anze with hepatitis neither A, no B and the control chimpanzees were increased using mainly reactions polymerase chain according to the method described by Saiki et al. (1986). Bare oligonucleotides were obtained from sequences of circular DNA virus hepatitis C in clone 81 or clones 36 and 37. Increased sequence were identified by electrophoresis through a gel and South patenier, using as samples suitable oligomer circular DNA with the sequence of the plot between the two blades, but not including it.

Samples of RNA sequences of hepatitis C virus to be tested by the system increase, were isolated from biopsies of the liver three chimpanzees, patients with hepatitis b neither A nor B, and from two control chimpanzees. The allocation fraction of RNA was performed using the thiocyanate guanidine, the method described in section IV.C.1.

RNA samples that were subject to investigation by the system increases, were also isolated from the plasma of two chimpanzees, patients with hepatitis b neither A nor B, and one control chimpanzees, as well as from a pool of plasma from control chimpanzees. One infected chimpanzees had infectious dose chimpanzees in 1 ml equal to or greater than 106and the other infected chimpanzees had infectionous way. Either 0.1 ml or 0.01 ml of plasma was diluted to a final volume of 1.0 ml solution TENB-proteinase K-sodium dodecyl sulphate (0.05 M Tris-HCl, pH 8.0, 0.001 M ethylenediaminetetraacetic acid, 0.1 M sodium chloride, 1 mg/ml proteinase K and 0.5% sodium dodecyl sulfate) containing 10 micrograms per 1 ml polyadenylic acid, and incubated at 37oC for 60 minutes. After this fermentation proteinase K obtained fractions of plasma were released from the protein by extraction with a mixture of TE [Tris-HCl-ethylenediaminetetraacetic acid (10.0 mm Tris-HCl, pH 8.0, 1 mm EDTA)] saturated with phenol. Phenolic phase was separated by centrifugation and re-extracted with a mixture of Tris-HCl EDTA-NaCl containing 0.1% sodium dodecyl sulfate. The resulting aqueous phase from each extraction were combined and extracted twice with equal volumes of a mixture of phenol-chloroform-isoamyl alcohol in the ratio of 1:1 (99:2) and two times with an equal volume mixture of chloroform-isoamyl alcohol in the ratio of 99:1. After phase separation by centrifugation the aqueous phase was concentrated to a final concentration of 0.2 M sodium acetate and nucleic acids precipitated with addition of two volumes of ethanol. Precipitated precipitated nucleic acids were isolated by ultracentrifugation in a SW 41 rotor at 38 K in impasse and accumulated control plasma alternative method Chomcyzski and Sacchi (1987) with 50 micrograms of poly A carrier. This process used the extraction of acidic thiocyanate guanidine. RNA was isolated by centrifugation at 10000 rpm for 10 minutes at 4oC microcentrifuge Eppendorf.

In two cases, prior to synthesis of circular DNA in polymerase chain reaction nucleic acid extracted from plasma proteinase K-sodium dodecyl sulphate-phenol method, were further purified by binding and elution from S and S Elutip-R-columns. The process was carried out according to instructions of the equipment suppliers.

The circular DNA is used as template for the polymerase chain reaction, were obtained from nucleic acids (either from the total nucleic acid, or RNA), prepared as described above. After precipitation with ethanol precipitated amino acids were dried and resuspendable in DEPC-treated distilled water. Secondary structure in nucleic acids tore by heating at 65oC for 10 minutes and samples were immediately cooled with ice. Circular DNA was synthesized using 1 to 3 micrograms of total RNA from the liver of a chimpanzee, or of nucleic acids (or RNA) extracted from 10-100 microliters plasma. In the synthesis used reverse transcriptase and its proalcool DNA were diluted used in the polymerase chain reaction, described below. All of the reaction mixture for the synthesis of circular DNA consisted of 23 units of ribonuclease inhibitor RNASINm(Fisher/Promega). After synthesis of circular DNA, the reaction mixture was diluted with water, boiled for 10 minutes and rapidly cooled in ice.

Polymerase chain reaction was performed mainly according to the manufacturer's instructions drugs (Cetus-Perkin-Elmer), except for the addition of one microgram of ribonuclease A. Reaction was performed in a final volume of 100 microliters. Polymerase chain reaction was carried out for 35 cycles using the 37oC, 72oC and 94oC.

The initiators of the synthesis of circular DNA and polymerase chain reactions were obtained from sequences of circular DNA virus hepatitis C or clone 81, clone 36 or clone 37b. (Sequence of circular DNA virus hepatitis C in clone 81, 36 and 37b shown in Fig. 4, 5 and 10 respectively). Sequence of two 16-membered initiators derived from clone 81, were as follows:

5' CAA TCA TAG CTG ACA G 3' and

5' GAT AAC CTC TGC CTG A-3'.

The sequence initiator of the clone 36 was:

5' GCA TGT CAT GAT GTA T 3'.

The sequence initiator of the clone 37b was:

5' ACA ATA Southernera from clone 81, or 16-membered sequence from clone 36 and 16-membered sequence from clone 37b.

The products of the polymerase chain reaction were analyzed by separation of the products by electrophoresis on alkaline gel and subsequent southern Platinium and increased definition sequences circular DNA virus hepatitis C breakdown labeled with phosphorus-32 internal oligonucleotide derived from a plot of circular DNA virus hepatitis C, which does not overlap sequence initiators. A mixture of chain reactions polymerase were extracted with a mixture of phenol-chloroform, and the nucleic acid was besieged from the aqueous phase salt or ethanol. Precipitated precipitated nucleic acids were collected by centrifugation and dissolved in distilled water. Aliquots of the samples were subjected to electrophoresis on gels containing 1.8% alkaline agarose. Agentcities DNA lengths 60, 108 and 161 nucleotides were subjected to parallel electrophoresis on gels as molecular weight markers. After electrophoresis of circular DNA in the gel was transferred onto paper Biorad Zeta Probem. Conditions pre-hybridization and hybridization and washing was determined by the specifications of the manufacturer (Biorad).

Samples used for hybridoscillation chain reaction polymerase were obtained from clone 81, the break served 108 nucleotide sequence corresponding to the sequence, which is localized in the area between the sequences of the two initiators. If a pair of initiators of the polymerase chain reaction obtained from clone 36 and 37b, the break was nick-translated insert circular DNA virus hepatitis C, obtained from clone 35. The initiators were obtained from nucleotide 155-170 insert of clone 37b and 206-268 nucleotide insert of clone 36. the 3'end of the insert circular DNA virus hepatitis C in clone 35 overlaps the nucleotides 1-186 insert in clone 36; and 5'-end of the insert of clone 35 overlaps the nucleotides 207-269 insert in clone 37b. (Compare Fig. 5, 8 and 10). Thus, the insert ring DNA in clone 35 overlaps part of the site between the sequence initiator, obtained from clone 36 and 37b, and is useful as a probe for increased sequence, which include these initiators.

Analysis of RNA from liver samples was conducted in accordance with the above procedure, using both sets of initiators and samples. RNA from the liver of three chimpanzees with hepatitis neither A nor gave positive hybridization results for extended sequences of the expected size (161 and 586 nucleotides to clone. the e same results were obtained when repeating the experiment three times.

Analysis of nucleic acids and RNA from plasma was carried out in accordance with the above procedure, using the initiators and the sample from clone 81. Plasma was obtained from two chimpanzees with hepatitis neither A nor B, the control chimpanzees and pooled plasma control chimpanzees. Both plasma sample chimpanzees, patients with hepatitis b neither A nor B contain a mixture of nucleic acids - RNA, which gave positive results in the response of growth polymerase chain while both control plasma gave negative results. These results were reproduced in repeated experiments.

IV. D. Radioimmunoassay analysis for the detection of antibodies of hepatitis C virus in serum antivirusnik individuals

Solid-phase radioimmunologically analysis for the detection of antibodies to antigens of the hepatitis C virus has been improved, based on the Tsu research and Herzenberg (1980). Plates microtitre (immulon 2, Removawell strips) covered purified polypeptides containing epitopes of hepatitis C. Coated plates were incubated with either human sera suspicious content of antibodies to epitopes of the virus is facilitated, contacted with the solid phase antigen. After removal of unbound material and washing microtitre plates was determined complexes virus antigen neither A nor B antibody man intubirovannam labeled with iodine-125 human immunoglobulin antisera sheep. Unbound labeled antibody is removed by aspiration and the plates washed. Determined the radioactivity in individual cells: the number of bound antibodies to the antigen of the hepatitis C virus human proportion of radioactivity in the sources.

IV.D.1. Purification of the polypeptide merge peroxydisulfate-neither A nor B5-1-1< / BR>
Polypeptide merge peroxydisulfate-neither A nor B5-1-1obtained by expression in recombinant bacteria, as described in section IV.B.1, was purified from rekombinantnyi E. coli differential extraction extracts of cells with urea followed by chromatography on anyone - and cation-exchange columns as follows.

The thawed cells from one liter of culture suspended in 10 ml of 20% (weight/volume) sucrose containing 0.01 M Tris-HCl, pH 8.0 and 0.4 ml) was added 0.5 M ethylenediaminetetraacetic acid, pH 8.0. After 5 minutes at 0oC the mixture was centrifuged at 4000 g for 10 minutes. The precipitate Srila and 1 micrograms/ml pepstatin A, with the subsequent addition of 0.5 ml of lysozyme (10 mg/ml) and incubated at 0oC for 10 minutes. After adding 10 ml of 1.0% (by volume) Triton-X-100 in 0.05 M Tris-HCl, pH 8.0, 1.0 mm ethylenediaminetetraacetic acid mixture incubated an additional 10 minutes at 0oC possible with shaking. The obtained viscous solution homogenized six passing through hypodermal needle 20 size and centrifuged at 13,000 g for 25 minutes. Dropped into the sediment material suspended in 5 ml of 0.01 Tris-HCl, pH 8.0, and the suspension was centrifuged at 4000 g for 10 minutes. The precipitate containing protein merge peroxydisulfate neither A nor B5-1-1, was dissolved in 5 ml of 6 M urea in 0.02 M Tris-HCl, pH 8.0, 1 mm dithiothreitol (buffer A) and was placed in a column with rapidly permeable Q-separate, balanced with buffer A. Proteins were suirable linear gradient from 0.0 to 0.3 M sodium chloride in buffer A. After elution fractions were analyzed by electrophoresis through a polyacrylamide gel in the presence of sodium dodecyl sulfate to determine the content peroxydisulfate-neither A nor B5-1-1. The fractions containing this polypeptide was collected and dialyzed with 6 M urea in 0.02 M buffer phosphate, pH of 6.0, 1 mm dithiothreitol (buffer B). Cialisovernight education is t 0.0 to 0.3 M NaCl in buffer B. Fractions were analyzed by electrophoresis through a polyacrylamide gel in the presence of the polypeptide peroxydisulfate-neither A nor B5-1-1and the appropriate fractions were collected.

The final receipt peroxydisulfate-neither A nor B5-1-1were tested by electrophoresis on polyacrylamide gels, which were attended by sodium dodecyl sulphate. The results of this analysis, the product had a purity of more than 80%.

IV.D.2. Purification of the polypeptide merge peroxydisulfate-neither A nor B81< / BR>
Polypeptide merge peroxydisulfate-neither A nor B81obtained by expression in recombinant bacteria, as described in section IV.B.2, was purified from recombinant E. coli by differential extraction of cell extracts urea followed by chromatography on anyone - and cation-exchange columns using the process described in section IV.D.1, when the selection of the polypeptide merge peroxydisulfate-neither A nor B5-1-1.

End getting polypeptide peroxydisulfate-neither A nor B81were checked by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Based on this analysis the product had a purity of more than 50%.

IV. D. 3. Determination of antibodies was epitopically hepatitis neither A, neither B, were subjected to radioimmunoassay analysis to determine whether antibodies to epitopes of the virus C polypeptides merge peroxydisulfate-neither A nor5-1-1or peroxydisulfate-neither A nor B81and their registration.

Microtitre plates were covered with a polypeptide peroxydisulfate-neither A nor B5-1-1or peroxydisulfate-neither A nor B81that had been partially purified by the methods described in sections IV. D. 1 and IV.D.2 respectively. The analysis was carried out as follows.

100-Microlitre aliquot of sample containing 0.1-0.5 micrograms peroxydisulfate-neither A nor B5-1-1or peroxydisulfate-neither A nor B81in 0,125 M borate-sodium buffer, pH 8,3, of 0.075 M sodium chloride (BBS) was added to each cell microtitre plates (Dynatech Immulon 2 Removawell Strips). The plate was incubated at 4oC, leaving overnight in a humid chamber, after which the protein solution was removed and cells were washed three times with BBS containing 0.02% of Triton X-100 (BBST). To prevent nonspecific binding, cells were covered with bovine serum albumin by adding 100 microliters solution of bovine serum albumin in BBS at a concentration of 5 mg/ml followed by incubation at room temperature for h which was Imedashvili serum adding 100 microlitres plasma samples, diluted in a ratio of 1:100 in 0.01 M phosphate buffer, pH of 7.2, 0.15 M sodium chloride (PBS) containing 10 mg/ml bovine serum albumin, and containing serum, the cells were incubated one hour at 37oC. After incubation, the serum samples were removed by aspiration and the cells washed five times borate buffer BBST. Antibodies against neither A nor B5-1-1and neither A nor B81was determined by binding labeled with iodine-125 F'(ab)2sheep IgG against human covered with cells. Aliquots of 100 microliters labeled probe (specific activity of 5-20 microcurie/micrograms) was added to each cell and the plate incubated at 37oC for one hour, followed by removal of excess sample suction and five washing borate buffer with Triton X-100 (BBST). The amount of radioactivity bound in each cell was determined by counting the number of decays per meter, detecting gamma radiation.

The results of the determination of anti-neither A nor B5-1-1and anti-neither A nor B81individuals with hepatitis b neither A nor B are presented in table. 1.

As can be seen from the table, the serum of patients 1, 19 and 32, have been diagnosed viral hepatitis neither A nor B were positive for the antibodies directed against epitopes of the virus is, however, serum samples, which were positive, unequal interacted with immunological peroxydisulfate-neither A nor B5-1-1and peroxydisulfate-neither A nor B81. Serum samples of the patient N 1 were positive to the polypeptide peroxydisulfate-neither A nor B81but were not positive for peroxidisulfate-neither A nor B5-1-1. Serum samples of patients N 10, 15 and 17 were positive for peroxidisulfate-neither A nor B5-1-1but were not positive for peroxidisulfate-neither A nor B81. Serum samples of patients N 3, 8, 11 and 12 reacted equally with both polypeptides merge, whereas serum samples of patients N 2, 4, 7 and 9 were 2-3 times greater in the reaction with peroxydisulfate-neither A nor B5-1-1than peroxydisulfate-neither A nor B81. These results suggest that neither A nor B5-1-1and neither A nor B81can contain at least three different epitope, i.e., it is possible that each polypeptide contains at least one unique epitope and that two polypeptide is divided between at least one epitope.

IV. D. 4. The specificity of solid-phase radioimmunoassay analysis for neither A nor B

The specificity of solid-phase radioimmunoassay analysis for virus neither A nor B was tested with Esplanade control individuals. The tests, which used a partially purified polypeptides peroxydisulfate-neither A nor B5-1-1and peroxydisulfate-neither A nor B81, was performed mainly as described in section IV.D.3, except that sera were obtained from patients who have previously determined the hepatitis A virus or hepatitis B, or individuals who are blood Bank donors. The results obtained with sera infected with hepatitis A and B, are presented in table. 1. Radioimmunoassay analysis was tested on 11 kinds serum of patients infected with the hepatitis A virus and 20 kinds serum of patients infected with hepatitis B. As shown in the table. 1, none of these sera gave a positive immunological reaction with the polypeptides of the fusion containing the epitopes of the virus BB-neither A nor B.

Radioimmunological analysis using antigen neither A nor B5-1-1was applied to determine the immunological reactogenicity serum control individuals. From 230 serum samples obtained from normal populations of donor blood, only two showed positive reaction radioimmunoassay analysis (data not shown). It is possible that two blood donor, who belonged to these two on-1
in the process of virus infection hepatitis neither A nor B

The presence of antibodies against either A or B5-1-1if during infection of hepatitis b neither A nor B have two sick people and 4 chimpanzees were determined using radioimmunoassay analysis, as described in section IV.D.3. In addition radioimmunoassay analysis was used to determine the presence or absence of antibodies against neither A nor B5-1-1for infections of hepatitis A virus and hepatitis B virus in infected chimpanzees.

The results presented in table. 2, show that antibodies against either A or B5-1-1it was discovered after the acute phase of hepatitis b infection neither A nor b Antibodies against either A or B5-1-1was not determined in serum samples from chimpanzees infected with either hepatitis A or hepatitis B. Thus, antibodies against peptide neither A nor5-1-1serve as a marker for individuals exposed to infection hepatitis C virus

IV.E. The purification of polyclonal serum antibodies to neither A nor B5-1-1< / BR>
On the basis of specific immunological reactogenicity polypeptide peroxydisulfate-neither A nor B5-1-1in respect of antibodies serum samples of patients with hepatitis neither A nor B was developed treatment method and the t affinity chromatography. The purified polypeptide peroxydisulfate-neither A nor B5-1-1(see section IV.D.1) attached to an insoluble substrate; attaching carried out so that the fixed polypeptide retained its affinity with antibody to neither A nor B5-1-1. Antibody in serum samples absorbed to may be put at risk-associated polypeptide. After removal of nonspecific related materials and unbound materials associated antibody released from the associated polypeptide peroxydisulfate - hepatitis C virus by changing pH and/or application of chaotropes reagent such as urea.

Nitrocellulose membranes containing the linked polypeptide peroxydisulfate-neither A nor B5-1-1, was prepared as follows. 2.1 cm Sartorius with a pore size of 0.2 micron was washed three minutes three times with buffer BBS (borate buffer). Polypeptide peroxydisulfate-neither A nor5-1-1was associated with the membrane by incubation of the pure drug in BBS buffer at room temperature for two hours; alternatively incubated at 4oC, leaving for the night. The solution containing unbound antigen was removed, and the filter three times washed with borate buffer (BBS) for three minutes each wash. Remaining active localization on the membrane yecenia 30 minutes. An excess of bovine serum albumin was removed by washing the membrane five times borate buffer (BBS) and three times with distilled water. The membrane containing viral antigen and bovine serum albumin, was treated with 0.05 M glycine hydrochloride, pH of 2.5, 0.10 M sodium chloride (Cly-HCI) for 15 minutes followed by three washing of phosphate-saline buffer solution, with a duration of three minutes.

Polyclonal antibodies against either A or B5-1-1were isolated by incubation of the membranes containing the polypeptide binding with sera from individuals infected with hepatitis neither A nor B, for two hours. After incubation, the filters were washed five times borate buffer solution (BBS) and twice with distilled water. Bound antibodies were suirable then from each filter five vymyvaniya hydrochloride for three minutes each elution. The pH of the eluates was maintained equal to 8.0 collecting each of the eluate in the experimental test tube containing 2.0 M Tris-HCl, pH 8.0. The output of antibodies against neither A nor B5-1-1after affinity chromatography was approximately 50%.

These nitrocellulose membrane containing the associated viral antigen, can be used several times without visible slimming is whether borate buffer solution three times for three minutes each. Then they are stored in borate buffer solution at 4oC.

IV. F. the Capture particles of the hepatitis C virus from infected plasma, using purified polyclonal human antibodies against hepatitis C virus; hybridization of nucleic acid in the captured particles with circular DNA virus hepatitis C

IV. F. 1. The capture particles of the hepatitis C virus from infected plasma, using polyclonal human antibodies against hepatitis C virus

Complexes of protein - nucleic acid present in infectious plasma chimpanzees with hepatitis neither A nor B were selected with the use of purified polyclonal antibodies against the hepatitis C virus, which were associated with a layer of polystyrene.

Polyclonal antibodies against either A or B5-1-1was purified from the serum of a sick person with hepatitis neither A nor B, using the polypeptide peroxydisulfate - hepatitis C virus encoded in clone 5-1-1. Method of purification was similar to the one described in section IV.E.

Purified antibodies against either A or B5-1-1were associated with polystyrene layers (about 6.5 mm in diameter, mirror processing. Precision Plastic Ball Co. , Chicago, Illinois) each incubation at room temperature, leaving Bali substrate once washed with TBST [50 mm Tris-HCl, pH 8.0, 0.150 mm NaCl, 0,05% (by volume) tween-20] and then saline solution, stabilized phosphate buffer (PBS) containing 10 mg/ml bovine serum albumin.

The control substrate was prepared in an identical manner, except that purified antibodies against either A or B5-1-1were replaced by the overall human immunoglobulin.

The capture of hepatitis C virus from infected plasma chimpanzees with hepatitis neither A nor associated with the substrate antibodies against either A or B5-1-1was carried out as follows. Plasma chimpanzees with hepatitis neither A nor B was used as described in section IV.A.1. An aliquot (1 ml) infected with hepatitis neither A nor B plasma chimpanzees were incubated for three hours at 37oC with each of the five substrates coated with either antibodies against either A or B5-1-1or control immunoglobulins. The substrate was washed three times with Tris-buffer solution (TBST).

IV. F. 2. Hybridization of nucleic acid in the captured particles with circular DNA virus neither A nor B

Component of nucleic acids released from particles captured by antibodies against either A or B5-1-1analyzed by hybridization with the circular DNA of hepatitis C virus obtained from a and B, as described in section IV.F.1. In order to release nucleic acids from the particles, the washed substrate was incubated for 60 minutes at 37oC with 0.2 ml of substrate solution containing proteinase k (1.0 mg/ml), 10 mm Tris-HCl, pH 7.5, 10 mm ethylenediaminetetraacetic acid, and 0.25% (weight by volume) sodium dodecyl sulfate, 10 micrograms/ml of soluble yeast RNA, and the supernatant layer was removed. The supernatant solution was extracted with phenol and chloroform, and nucleic acids precipitated with ethanol, leaving the solution overnight at -20oC. the Precipitate nucleic acids were collected by centrifugation, dried and dissolved in 50 mm HEPES, pH 7.5. Double aliquots of the soluble nucleic acids from samples obtained on substrates coated with antibodies against either A or B5-1-1and with control substrates containing the total human immunoglobulin, filtered on nitrocellulose filters. Filters were hybridisable labeled with phosphorus-32, nick-translated breakdown obtained from the purified fragment circular DNA virus hepatitis C from clone 81. Methods of obtaining samples and conducting hybridization are described in section IV.C.1.

Autoradiography investigated filters containing nucleic acids from the particles trapped in the layers of the body against any A, neither B5-1-1(A1, A2), gives a clear hybridization signals relative to a reference extract (A3, A4and control yeast RNA (B1B2). Standards containing 1 PG, 5 PG, and 10 PG (picograms) of purified fragment circular DNA of clone 81 shown in Cl-3, respectively.

These results show that the particles captured from plasma of infected hepatitis neither A nor B antibodies against either A or B5-1-1contain nucleic acids that hybridizing with circular DNA virus hepatitis C in clone 81, and present further evidence that the circular DNA of these clones obtained from the etiologic agent for hepatitis neither A nor B.

IV. G. Immunological reactogenicity C100-3 with purified antibodies against either A or B5-1-1< / BR>
Immunological reactogenicity C100-3 polypeptide merge with antibodies against either A or B5-1-1was determined by radioimmunoassay analysis, in which the antigens that were associated with the solid phase, provoked purified antibodies against either A or B5-1-1and determined the complex antigen - antibody labeled with iodine-125 sheep antibodies against the antigen of human rights. Immunological reactivity of the polypeptide C100-3 was compared with reacciona, as described in section IV. B. 5 and in section IV.B.6, respectively. Polypeptide merge peroxydisulfate-neither A nor B5-1-1synthesized and purified as described in section IV.B.1 and in section IV.D.1 respectively. Purified antibodies against either A or B5-1-1received as described in section IV.E.

Stmicroelectroni aliquots containing various amounts of purified antigen C100-3 in 0.125 M buffer sodium borate, pH 8,3, of 0.075 M sodium chloride (BBS) was added to each cell microtitre plates (Dynatech Immulon 2 Removawell Strips). The plate was incubated at 4oC, leaving overnight in a humid chamber, after which the protein solution was removed and cells were washed three times with borate buffer solution containing 0.02% of Triton X-100 (BBST). To prevent nonspecific binding, cells were covered with bovine serum albumin by adding 100 microliters 5 mg/ml solution of bovine serum albumin in borate buffer solution, followed by incubation at room temperature for one hour, after which the excess solution of bovine serum albumin was removed. Polypeptides in coated cells were subjected to interaction with purified antibodies against either A or B5-1-1adding one microgram of antibody on ACE and the cells were washed five times BBST. Antibodies against either A or5-1-1associated with the polypeptides of the fusion was determined by binding labeled with iodine-125 F'(ab)2sheep IgG against human covered in albumin bovine serum to the cells. Aliquots hundred microlitres labeled probe (specific activity of 5-20 microcurie/micrograms) was added to each cell and the plate incubated at 37oC for one hour, followed by removal of excess sample suction and five times of washing buffer solution BBST. The amount of radioactivity in each cell was determined by counting pulses in the counter, which detects gamma radiation.

The results of immunological reactogenicity C100 with purified antibodies against either A or B5-1-1with purified antibodies are presented in table. 3.

The results presented in table. 3, show that antibodies against either A or B5-1-1recognize the epitope(s) in part C100 polypeptide C100-3. Thus, neither A nor B5-1-1and C100 share one common epitope(s). The result suggests that the ring sequence of the DNA encoding that neither A nor B-epitope(s) is a sequence that is present in clone 5-1-1, and in clone 81.

IV.N. Characterization of hepatitis C virus

IV.H the AI its nakatoshi isolating fractions of nucleic acids from the particles, captured by the antibody against neither A nor B5-1-1the coated polystyrene layers, and determined that it hybridises whether the selected nucleic acid with plus and/or minus-strands of circular DNA virus of hepatitis C.

Particles were captured from plasma chimpanzees infected with hepatitis C, using polystyrene layers, covered with immunoenzyme antibodies against either A or B5-1-1as described in section IV.F.1. Component nucleic acid particles were released using the method described in section IV.F.2. The selected aliquots of genomic nucleic acid, equivalent to 3 ml plasma high titer, put spots on nitrocellulose filters. Control aliquots were denatured circular DNA virus hepatitis C from clone 81 (two picograms), which also inflicted spots on the same filters. The filters were tested labeled with phosphorus-32 mixture of plus or a mixture of minus-strands ednonachalie DNA cloned from circular DNA virus hepatitis C; circular DNA are cut out of the clones 40b, 81 and 25c.

Agentcities samples were obtained by cutting the circular DNA of hepatitis C virus from clone 81, 40b and 25c with EcoRI and cloning of fragments of circular DNA in M13 vectors mp18 and mp19 (Messing, 1983). M13 clones of powergas hepatitis C. Determination of the sequences was performed by the method of finishing detoxicate by Sanger et al. (1977).

Each kit dual filters containing aliquots of the genome of hepatitis C virus isolated from trapped particles were hybridisable either plus-or minus-filamentous samples of circular DNA virus of hepatitis C. Autoradiography obtained from the test genome of hepatitis C virus with a mixture of samples derived from clone 81, 40b and 25c, as shown in Fig. 41. This mixture was used to increase the sensitivity of the hybridization assay. The samples in the list I was hybridisable mixture samples plus-strands. Samples in list II were tested by hybridization with a mixture of samples minus-strands. The composition of the samples in the lists immune stains are presented in table. 4.

As can be seen from the results in Fig. 41, only samples of minus-strand DNA hybridizing with the selected genome of hepatitis C. This result combined with the result indicating that the gene is sensitive to ribonuclease and insensitive to desoksiribonukleaza (see section IV.C.2) suggests that the genome of the virus neither A nor b is a positive filamentous RNA.

These data and data obtained in other laboratories regarding physical-Henichesk a new class of viral agent, and do not exclude such a possibility.

IV. H. 2. Sequencing caught in the particles, which, when increased by the polymerase chain reaction, was hybridisable with circular DNA virus hepatitis A, derived from clone 81

RNA in the captured particles were obtained as described in section IV.N. 1. Determination of sequences that hybridizing with the circular DNA of hepatitis C virus derived from clone 81 was carried out using the process of increasing underlying the polymerase chain reaction, described in section IV. C. 3, except that hybridization break served kinesiology oligonucleotide derived from the sequence of circular DNA elephant 81. The results show that increased sequence hybridizing with a sample of circular DNA of hepatitis C virus derived from clone 81.

IV. N. 3. Homology between the non-structural protein of Dengue-flavivirus (MNWWVD1) and polypeptides of hepatitis C virus encoded United openly readable structures in the clones 14i to 39c

Joint circular DNA virus hepatitis C in clones 14i to 39c contain one continuous openly readable structure, as shown in Fig. 26. Encoded in it polyplacophora (MNWWVD1). The analysis used a database of proteins Dayhoff and processed on the computer. The results are presented in Fig. 42, where the symbol (:) means the exact homology and the symbol (.) indicates a conservative substitution in the sequence; the dashed lines show the space introduced in sequence to achieve the greatest homology. As can be seen in the figure, there is considerable homology between the sequence encoded in the circular DNA of hepatitis C virus and non-structural polypeptide(s) Dengie-virus. In addition to the homology shown in Fig. 42, the analysis of the polypeptide segment encoded on the site towards the 3'-end of the DNA also contains sequences homologous to sequences in Dingle-polymerase. The consequence of this finding is that the canonical Cly-Asp-Asp (GDD) sequence, although essential for RNA dependent RNA polymerase, contained in the polypeptide encoded in the circular DNA of hepatitis C virus in localization, which is consistent with localization in Dingle-2 virus (data not shown).

IV.H.4. DNA of the hepatitis C virus is not detected in infected hepatitis neither A nor B tissues

Two types of studies are neither B. These results in combination with the results described in sections IV.C, IV. H. 1 and IV.H.2, is evidence that the hepatitis C virus is a DNA-containing virus and its replication does not involve circular DNA.

IV.N. 4.a. Is the process "southern" patania

To determine whether liver infected with hepatitis neither A nor B chimpanzees defined DNA of hepatitis C virus (or circular DNA (hepatitis C virus), fragments limiting enzyme DNA were isolated from this source and subjected to a "southern" patania and stains investigated labeled with phosphorus-32 circular DNA virus of hepatitis C. the Results indicate that the tagged circular DNA virus hepatitis C is not it hybridises with the test DNA in the spots from the liver of an infected chimpanzee. It also produces hybrids with the control DNA in the spots from the liver of a normal chimpanzee. Conversely, positive control labeled sample gene beta-interferon was hybridisable South stains limiting enzyme digested placental DNA of the person. These systems were designed to detect single copy of a gene that would be the definition of a labeled break.

DNA was isolated from the liver of two chimpanzees with hepatitis neither A nor B. Conte DNA was carried out, basically, according to the method of Maniatis et al. (1982), and DNA samples were treated with ribonuclease in the allocation process.

Each DNA sample was treated with either EcoRI, MboI or HincII (12 micrograms) according to the manufacturer's specications. Digested DNA was subjected to electrophoresis on 1% neutral agarose gel, put "South" spots on nitrocellulose and spotted the material was hybridisable with eligible nick-translated breakdown circular DNA (3106registrations CPM per ml of hybridization mixture). DNA liver of infected chimpanzees and from normal liver was hybridisable labeled with phosphorus-32 circular DNA virus hepatitis C from clones 36 plus 81; DNA from human placenta was hybridisable labeled with phosphorus-32 DNA of the gene of beta-interferon. After hybridization spots were washed in stringent conditions, i.e., a solution containing 0.1 x SSC, 0.1% sodium dodecyl sulfate at 65oC.

DNA gene beta-interferon was obtained by the method Honghton and collaborators (1981).

IV.N. 4.b. The increase method of the polymerase chain reaction

To determine whether DNA hepatitis C virus be detected in the liver of chimpanzees, patients with hepatitis b neither A nor B, DNA was extracted from tissue and subjected to a chain reaction of hepatite C in clone 81. Negative controls were samples of DNA isolated from uninfected tissue culture cells HepG2 and probably uninfected human placenta. Positive controls were samples negative control DNA, to which was added a known relatively small amount (250 molecules) circular DNA of hepatitis C virus in the form of an insert from clone 81.

In addition, to confirm that the fraction of RNA extracted from the same disease chimpanzees with hepatitis neither A nor B contain the sequence complementary to the sample DNA hepatitis C virus, used the system to increase the detection reaction growth polymerase chain on selected samples of RNA.

In these studies, DNA were isolated by the method described in section IV. N. 4. a, RNA was extracted, mainly, as described in Chirgwin et al. (1981).

DNA samples were isolated from two disease infected chimpanzees from uninfected HepG2 cells and from human placenta. One microgram of each DNA was spaziali with HindIII according to the manufacturer's instructions. Digested samples were subjected to increase in response to growth polymerase chain and defining an enlarged circular DNA virus hepatitis C, essentially as described reaction growth polymerase chain and the sample were obtained from the circular DNA of hepatitis C virus clone 81 and described in section IV.C.3. To increase the positive control one microgram of each sample DNA was "sharpened" by adding 250 molecules insert circular DNA of hepatitis C virus isolated from clone 81.

To determine the presence of a sequence of hepatitis C virus in RNA extracted from the liver of chimpanzees with hepatitis neither A nor B, the samples containing 0.4 micrograms of total RNA were subjected to the scaling process, basically as described in section IV. C. 3, except that neglected reverse transcriptase in some samples as a negative control. The initiators of the reaction growth polymerase chain and the sample were obtained from the circular DNA of hepatitis C virus clone 81, as described above.

The results show that increased sequence, complementary to the sample circular DNA of the hepatitis C virus was not detected in DNA from the liver of infected chimpanzees, they were not determined in the negative controls. Conversely, if the sample comprising DNA from the liver of infected chimpanzees were "sharpened" circular DNA virus hepatitis C to increase, the sequence of clone 81 was detected in all positive control samples. In addition, in studies of RNA increased sequence Kohl is about safely conclude, that the results are not due to DNA contamination.

These results indicate that hepatocytes from chimpanzees with hepatitis neither A nor B do not contain or contain unrecorded levels of DNA of hepatitis C virus Based on the study of "witness" if the DNA of the hepatitis C virus is present, it has a lower level of 0.06 copies on hepatocyte, and, on the contrary, the sequence of hepatitis C virus in the total RNA from the same samples was easily determined by the growth response polymerase chain.

IV.I. Definition ELISA analysis of infection of hepatitis C virus using the C100-3 as an experimental antigen

All samples were analyzed using solid-phase enzyme-linked immunosorbent assay (ELISA) antigen C100-3 hepatitis C virus (which was synthesized and purified as described in section IV.B.5) and a conjugate of horseradish peroxidase and mouse monoclonal antibodies against human IgG.

Plates coated with antigen C100-3 hepatitis C, were prepared as follows. The solution containing the coating buffer (50 mm sodium borate, pH 9,0), 21 ml/plate, bovine serum albumin (25 micrograms/ml), C100-3 (2.50 micrograms/ml) was prepared directly before adding to the plates (removable cells were incubated for two hours at 37oC, after which the solution was removed by suction. Cells were washed once with 400 Microlitre wash buffer (100 mm phosphate, pH of 7.4, 140 mm NaCl, 0,1% (weight by volume) of casein, 1% (weight by volume) Triton X-100, 0.01 percent (weight/volume) of thimerosal). After removal of the wash solution was added 200 microliters/cell poslepechatnogo solution (10 mm phosphate, pH of 7.2, 150 mm NaCl, 0,1% (weight/volume) of casein and 2 mm fluoride phenylmethylsulfonyl), a plate loosely covered to prevent evaporation and kept at room temperature for 30 minutes. Then cells was aspirated to remove the solution and liofilizirovanny drying, leaving at night without heat shells. The obtained plates were stored at 2-8oC in sealed aluminium bags.

To perform the ELISA test, 20 microliters of serum sample or control sample was added to the cell containing 200 microliters sample diluent (100 mm phosphate, pH of 7.4, 500 mm sodium chloride, 1 mm ethylenediaminetetraacetic acid, 0.1 percent (weight/volume) of casein, 0,015 (weight/volume) of tarsala, 1% (weight/volume) Triton X-100, 100 micrograms/ml yeast extract). Plates were corked, and incubated at 37o3Fe(CN)6, 0,05% (weight/volume) tween-20, 0.02 percent (weight/volume) of thimerosal). Treatment was continued for one hour at 37oC, the solution was removed by aspiration and cells were washed with wash buffer, which was also removed by suction. To determine the amount of bound enzyme conjugate was added 200 microliters substrate solution (10 mg of orthophenylphenol dihydrochloride in 5 ml of developing solution). Developing solution contains 50 mm sodium citrate with a pH of 5.1, installed phosphoric acid, and 0.6 microliters/ml 30% hydrogen peroxide. Plates containing the substrate solution were incubated in the dark for 30 minutes at room temperature. The reaction was stopped by adding 50 microliters/ml 4 N. sulfuric acid and was determined by optical density.

The examples below show that the study microtitre panels ELISA is there is the initial velocity of the reactivity of about 1% with re-rate reactivity about 0.5% on random donors. The analysis is able to determine the immune response as in after the acute phase of the current infection and chronic stages of the disease. In addition, the analysis may identify some samples, which are regarded as negative in tests-substitutes for hepatitis neither A nor B; these samples come from individuals with a history of hepatitis neither A nor B, or from donors involved in the transfer of hepatitis neither A nor B.

In the examples described below use the following abbreviations:

ALT - alanine aminotransferase

Anti-HBc - HBc antibody against

Anti-HBsAg antibody against HBsAg

HCV - core antigen of hepatitis B

HBsAg surface antigen of hepatitis B

IgG - immunoglobulin G

IgM - immunoglobulin M

IU/L (international units per liter

NA - not rated

NT - not tested

N - sample size

Neg - negative

OD - optical density

Pos - positive

S/CO - signal/threshold

SD - standard deviation

x is the average value

WNL - within normal limits

IV.I.1. Hepatitis C infection in a population of random blood donors

The group of samples in the number 1056 (fresh serum) was obtained from random blood donors from memorial blood Bank Irvine, San Franz is adelene values of optical density (Fig. 43). As can be seen in Fig. 43, 4 specimens show more than 3, one sample shows between 1 and 3, five samples show between 0.4 and 1, and the rest 1046 samples show less than 0.4, and more than 90% of these samples show the optical density of less than 0.1 nanometer.

The test results on the reactivity random samples are presented in table. 5. Using a threshold value equal to the average plus 5 standard deviations of 10 samples from 1056 (0,95%) were initially reactive. Five of the samples (0,47%) confirmed its reactivity with repeated analysis of the ELISA method. In table. 5 shows the values of alanine aminotransferase and status antibodies against the core antigen of hepatitis B virus for each of the samples, again showing reactivity. Of particular interest is the fact that all five of the repeatedly reactive samples were negative in tests-substitutes for hepatitis neither A nor B, while behaved as positive in the ELISA analysis.

IV.I.2. Serum samples chimpanzees

Serum samples eleven chimpanzees were tested on the C100-3 of hepatitis C virus ELISA analysis. Four of these chimpanzees ustanovlennoy procedure in collaboration with Dr. Daniel Bradley in the Center for disease control. To control 4 other chimpanzees infected with hepatitis A and three by the virus of hepatitis B. serum Samples were obtained at different periods of the current infection.

The results are summarized in table. 6 shows the documented appearance of serum specific antibodies in all cases of infection in the chimpanzee strain Hutchinson hepatitis neither A nor B. After the acute phase of the infection (as evidenced by a significant increase and a subsequent return to normal levels of alanine aminotransferase) antibodies to C100-3 of hepatitis C virus became detectable in the serum of four out of four infected with hepatitis neither A nor B chimpanzees. These samples, as discussed in section IV.B.3, was determined previously as positive in the analysis of "Western" patania and radioimmunoassay analysis. In contrast, none of the control chimpanzees infected with hepatitis A virus or hepatitis B virus, showed no evidence of their reactivity in the ELISA analysis.

IV.I.3. Panel 1: confirmed infectious serum chronic carriers of hepatitis b neither A nor B

A coded panel contains 22 of a single sample, each of which was presented in two instances, the diagnostically, of infectious serum of patients with hepatitis neither A nor B in the acute phase. In addition samples were presented closely related negative control and control of diseases. This panel developed by Dr. H. Alter from Department of health and maintenance of the National Institute of health, Bethesda, Maryland, Maryland. The panel was designed by Dr. Alter a few years ago and was used as a qualified panel for prospective analyses of hepatitis neither A nor B.

The entire panel was subjected to ELISA (ELISA analysis) twice and the results were sent to Dr. Alter to decipher. The results of the evaluation are presented in table. 7. Although the table presents the results of one set of samples, the same results were obtained from each of the double sample.

As shown in table. 7, six sera that were reliably infectious model chimpanzees were strictly positive. Seven infectious sera corresponding to the sample with acute hepatitis neither A nor B was directionspanel in this analysis, ELISA. A sample from the involved donor as with normal aminotransferase levels, and with dubious results is that neither A, nor B were also directionspanel. All samples from closely related negative controls obtained from donors who had at least 10 blood donations, without the involvement of hepatitis, were non-reactive in the ELISA analysis. In conclusion, four of the tested sample, previously assenivaviese as positive in the analysis of the proposed hepatitis b neither A nor B, developed by other researchers, these analyses have not been confirmed. These samples are considered negative ELISA analysis of hepatitis C.

IV.I.4. Panel 2: the donor-recipient hepatitis neither A nor B

A coded panel contained 10 valid cases associated with transfusion hepatitis neither A nor B, a total of 188 samples. Each case contains samples of some or all of the donor to the recipient and serial samples were taken 3, 6 and 12 months after transfusion) from the recipient. Also included samples from individuals who had blood transfusions prior to the transfusion. A coded panel was proposed by Dr. Alter of the National institutes of health and the results were sent to him for evaluation.

The results are summarized in table. 8, show that immunother from case 4 (which was not determined seroconversion) always reacted poorly when performing enzyme linked immunosorbent assay. Two out of ten serum samples recipients were reactive three months after transfusion. After 6 months of 8 serum samples recipients were reactive and 12 months, except 4, all samples were reactive. In addition at least positive for antibody donor was found in 7 of 10 cases, while in the case 10, there were two positive donor. So, if 10 recipient before transfusion of blood was positive for antibodies against hepatitis C virus Through the blood of this month the recipient fell to the boundary values of the reactive levels, whereas it increased to positive after 4 and 10 months after transfusion. Thus, this case may be a primary infection of an individual with hepatitis C.

State aminotransferase and core antigen of hepatitis B virus for all reactive, i.e., positive samples are summarized in table. 9. As can be seen from the table, samples of serum donors 1-8 were negative for markers substitutes and reactive in the ELISA analysis of antibodies to hepatitis C. on the other hand, serum samples recipients (taken 12 months after transfusion) had a high content of al is CLASS="ptx2">

IV.I.5. Definition infection of hepatitis C virus in high-risk group

Serum samples from high-risk groups cheated using ELISA analysis to determine reactivity to the antigen C100-3 hepatitis C. These samples were obtained from Dr. Gary Tegtmeier, Community Blood Bank, Kansas City. The results are summarized in table. 10.

As can be seen from the table, the samples with the highest reactivity were obtained in hemophilia patients (76%). In addition, samples from individuals with a high content of alanine aminotransferase and positive for antibodies against the core antigen of hepatitis B virus was estimated at 51% reactivity, a value that is consistent with the value of clinical data, and the prevalence of hepatitis b neither A nor B in this group. The appearance of antibodies to hepatitis C virus was also higher than in blood donors with elevated alanine aminotransferase only in blood donors positive for antibodies to the core antigen of hepatitis B virus in blood donors rejected for reasons other than the high content of alanine aminotransferase or the presence of antibodies against the core antigen of hepatitis B virus in comparison with a random volunteer donors.

IV.I.6. Compare the x antibody as a second antibody in the ELISA analysis of antigen C100-3 of hepatitis C virus

The sensitivity of ELISA method using a conjugate of a monoclonal antibody with antiimmunoglobulin G was compared with the sensitivity obtained using conjugate monoclonal antibodies to antiimmunoglobulin M or replacement of monoclonal antibodies, polyclonal anticorodal, which, as reported, with specific heavy and light chain. Following studies were conducted.

IV.I.6.a. Serial samples from seroconverters

Serial samples from three cases of seroconverters virus neither A nor B were investigated by enzyme immunoassay in the solid phase antigen C100-3 of hepatitis C virus using enzyme conjugate or with a monoclonal antibody to immunoglobulin G, or in combination with a monoclonal antibody against immunoglobulin M, or using the polyclonal anticigarette. The samples were provided by Dr. Cladd Stevens, N. Y. Blood Center, N. Y. C., N. Y. History of the samples shown in the table. 11.

The results obtained with the enzyme conjugated with a monoclonal antibody against immunoglobulin G, are presented in table. 12. These data suggest that strong reactivity was first identified in samples 1-4, 2-8 3-5 cases 1, 2 and 3, respectively.

The results obtained by ELISA using monoclonal conjugate antibodies against immunoglobulin G (dilution 1:10000) or with polyclonal conjugate Tago (dilution 1:80000), or polyclonal conjugate Jackson (dilution 1:80000), shown in the table. 14. These data show that the initial strong reactivity was determined in samples 1-4, 2-8 and 3-5 using all three configurations: polyclonal antibodies Tago gave the lowest signals.

The above results show that all three configurations define reactive samples at the same time after the acute phase of the disease (as evidenced by the increased level of alanine aminotransferase). In addition, the results show that the sensitivity of enzyme-linked immunosorbent tverdova against immunoglobulin G is equal to or higher sensitivity, obtained using other test configurations enzyme conjugates.

IV.I.6.b. Samples from random blood donors

Samples from random blood donors were investigated for infection with hepatitis C virus (see section IV.I.1), using the ELISA test C100-3 of the hepatitis C virus, in which a conjugate of the antibody - enzyme was either monoclonal conjugate of antiimmunoglobulin G, or polyclonal conjugate. The total number of specimens tested was 1077 and 1056 for polyclonal conjugate and monoclonal conjugate, respectively. The overall result of the screening are presented in table. 15 and sample distribution shown in the histogram in Fig. 44.

Calculations of the average values and standard deviations were conducted, excluding the samples that gave a signal higher than 1.5, i.e., 1073 absorbance values were used in calculations when using polyclonal conjugate and 1051 value for monoclonal conjugate of antiimmunoglobulin G. As can be seen from the table. 15, if used polyclonal conjugate, then the average value deviated from 0,0493 to 0,0931, and the standard deviation increased from 0,074 to 0,0933. In addition, the results also show CityLine antibody the enzyme conjugate requires ELISA higher threshold values. This indicates a reduced specificity in comparison with monoclonal system. In addition, as shown in the histogram in Fig. 44, a greater separation between the positive and negative distributions you receive, if random blood donors tested by ELISA analysis using monoclonal conjugate of antiimmunoglobulin G compared with analysis using commercial polyclonal tag.

IV.J. The definition of seroconversion of hepatitis C virus in patients with hepatitis neither A nor B from different geographical locations

Serum of patients suspected hepatitis neither A nor B on the basis of increased levels of alanine aminotransferase and have had negative tests for hepatitis A and hepatitis B, was tested using radioimmunoassay analysis mainly as described in section IV.D, except that the antigen C100-3 of the hepatitis C virus was used as a screening antigen in microtitre panels. As can be seen from the results shown in table. 16, radioimmunoassay analysis identified positive samples in a high percentage of cases.


Sera obtained from 100 patients with hepatitis neither A nor B, with no obvious way of transfer (i.e., no blood transfusion, intravenous drugs, promiscuity and so on, which have been identified as risk factors), were provided by Dr. M. Alter from the Center for disease control and Dr. J. Dienstag from Harvard University. These samples were tested using radioimmunoassay analysis mainly as described in section IV.D, except that the used antigen C100-3 of the hepatitis C virus as excluding the antigen associated with microturbine panels. The results show that out of 100 samples of 55 serum samples contained antibodies that react immunologically with antigen C100-3 of hepatitis C virus

The above results suggest that the "utility" hepatitis neither A nor B is caused by hepatitis C. in Addition, since it has been demonstrated here that the hepatitis C virus belongs to flaviviruses, most of whom migrated arthropods, it can be assumed that the transmission of hepatitis C virus in "domestic" cases is also the result of the migration arthropods.

IV. L. Comparison of frequency of occurrence
A prospective study was conducted to determine whether recipients of blood donors, suspicious for hepatitis neither A nor B who are hepatitis b neither A nor B, seroconvertion positive for antibodies against hepatitis C. blood tested other markers anormality, i.e. elevated levels of alanine aminotransferase and the presence of antibodies against the core antigen. In addition, the donors were also tested for the presence of antibodies against hepatitis C virus, was performed using a radioimmunoassay analysis, as described in section IV.K. The results of the study are presented in table. 17, which shows the number of patients (column 1); the presence of antibodies against hepatitis C virus in the serum of patients (column 2); the number of injections received by patients with each transfusion received from various donors (column 3); the presence of antibodies in the serum of the donor (column 4) and deviations from the norm, certain other markers in blood donors (column 5) (NT or -- "means" not tested") (ALT means "increased transaminase and ANTI-HBc indicates antibody against the core antigen of hepatitis B virus).

The results presented in table. 17, show that the texts markers-substitutes. Nine out of ten patients who manifested symptoms of hepatitis neither A nor B, gave a positive test for seroconversion to antibodies against hepatitis C virus From 11 suspicious donor (patient 6 received blood from two different individuals, suspected carriers of hepatitis b neither A nor B), nine were positive for antibodies against hepatitis C virus and one patient was in the lower range of positive and therefore questionable (donor to patient 1). In contrast, using the test increased transaminase six out of ten donors have given a negative test result, and using the test antitumorigenic antigen five out of ten donors showed a negative test result. More consistent test for antibodies against the core antigen of hepatitis B virus, gave inconsistent results.

IV. M Increase cloning sequences circular DNA of hepatitis C virus using the growth response and polymerase chain initiators derived from conserved sections of the genomic sequences of flavivirus

The results presented above point to the fact that hepatitis C is flaviviruses or flaviopolis virus, and allow the strategy cloa and polymerase chain initiators, obtained from the plots, the coding sequence of a conserved amino acids of flaviviruses. In General, one of the initiators from a specific genomic sequence of the hepatitis C virus, and the other initiator, which is adjacent to the site inconsistent polynucleotide of hepatitis C virus derived from a conserved region of the genome of flaviviruses. The genomes of flavivirus known to contain conserved sequence in NS1 and E polypeptides that are encoded in the 5'region of the genome of flaviviruses. The corresponding sequences encoding these areas lie on rising from a sequence of circular DNA virus hepatitis C shown in Fig. 26. Thus, to select a sequence of circular DNA, derived from this region of the genome of hepatitis C virus, designed ascendant initiators, which are derived from conserved sequences in these polypeptides of flaviviruses. Ascending initiators received from the upstream end of the famous sections of circular DNA virus of hepatitis C.

Because of violations of the code of the likely discrepancy between the samples flavivirus and the corresponding genomic sequence of the virus of hepatitis C. Therefore, use plementary products reverse translation of amino acid sequences; sequences in mixed initiators take into account every breach of the codon for conserved amino acid sequence.

Were generated by three composition initiator mixtures, based on the amino acid homologues found in different flaviviruses, including Dengue-2,4 (D-2,4), Japanese encephalitis virus, yellow fever virus and West Nile virus. Initiating a mixture of mostly rising conserved sequence (5'-1), based on amino acid sequence glycine-tryptophan-glycine, which is part of the conserved sequence asparagine-arginine-glycine-tryptophan-glycine-asparagine, found in the E protein of the virus Dangle, Japanese encephalitogen virus, yellow fever virus and West Nile virus. The following initiator mixture (5'-2) is based on the falling conserved in the sequence of the E protein, i.e. phenylalanine - asparagine-glycine-asparagine-serine-theronin-isoleucine-phenylalanine - glycine-asparagine-serine-theronin-isoleucine, and derived from phenylalanine-glycine-asparagine; canned sequence is present in viruses Dangle, Japanese encephalites, yellow fever and Zapadno is which is part of the conserved sequence cystine-cystine-arginine-serine-cystine in the protein NS 1 viruses Dangle-2, D-4, Japanese encephalitogen, yellow fever and West Nile. Separate initiators which form the mixture into 5'-3 shown in Fig. 45. In addition to the various sequences derived from conserved areas, each initiator in each mixture also contains a permanent site in the 5'-end, which contains a sequence encoding a localization for limiting enzymes: HindIII, MboI and EcoRI.

Descending the initiator ssc5h20A derived from the nucleotide sequence of the clone 5h, which contains a sequence of circular DNA virus hepatitis C, overlapping sequences in the clones 14i and 11b. The sequence ssc5h20A is:

5' GTA ATA TGG TGA CAG AGT CA-3'.

Can be obtained also alternative initiator ssc5h34A. This initiator is obtained from the sequence in clone 5h, in addition contains nucleotides in the 5'-end, which form the localization limiting enzymes, thus facilitating cloning. The sequence ssc5h34A the following:

5' GAT CTC TAG AGA AAT CAA TAT GGT GAC AGA GTC A-3'.

The polymerase chain reaction, which originally was described by Saiki et al. (1986), conducted mainly by the method of Lee et al. (1988), except that the matrix for circular DNA suitable for all kinds of the viral particles, isolated from the serum of infected with hepatitis C, a chimpanzee, as described in section IV.A.1. B addition of renaturation conditions less restrictive in the first round of increase (0.6 M sodium chloride and 25oC) as part of the initiator, who will denaturiruet to the sequence of the hepatitis C virus, is a sequence of only 9 nucleotides, and there can be discrepancies. In addition, if you use ssc5h34A, the additional sequence not derived from the genome of the hepatitis C virus, tend to destabilize the hybrid matrix - initiator. After the first stage, increasing the renaturation conditions may be more stringent (of 0.066 M sodium chloride and 32-37oC) because of the increased sequence contain areas that are complementary to the initiators or duplicating them. In addition, the first 10 cycles of increasing proceed with enzyme 1 maple under suitable conditions for this enzyme. After these cycles, the samples were extracted and dispersed Tag polymerase in accordance with instructions developed Cetus/Perkin-Elmer.

After increasing the enlarged ring sequence of the DNA of the hepatitis C virus was determined by hybridization with the use of the sledovatelnot, used to obtain the initiator, and does not overlap the sequence initiator derived from clone 5h. The sequence of samples is as follows:

5' CCC AGC GGC GTA CGC GCT GGA CAC GGA GGT GGC CGC GTC GTG TGG CGG TGT TGT TCT CGT CGG GTT GAT GGC GC 3'.

IV.N.1. Creating a pool of circular DNA of hepatitis C virus from the liver of a chimpanzee infectious hepatitis neither A nor B

The pool is circular DNA of the hepatitis C virus was created from the liver of a chimpanzee, which had created a pool of circular DNA virus hepatitis C in section IV.A.1. The method of creating the pool was similar to that described in section IV.A.24, except that an excellent source of RNA and that was used by the initiator based on the sequence in the circular DNA of hepatitis C virus in clone 11b. The sequence initiator was the following:

5' CTG GCT TGA AGA ATC 3'.

IV. N. 2. The selection of the nucleotide sequence of the circular DNA of hepatitis C virus in the clone k9-1, overlapping circular DNA in clone 11b

Clone k9-1 was selected from a pool of circular DNA virus hepatitis C, a liver infected with hepatitis neither A nor B chimpanzees, as described in section IV. A. 25. Pool were tested clones that overlap the sequence in clone 11b, by using the clone, which perekryvatel with a frequency of one clone at 500000. One isolated clone k9-1, were subjected to further study. The nature of overlapping circular DNA virus hepatitis C in clone k9-1 in the 5'-end sequence of circular DNA virus hepatitis C in Fig. 26 was confirmed by the test clone with clone Al46; this clone contains a sequence of circular DNA virus hepatitis C in 30 base pairs, which corresponds to the sequence of the base pairs at the 5' terminal circular DNA virus hepatitis C in clone 14i described above.

The nucleotide sequence of the circular DNA of hepatitis C virus from clone k9-1 was determined using the method described above.

The sequence of the circular DNA of hepatitis C virus in the clone k9-1, overlapping with the circular DNA virus hepatitis C in Fig. 26 and encoded in its amino acid shown in Fig. 46.

The sequence of the circular DNA of hepatitis C virus in the clone k9-1 was ordered in line with the sequences of the clones described in section IV.A. 19 to create a composite sequence of circular DNA of hepatitis C virus sequence k9-1, placed at the top in the direction of the sequence shown in Fig. 32. Composite circular DNA virus hepatitis C, which is an amino acid Sequence that encoded in the 5'section of the ring virus DNA, hepatitis C, shown in Fig. 47, compared with the corresponding plot of one of the strains of Dengue virus, described above, with respect to the profile plots of hydrophobicity and hydrophilicity. This comparison showed that the polypeptides of hepatitis C virus and Dengue virus encoded in this area, which corresponds to the segment encoding protein NS1 (or part thereof), have similar hydrophobic/hydrophilic profile.

The information below allows you to identify strains of hepatitis C. the Selection and characteristics of other strains of the hepatitis C virus can be carried out by separating the nucleic acids from body components containing viral particles, creating a pool of circular DNA using polynucleotide samples based on samples of circular DNA virus hepatitis C described above, the research pools to the screening of clones containing sequences circular DNA virus hepatitis C, described below, and compare the circular DNA of hepatitis C virus from the newly selected samples with circular DNA, described below. Polypeptides encoded in them or in the viral genome, can be registered and verified on immunologically meet the parameters of the hepatitis C virus, as described in the definitions above, it is easily identifiable. Other methods of identification of strains of the hepatitis C virus will be obvious to experts in this field on the basis of the information provided here.

Industrial applicability

The present invention, disclosed in various forms in the description, has various industrial applications, which may include the following. Circular DNA virus hepatitis C can be used to design assays to detect the nucleic acid of hepatitis C virus in the samples. Samples were obtained from circular DNA can be used to identify nucleic acid of hepatitis C virus, for example, in the reactions of chemical synthesis. They can also be used in the screening of antiviral agents to identify agents through the inhibition of viral replication and cell culture systems and in animal model systems. Polynucleotide samples of hepatitis C virus is also useful for the detection of viral nucleic acids in humans, and thus can serve as a basis for the diagnosis of infection of hepatitis C virus in humans.

In addition to the above described here circular DNA is tidy useful for the detection of antibodies to antigens of hepatitis C. A number of immunological tests for the infection of hepatitis C virus-based recombinant polypeptides containing epitopes of hepatitis C virus, is given in the description and can find commercial use in the diagnosis of hepatitis C caused by the hepatitis b neither A nor B, and the study (screening) blood Bank donors to identify caused by hepatitis C virus hepatitis, as well as for the determination of blood contamination from infectious blood donors. Viral antigens can also be useful in assessing the effectiveness of antiviral agents in animal model systems. In addition, the polypeptides derived from the circular DNA of hepatitis C virus, as described in the application, can be used as vaccines for the treatment of infections of hepatitis C virus

Polypeptides derived from circular DNA, in addition to the previously mentioned applications, can also be useful for stimulating the appearance of antibodies against hepatitis C. Thus, they can be used in vaccines against hepatitis C. of Course, and antibodies generated by immunization with peptides of hepatitis C virus can also be useful for determining the presence of viral antigens in the samples. Therefore, they maguth. Antibodies against hepatitis C virus can also be used to assess the efficacy of antiviral agents in the implementation of screening programs in which these agents experience in tissue culture. They can also be used for passive immunotherapy and diagnosis of hepatitis b neither A nor B is caused by hepatitis C virus, allowing the detection of viral antigen(s) in blood donors and recipients. Another important application is found antibodies against hepatitis C virus in affinity chromatography for the purification of virus and viral polypeptides. These purified preparations of virus and viral polypeptides can be used in vaccines. Of course that purified viruses can be useful for the development of cell culture systems in which replicates the hepatitis C virus

Cell cultures containing infected with hepatitis C cells, can find various applications. They can be used for large-scale production of hepatitis C virus, which normally is a low virus titer. These systems can be useful for clarifying the molecular biology of the virus and lead to the development of antiviral agent(s). System cultures klepetar, drilled for hepatitis C, useful for the production of attenuated strains of hepatitis C.

For the convenience of antibodies against hepatitis C virus and polypeptide of hepatitis C virus, natural or recombinant, may be Packed in sets (sets).

The method used to allocate circular DNA virus hepatitis C, which includes obtaining the pool of circular DNA obtained from infected tissue of individuals in the expression vector, and selecting clones that produce the products of expression of reacting immunologically with antibodies containing antibodies components of the body from another(their) infected individuals, but not from uninfected individuals, can also be used to highlight the circular DNA obtained from other non-characterized-related disease agents that contain genomic component. This, in turn, can lead to isolation and characterisation of these agents and to diagnostic reagents and vaccines against these other disease agents.

1. Polypeptide having antigenic properties of HBV With having the amino acid sequence shown in Fig.47.

2. The polypeptide under item 1, otlichaet a sequence of beta-galactosidase, or superoxide dismutase.

3. A fragment of the polypeptide described in paragraph 1, containing at least 10 amino acid residues.

4. A fragment of the polypeptide under item 3, wherein the amino acid sequence of HCV merged with the sequence of merge, which is a sequence of beta-galactosidase, or superoxide dismutase.

5. A fragment of a polypeptide having antigenic properties of the hepatitis C virus, characterized in that it contains 363 amino acid residue HCV, the sequence of which is shown in Fig.36 associated with the sequence of superoxide dismutase.

6. Polypeptide having antigenic properties of hepatitis C virus comprising the amino acid sequence shown in Fig.4.

7. The polypeptide under item 6, characterized in that the amino acid sequence of HCV merged with the sequence of merge, which is a sequence of beta-galactosidase, or superoxide dismutase.

8. A fragment of the polypeptide described in paragraph 6, comprising at least 10 amino acid residues.

9. A fragment of the polypeptide under item 8, wherein the amino acid sequence of HCV merged with poledouris.

10. Polypeptide having antigenic properties of hepatitis C virus comprising the amino acid sequence shown in Fig.14.

11. The polypeptide under item 10, wherein the amino acid sequence of HCV merged with the sequence of merge, which is a sequence of beta-galactosidase, or superoxide dismutase.

12. A fragment of the polypeptide described in paragraph 10, characterized in that it contains at least 10 amino acid residues.

13. A fragment of the polypeptide under item 12, characterized in that the amino acid sequence of HCV merged with the sequence of merge, which is a sequence of beta-galactosidase, or superoxide dismutase.

14. Diagnostic reagent for the detection of antibodies against hepatitis C virus with immune assay containing polypeptide, described in any of paragraphs.1 and 2 or a polypeptide fragment described in any of paragraphs.3 - 5.

15. Diagnostic reagent under item 14, characterized in that the polypeptide or its fragment is mixed with the solid phase.

16. Diagnostic reagent for the detection of antibodies against hepatitis C virus using eastersunday in any of paragraphs.8 and 9.

17. Diagnostic reagent under item 16, characterized in that the polypeptide or fragment of the polypeptide is mixed with the solid phase.

18. Diagnostic reagent for the detection of antibodies against hepatitis C virus using immunological analysis, including the polypeptide described in any of paragraphs.10 and 11, or a fragment of the polypeptide described in any of paragraphs.12 and 13.

19. Diagnostic reagent under item 18, characterized in that the polypeptide or fragment of the polypeptide is mixed with the solid phase.

20. Diagnostic reagent for the detection of antibodies against hepatitis C virus using immunological analysis, including the polypeptide described in paragraph 10, or a fragment of the polypeptide described in any of paragraphs.12 and 13.

21. Diagnostic reagent according to p. 20, characterized in that the polypeptide or fragment of the polypeptide is mixed with the solid phase.

22. Kit for detection of antibodies against hepatitis C virus by immune analysis, including diagnostic reagent described in any of paragraphs.14 and 15, and control reagents.

23. Kit for detection of antibodies against hepatitis C virus by immune assay containing the diagnostic reagent, karavirta hepatitis C by immunological analysis, containing the diagnostic reagent described in any of paragraphs.18 - 21, and control reagents.

25. The detection of antibodies against hepatitis C virus by the immune analysis, providing incubation diagnostic system with biological way, presumably containing antibodies against hepatitis C virus, in conditions which ensure the formation of complex antiheretical, and subsequent detection of the specified complex, wherein the diagnostic system using the reagent described in any of paragraphs.1 4 and 15, or set, as described in paragraph 22.

26. The method according to p. 25, wherein detection of the complex antigen-antibody carried out using labeled antibodies against human immunoglobulins.

27. A method for detecting antibodies to hepatitis C virus by the immune analysis, providing incubation diagnostic system with a biological sample suspected of containing antibodies against hepatitis C virus, in conditions which ensure the formation of the complex antigen-antibody, and subsequent detection of the specified complex, wherein the diagnostic system ispab on p. 27, wherein detection of the complex antigen-antibody carried out using labeled antibodies directed against human immunoglobulins.

29. A method for detecting antibodies to hepatitis C virus by the immune analysis, providing incubation diagnostic system with a biological sample suspected of containing antibodies against hepatitis C virus, in conditions which ensure the formation of the complex antigen-antibody, and subsequent detection of the specified complex, wherein the diagnostic system using the reagent described in any of paragraphs.18 - 21, or set, as described in paragraph 24.

30. The method according to p. 29, wherein detection of the complex antigen-antibody carried out using labeled antibodies directed against human immunoglobulins.

Priority points:

18.11.87 - PP.6-9, 16, 17, 22, 26, 27;

06.05.88 - PP.10-13, 18, 19, 20, 23, 28, 29;

18.11.88 - PP.1-5, 14, 15, 21, 24, 25.

 

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