JP3848663B2 - Non-A non-B non-C non-D non-E hepatitis reagents and methods for their use - Google Patents

Description

  The present invention includes a group of infectious viral substances that cause hepatitis in humans, in particular polynucleotides derived from such viruses, polypeptides encoded by them, antibodies that specifically bind to such polypeptides, and It relates to diagnostics and vaccines using such materials.

  Hepatitis is one of the most important diseases transmitted from donors to recipients by blood product transfusion, organ transplantation and hemodialysis. Hepatitis can also be transmitted by ingestion of contaminated food and water, or contact between people. Viral hepatitis contains a group of viral substances with unique viral genes and replication modes, and is known to cause hepatitis, although the degree of liver damage varies with different infection routes. Acute viral hepatitis is clinically diagnosed by jaundice, liver tenderness, and specific patient symptoms including elevated levels of liver transaminase such as aspartate transaminase (AST), alanine transaminase (ALT) and isocitrate dehydrogenase (ISD) However, it may not be clinically manifested. As viral hepatitis substances, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis δ virus (HDV), hepatitis E virus (HEV), Epstein-Barr Virus (EBV), and cytomegalovirus (CMV).

A specific serum assay that screens blood donated blood for the presence of HBV surface antigen (HBsAg), available in the late 1960s, has shown good results in reducing the incidence of post-transfusion hepatitis (PTH) in blood recipients. Although given, PTH is still occurring at a significant rate. H. J. et al. Alter et al . , Ann. Int. Med . 77: 691-699 (1972); J. et al. Alter et al., Lancet ii: 838-841 (1975). “Non-A non-B hepatitis”, which causes viral hepatitis regardless of exposure to viruses previously known to cause hepatitis in humans (HAV, HBV, CMV and EBV) We began looking for a new substance named (NANBH). For example, S.W. M.M. Feinstone et al., New Engl. J. et al. Med . 292: 767-770 (1975); Editor's Anonymous, Lancet ii: 64-65 (1975); N. Fields' F.R. B. Hollinger and D.H. M.M. See Knipe et al., Virology , Raven Press, New York, pp 2239-2273 (1990).

Multiple occurrences of acute NANBH in intravenous drug users; inherent post-infection latency of patients who have acquired post-transfusion NANBH; results of a chimpanzee cross-challenge experiment; ultrastructural liver pathological analysis of infected chimpanzees; Some epidemiological and experimental evidence, such as differences in resistance to chloroform in sham patients and sham patients, indicate the presence of one or more parenterally infected NANB substances. J. et al. L. Dientag, Gastroenterology 85: 439-462 (1983); L. Dientag, Gastroenterology 85, 743-768 (1983); B. Hollinger et al., J. MoI. Infect. Dis . 142: 400-407 (1980); Chisari's D.C. W. Bradley, Advances in Hepatitis Research , Masson, New York, pp. 268-280 (1984); W. Bradley et al., J. MoI. Infect. Dis . 148: 254-265 (1983).

Serum samples obtained from surgeons who have developed acute hepatitis have been shown to induce hepatitis when inoculated into tamarins (Saguinus species). Of the 4 tamarins, 4 developed liver enzyme elevations within a few weeks after inoculation, which indicates that the substances in the surgeon's serum may have induced hepatitis in the tamarins. Through successive passages in various non-human primates, this hepatitis was found to be caused by an infectious substance, and filtration experiments indicated that this substance was viral. The infectious substance that caused hepatitis in the surgeon and tamarin was named “GB agent”. F. Deinhardt et al., J. MoI. Exper. Med . 125: 673-688 (1967); Dienhardt et al., J. MoI. Exper. Med . , Supra; Tabor et al . Med. Virol . 5: 103-108 (1980); O. Whittington et al., Viral and Immunological Diseases in Nonhuman Primates , Alan R., et al. Liss, Inc. , New York, pp. 221-224 (1983).

GB substances can be NANBH-inducing substances in humans, and GB substances have been shown to be unrelated to known NANBH substances studied in various laboratories, but are critical for GB substances Also, the final study is unknown, no viral material has been discovered and molecularly characterized. F. Deinhardt et al . , Am. J. et al. Med. Sci . 270: 73-80 (1975); L. Dienstag et al., Nature 264: 260-261 (1976). E. Tabor et al . Med. Virol . , Supra; Tabor et al . Infect. Dis . 140: 794-797 (1979); O. Whittington et al., Supra; See Karayiannis et al., Hepatology 9: 186-192 (1989).

Early studies indicated that GB material was unrelated to any known human hepatitis virus. S. M.M. Feinstone et al., Science 182: 1026-1028 (1973); J. et al. Provost et al . , Proc. Soc. Exp. Biol. Med . 148; 532-539 (1975); L. Melnick, Intervirology 18: 105-106 (1982); W. Holmes et al., Nature 243: 419-420 (1973); Deinhardt et al . , Am. J. et al. Med. Sci . , Supra. However, a potential tamarin that is a virus that induces hepatitis infection in humans or that is activated by GB serum and, once activated, easily passes to other tamarins and induces hepatitis in them. The problem was whether it was a virus. In addition, a small portion of marmoset inoculated with GB-positive serum did not clinically develop hepatitis (4 out of 52 animals, 7.6%), and such animals may have had innate immunity Thus, it has also been shown that the GB material can be a marmoset virus. W. P. Parks et al . Infect. Dis . 120: 539-547 (1969); P. Parks et al . Infect. Dis . 120: 548-559 (1969). Morphological studies are ambiguous, and an immunoelectron microscopy study in one report shows that GB material forms an immune complex with a size distribution of 20-22 nm, similar to the parvovirus sphere structure. However, in other studies, 34-36 nm aggregates of icosahedral symmetry were detected from immunoelectron microscopic data obtained from liver homogenates of GB-positive tamarins, and the GB substance was a calicilike virus. It is reported that. For example, J. et al. D. Almeida et al., Nature 261: 608-609 (1976); L. See Dienstag et al., Nature , supra.

Two types of viruses that cause hepatitis, HCV mainly caused by parenteral infection and HEV intestinal infection, were recently discovered and reported. For example, Q.I. L. Choo et al., Science 244: 359-362 (1989), G.M. Kuo et al., Science 244: 362-364 (1989), European Patent Application Publication No. 0318216 (published on March 31, 1989), G.M. R. See Reyes et al., Science 247: 1335-1339 (1990). HCV is responsible for most of the PTH attributed to NANBH substances and many of the acute NANBHs that are not due to blood transfusions. Editor's Anonymous, Lancet 335: 1431-1432 (1990); L. Dientag, Gastroenterology 99: 1177-1180 (1990); J. et al. Alter et al., JAMA 264: 2231-2235 (1990).

Although detection of HCV antibodies in a donor sample excludes 70-80% of NANBH-infected blood in the blood supply system, the discovery and detection of HCV does not completely prevent hepatitis infection. H. Alter et al., New Eng. J. et al. Med . 321: 1494-1500 (1989). Recent literature has questioned whether another hepatitis substance may be responsible for PTH and population acquired acute and / or chronic hepatitis unrelated to PTH. For example, out of 181 patients monitored in a predictive clinical specimen survey conducted in France from 1988 to 1990, the investigator has described a total of 18 PTH cases. Thirteen of these 18 patients were negative for anti-HCV antibodies, HBsAg, HBV and HCV nucleic acids. The authors have inferred the potential importance of non-A, non-B, non-C materials that induce PTH. V. Thiers et al . Hepatology 18: 34-39 (1993). Of the 1,476 patients monitored in another study conducted in Germany in 1985-1988, 22 recorded PTH cases were not associated with HBV or HCV infection. T.A. Peters et al . Med. Virol . 39: 139-145 (1993).

  Substances derived from a novel, unique group of viruses that cause hepatitis, such as polynucleotides, recombinant and synthetic polypeptides encoded therein, antibodies that specifically bind to such polypeptides, and such substances It would be advantageous to identify and provide diagnostic agents and vaccines. Such substances significantly improve the ability of medical institutions to more accurately diagnose acute and / or chronic viral hepatitis and detect non-A, non-B and non-C hepatitis in the blood and organs provided. Could provide safer blood and organs.

(Summary of the Invention)
The present invention relates to a purified polynucleotide derived from HGBV that can selectively hybridize to the genome of hepatitis GB virus (HGBV) or its complement, or a fragment thereof, comprising HGBV-A, HGBV-B and HGBV-C. An amino acid sequence having at least 35% identity, more preferably 40% identity, more preferably 60% identity, and most preferably 80% identity with an amino acid sequence selected from the group consisting of Purified polynucleotides or fragments thereof characterized by a positive-strand RNA genome comprising an open reading frame (ORF) encoding a polyprotein comprising Further provided is a recombinant polynucleotide or fragment thereof derived from HGBV that can selectively hybridize to the genome of hepatitis GB virus (HGBV) or its complement, wherein said nucleotide is at least one of HGBV. The recombinant nucleotide comprises an amino acid sequence having an identity of at least 35% with an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C. Characterized by a positive-stranded RNA genome containing an open reading frame (ORF) encoding a polyprotein. Such a recombinant polynucleotide is contained in a recombinant vector and further constitutes a host cell transformed with the vector.

  The present invention further relates to an HGBV recombinant polynucleotide comprising a nucleotide sequence derived from the hepatitis GB virus (HGBV) genome, or a fragment thereof, which is contained in a recombinant vector and further transformed with said vector. A host cell is provided, which provides an HGBV recombinant polynucleotide or fragment thereof wherein the sequence encodes an epitope of HGBV. An HGBV recombinant polynucleotide is an open reading frame (ORF) encoding a polyprotein comprising an amino acid sequence having at least 35% identity with an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C Is characterized by a positive-strand RNA genome comprising The present invention relates to a recombinant expression system comprising an open reading frame of DNA or RNA derived from hepatitis GB virus (HGBV), wherein the open reading frame comprises a sequence of HGBV genome or cDNA, and the open reading frame comprises Provided is a recombinant expression system operably linked to a control sequence compatible with the desired host, and further includes a cell transformed with the recombinant expression system and a length produced by the cell. Provides a polypeptide of at least about 8 amino acids.

  The present invention further provides a preparation of hepatitis GB virus (HGBV) polypeptide or fragment thereof, ie, an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C, with a percent identity of at least 35%; More preferably, it comprises an amino acid sequence characterized by a positive strand RNA genome comprising an open reading frame (ORF) encoding a polyprotein comprising an amino acid sequence having 40% identity, more preferably 60% identity, or a fragment thereof. Purified HGBV comprising the recombinant polypeptide is provided. Polyclonal and monoclonal antibodies, and fusion polypeptides comprising at least one hepatitis GB virus (HGBV) polypeptide or fragment thereof, amino acid sequences that can form particles when produced in eukaryotic or prokaryotic hosts From a group consisting of HGBV-A, HGBV-B, and HGBV-C, and a particle that is immunogenic against hepatitis GB (HGBV) infection, comprising a non-HGBV polypeptide having: and at least one HGBV epitope A polynucleotide probe for hepatitis GB virus (HGBV), characterized by a positive strand RNA genome comprising an open reading frame (ORF) encoding a polyprotein comprising an amino acid sequence having at least 35% identity with a selected amino acid sequence Provided.

  An assay kit and method for producing a polypeptide comprising at least one hepatitis GB virus (HGBV) epitope, comprising at least 35 amino acid sequences selected from the group consisting of HGBV-A, HGBV-B and HGBV-C A host cell transformed with an expression vector comprising a sequence encoding a polypeptide characterized by a positive-strand RNA genome comprising an open reading frame (ORF) encoding a polyprotein comprising an amino acid sequence having a percent identity. Also provided are assay kits and methods consisting of incubating. Further provided are methods for detecting HGBV nucleic acids, antigens and antibodies in a test sample, including methods using solid phases, recombinant or synthetic peptides, or probes. In addition, an individual is administered a vaccine, tissue culture proliferating cells infected with hepatitis GB virus (HGBV), an isolated immunogenic polypeptide or fragment thereof containing an amount of at least one HGBV epitope sufficient to produce an immune response A method for producing an antibody against hepatitis GB virus (HGBV) is also provided. Further provided are diagnostic reagents comprising polynucleotides or polypeptides or fragments thereof.

(Details of the invention)
The present invention relates to the characterization of non-A, non-B, non-C, non-D, and non-E hepatitis that are newly identified etiological agents, generically "hepatitis GB virus" or "HGBV" Provide law. The present invention provides a method for determining the presence of an HGBV etiological agent, an etiologic nucleic acid produced from infected serum, plasma or liver homogenate from a human or tamarin individual infected with HGBV and has been isolated so far. Provided is a method for detecting newly synthesized antigens from the genome of no viral material and selecting a clone that produced a product that is found only in infected individuals compared to non-infected individuals.

  The portion of the nucleic acid sequence derived from HGBV is useful as a probe to determine the presence of HGBV in a test sample and to isolate a natural variant. Such sequences can provide the polypeptide sequence of the HGBV antigen encoded within the HGBV genome, or can produce polypeptides useful as diagnostic test standards or reagents and / or as components of vaccines. Monoclonal and polyclonal antibodies against at least one epitope contained within such polypeptide sequences are also useful for diagnostic tests and therapeutics, for screening for antiviral substances, and for isolating HGBV substances from which such nucleic acid sequences are derived. It is. Isolation and sequencing of other parts of the HGBV genome can also be performed by using probes or PCR primers derived from such nucleic acid sequences, and thus prophylactic and therapeutic agents in the diagnosis and / or treatment of HGBV Other probes and polypeptides of HGBV that would be useful as can be produced.

  According to one aspect of the invention, a purified HGBV polynucleotide, a recombinant HGBV polynucleotide, a recombinant polynucleotide comprising a sequence derived from the HGBV genome, a recombinant polypeptide encoding an epitope of HGBV, an epitope of HGBV Synthetic peptides encoding, recombinant vectors containing any of the above recombinant polypeptides, and host cells transformed with such vectors are provided. These recombinant polypeptides and synthetic peptides can be used alone or in combination, or can be used in combination with other substances that give an epitope of HGBV.

  In another aspect of the invention, there is provided purified HGBV, a preparation of purified HGBV-derived polypeptide, purified HGBV polypeptide, purified polypeptide comprising an epitope that is immunologically identical to an epitope contained in HGBV .

  In yet another aspect of the invention, recombinant expression comprising an open reading frame (ORF) of DNA derived from the HGBV genome or HGBV cDNA operably linked to control sequences that are compatible with the desired host. Systems, cells transformed with the recombinant expression system, and polypeptides produced by the transformed cells are provided.

  Another aspect of the present invention relates to at least one recombinant polypeptide comprising a sequence derived from at least one recombinant HGBV polypeptide, HGBV genome or HGBV cDNA, at least one recombinant polypeptide comprising an HGBV epitope, HGBV At least one fusion polypeptide comprising the polypeptide.

  Furthermore, the present invention provides a method for producing a monoclonal antibody that specifically binds to at least one epitope of HGBV, a purified preparation of a polyclonal antibody that specifically binds to at least one HGBV epitope, and diagnostic, prophylactic and therapeutic uses A method of using such an antibody is provided.

  In yet another aspect of the invention, an immunogenicity against HGBV infection comprising a non-HGBV polypeptide having an amino acid sequence capable of forming particles when produced in a eukaryotic host and an HGBV epitope. Of particles are provided.

  In addition, a polynucleotide probe for HGBV is also provided.

  The present invention is a reagent that can be used to detect the presence and / or amount of a polynucleotide derived from HGBV in a suitable container, which is derived from HGBV consisting of about 8 or more nucleotides. A reagent comprising a polynucleotide probe comprising a nucleotide sequence; a reagent for detecting the presence and / or amount of HGBV antigen in an appropriate container, comprising an antibody against the HGBV antigen to be detected; and suitable There is provided a kit comprising a reagent for detecting the presence and / or amount of an antibody against HGBV antigen in a suitable container, the reagent comprising a polypeptide comprising an HGBV epitope present in HGBV antigen. Other kits for various assay formats are also provided by the invention described herein.

  Other aspects of the invention include a polypeptide comprising at least one HGBV epitope attached to a solid phase and an antibody against the HGBV epitope attached to the solid phase. Furthermore, a polymorphism comprising an HGBV epitope comprising incubating a host cell transformed with an expression vector comprising a sequence encoding a polypeptide comprising an HGBV epitope under conditions that allow expression of the polypeptide. A method for producing a peptide and a polypeptide comprising an HGBV epitope produced by the method are also included in the present invention.

  The invention further provides assays that use the recombinant or synthetic polypeptides of the invention and the antibodies of the invention in a variety of formats, both of which can use signal-generating compounds. Also provided are assays that do not use signal-generating compounds to provide detection means. All described assays will usually detect either or both antigens or antibodies and the test sample is contacted with at least one reagent of the present invention to form at least one antigen / antibody complex. And detecting the presence of the complex. Such an assay is described in detail.

  Use of an immunogenic peptide comprising an HGBV epitope, an inactivated preparation of HGBV, or a vaccine for the treatment of HGBV infection comprising an attenuated preparation of HGBV, or a recombinant vaccine expressing an HGBV epitope, and / or a synthetic peptide Is also included in the present invention. For effective vaccines, combinations of these immunogenic peptides may be used (eg, cocktails of recombinant antigen, synthetic peptide and natural viral antigen are administered simultaneously or at different times). Some of these can be used alone, while others can be supplemented later with separate immunogenic epitopes. Furthermore, the present invention provides a method for producing antibodies against HGBV, comprising administering to an individual an isolated immunogenic polypeptide comprising an amount of an HGBV epitope sufficient to generate an immune response in the inoculated individual. Is also included.

  Furthermore, tissue culture proliferating cells infected with HGBV are also provided by the present invention.

  In yet another aspect of the invention, a method is provided for isolating DNA or cDNA derived from the genome of an unidentified infectious agent, the method comprising representational difference analysis (RDA). This is an unprecedented improvement method, which will be described later.

(Definition)
As used herein, the term “hepatitis GB virus” or “HGBV” refers to viral species that cause non-A, non-B, non-C, non-D, non-E hepatitis in humans and derived therefrom. Attenuated strains or defective interfering particles are generically indicated. This is further due to acute viral hepatitis infected by contaminated food, drinking water, etc .; including HGBV infected by human contact (including sexual activity, respiratory and parenteral route transmission) or intravenous drug use Hepatitis is also included. The method of the present invention makes it possible to identify an individual who has acquired HGBV. Individually, HGBV isolates are designated specifically as “HGBV-A”, “HGBV-B” and “HGBV-C”. As used herein, the HGBV genome consists of RNA. From analysis of the nucleotide sequence and deduced amino acid sequence of HGBV, it is clear that this group of viruses has a similar genomic organization as the Flavividae family. Basically, but not absolutely, based on the similarity of genomic organization, the International Committee on the Taxonomic of Viruses recommends that this family consist of three genera: Flavivirus, Pestivirus and Hepatitis C group . Looking for similarity at the amino acid level, it can be seen that the sequence of the hepatitis GB virus subclone is somewhat similar to that of the hepatitis C virus. The information given here is sufficient to classify other strains of HGBV.

There is some evidence to show that HGBV-C is not a genotype of HCV. First, the presence of HCV antibodies was tested in sera containing HGB-C sequences. Conventional detection methods for individuals exposed to or infected with HCV rely on antibody tests using antigens derived from more than two regions derived from HCV-1. Such tests can detect antibodies against known genotypes of HCV (eg, Sakamoto et al ., J. Gen. Virol. 75: 1761-1768 (1994) and Stuyver et al ., J. Gen. Virol. 74: 1093-1102). (1993)). HCV-specific ELISA failed to detect serum containing GB-C sequences in 6 out of 8 cases. Second, some human sera that were seronegative for HCV antibodies were found to be positive for HCV genomic RNA by a sensitive RT-PCR assay (Sugitani, Lancet 339: 1018-1019). (1992)). This assay failed to detect HCV RNA in 7 out of 8 sera containing HGB-C sequences (Table A). That is, HGBV-C is not a genotype of HCV by both serological and molecular assays.

When a portion of the predicted translation product of HGB-C in the helicase region is aligned with the homologous region of another member of HGBV-A, HGBV-B, HCV-1, and Flaviviridae, the matched sequence is analyzed phylogenetically It can be seen that HGBV-C is more closely related to HGBV-A than other members of the HCV group. The sequences of HGBV-C and HGBV-A showing an evolutionary distance of 0.42 are not as far apart as HGBV-C and HGBV-B showing an evolutionary distance of 0.92 (Table 33, below). That is, HGBV-A and HGBV-C are members of one subgroup of GB viruses, and HGBV-B is considered to be a member of one subgroup by itself. Phylogenetic analysis of helicase sequences from various HCV isolates reveals that they form a small group of branches with a maximum evolutionary distance of 0.20 (Table 32, below). Comparing the HCV group and the HGBV group, it can be seen that the minimum evolutionary distance of any two sequences from each group is 0.69. Using the above distance values, a phylogenetic tree shown in FIG. 42 was created. The relatively high degree of branching of these viruses indicates that GB viruses do not simply form a type or subtype within the hepatitis C group, but constitute a unique phylogenetic group. Phylogenetic analysis using sequence information derived from a small portion of the HCV virus genome has been shown to be an acceptable method of assigning new isolates to genotype groups (Simmonds et al., Hepatology). 19: 1321-1324 (1994)). In recent analyses, using sequences of 110 amino acids within a helicase gene from a representative HCV isolate, they are appropriately classified for each genotype (Simmonds et al., J. Gen. Virol .75: 1053-1061 (1994)). Thus, the indicated evolutionary distance probably correctly reflects the high degree of branching between GB virus and hepatitis C virus.

  In the prior patent application, “HGBV strains can be identified at the polypeptide level, and HGBV strains are at least 40% homologous, preferably at least about 60% homologous, more preferably at least about 80% homologous at the polypeptide level. There was. The term “homologous”, although used, is ambiguous when referring to the degree of association between two polynucleotide or polypeptide sequences, and in fact suggests an evolutionary relationship. According to recent practice in the art, the term “homology” is no longer used, but instead uses the terms “similarity” and / or “identity” to The degree of association of the polynucleotide or polypeptide sequence is indicated. Methods for determining amino acid sequence “similarity” and / or “identity” are known in the art, for example, determining amino acid sequences directly and comparing them with the sequences given herein. , Determining the nucleotide sequence of the putative HGBV genomic material (usually via a cDNA intermediate), determining the amino acid sequence encoded therein, comparing the corresponding regions, and the like. Usually, “coincidence” means that the nucleotide sequence of HGBV and another strain's nucleotide sequence, or the amino acid sequence of HGBV and another strain's amino acid sequence exactly match at the appropriate place on each genome. . Usually, “similarity” means that the amino acid sequence of HGBV and another strain's amino acid sequence exactly match each other at an appropriate place, in which case the amino acids are the same or similar chemical and / or physical. Characteristics such as charge or hydrophobicity. Programs available in the Wisconsin Sequence Analysis Package, version 8 (available from the Genetics Computer Group, Madison, Wisconsin, 53711), such as the percent identity between sequences of two polynucleotides or two polypeptides, eg, the GAP program A similarity rate can be calculated. Other programs that calculate percent identity and similarity between two sequences are also known in the art.

  Furthermore, in identifying strains of HGBV-A, HGBV-B or HGBV-C, the following parameters may be used alone or in combination. Since HGBV strains may preferably have a genetic association of about 60% or more, more preferably about 80% or more, HGBV-A, HGBV-B or HGBV-C and one of these hepatitis GB viruses The overall nucleotide sequence identity between the two strains is estimated to be about 45% or more.

  Furthermore, since the HGBV strain may have a genetic association of preferably about 40% or more, more preferably about 60% or more, and even more preferably about 80% or more, the HGBV-A genome and HGBV-A It is estimated that the overall sequence identity at the amino acid level with the genome of certain strains will be about 35% or more. In addition, there is a corresponding contiguous sequence of at least about 13 nucleotides that would be given in combination of two or more contiguous sequences. In addition, since it is considered that the HGBV strain may have a genetic association of preferably about 40% or more, more preferably about 60% or more, and further preferably about 80% or more, the HGBV-B genome and HGBV-B It is estimated that the overall sequence identity with the genome of a certain strain at the amino acid level is about 35% or more. In addition, there is a corresponding contiguous sequence of at least 13 nucleotides that would be given in combination of two or more contiguous sequences. In addition, since it is considered that the HGBV strain may have a genetic association of preferably about 40% or more, more preferably about 60% or more, and still more preferably about 80% or more, the HGBV-C genome and HGBV-C It is estimated that the overall sequence identity at the amino acid level with the genome of certain strains will be about 35% or more. In addition, there is a corresponding contiguous sequence of at least about 13 nucleotides that would be given in combination of two or more contiguous sequences.

  The compositions and methods of the present invention allow the growth, identification, detection and isolation of HGBV and its possible strains. Furthermore, the composition and method of the present invention make it possible to produce diagnostic agents and vaccines against various strains in which HGBV can be present, and are useful for screening treatment of antiviral substances. The information will be sufficient for a virus taxonologist to identify other strains belonging to the species. We believe that HGBV encodes the sequences included herein. Methods for assaying for the presence of such sequences are known in the art and include amplification methods such as ligase chain reaction (LCR), polymerase chain reaction (PCR) and hybridization. In addition, such sequences include an open reading frame that can find immunogenic viral epitopes. This epitope is unique to HGBV compared to other known hepatitis-inducing viruses. Epitope uniqueness can be determined by immunological reactivity to HGBV and lack of immunological reactivity to hepatitis A, B, C, D and E viruses. Methods for determining immunological reactivity are known in the art and include, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), hemagglutination reaction (HA), and fluorescence polarization immunoassay (FPIA). Some examples of suitable methods are described herein.

  A specific sequence, eg, a polynucleotide “derived from” an HGBV cDNA or HGBV genome, corresponds to a region of the specific nucleotide sequence, ie, approximately at least about 6 nucleotides that are similar or complementary, Preferably, it refers to a polynucleotide sequence consisting of a sequence of at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides. The sequence of the region from which the polynucleotide is derived is preferably similar or complementary to a sequence unique to the HGBV genome. Whether a sequence is complementary or similar to a sequence unique to the HGBV genome can be determined by methods known to those skilled in the art. As a method for determining the uniqueness of a particular sequence, for example, a comparison with sequences in a data bank can be performed. The region from which the sequence can be derived includes, but is not limited to, a region encoding a specific eptop, and a non-translated and / or non-transcribed region.

  The derived polynucleotide need not be physically derived from the nucleotide sequence of HGBV, and is not limited to chemical synthesis, replication, or inversion based on information provided by the nucleotide sequence of the region from which the polynucleotide is derived. It can be generated by any method including copying or transcription. Furthermore, the combination of regions corresponding to a particular sequence can be varied depending on the intended use in a manner known in the art.

  A “polypeptide” or “amino acid sequence” derived from a specific nucleic acid sequence or HGBV genome is a polypeptide having the same amino acid sequence as the polypeptide encoded in the sequence, or at least 3-5 An amino acid, more preferably at least 8 to 10 amino acids, more preferably 15 to 20 amino acids, and refers to a part of the polypeptide that can immunologically match the polypeptide encoded in the sequence .

  As used herein, a “recombinant polypeptide” is at least unrelated to all or part of a related polypeptide in natural or library form and / or is naturally occurring, depending on origin or manipulation. By genomic, semi-synthetic or synthetic polypeptide linked to a polynucleotide other than the linked one. A recombinant or derived polypeptide need not necessarily be translated from a specific nucleic acid sequence of HGBV or from the HGBV genome. Recombinant or derived polypeptides can be produced by any method, including chemical synthesis or expression in a recombinant expression system, or isolation from mutant HGBV.

  The term “synthetic peptide” as used herein refers to any length of polymerized form of amino acids that can be chemically synthesized by methods known to those skilled in the art. Such synthetic peptides are useful for various applications.

  The term “polynucleotide” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. The term thus includes double- and single-stranded DNA and double- and single-stranded RNA. Further, modified forms of polynucleotides by methylation and / or capping, as well as unmodified forms of polynucleotides are also included.

  “HGBV comprising a sequence corresponding to a cDNA” means that the HGBV comprises a polynucleotide sequence that is similar or complementary to a sequence in a particular DNA. The degree of similarity or complementarity to the cDNA is about 50% or more, preferably at least about 70%, more preferably at least about 90%. The corresponding sequence length is at least about 70 nucleotides, preferably at least about 80 nucleotides, more preferably at least about 90 nucleotides. The correspondence between HGBV and cDNA can be determined by methods known in the art, for example, comparing the sequenced material directly with the described cDNA, or by hybridizing and digesting with a single-stranded nuclease, For example, determining the size.

  “Purified viral polynucleotide” means that the viral polynucleotide is substantially free of naturally associated polypeptides, ie, less than about 50%, preferably less than about 70%, more preferably less than about 90% thereof. Refers to the HGBV genome or a fragment thereof. Methods for purifying viral polynucleotides are known in the art, for example, disrupting particles using chaotropic agents and separating polynucleotides and polypeptides by ion exchange chromatography, affinity chromatography, density sedimentation. Can be mentioned. A “purified viral polypeptide” is substantially free of cellular components with which the viral polypeptide is naturally associated, ie, less than about 50%, preferably less than about 70%, more preferably less than about 90%. Means HGBV polypeptide or fragment thereof. Purification methods are known to those skilled in the art.

  As used herein, “polypeptide” refers to a molecular chain of amino acids and not a product of a particular length. Thus, peptides, oligopeptides and proteins are included in the definition of polypeptide. However, this term does not include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation and the like.

  “Recombinant host cell”, “host cell”, “cell”, “cell line”, “cell culture”, and other similar terms referring to a microorganism or higher eukaryotic cell line cultured as unicellular material are: Refers to a cell that can or can be used as a recipient for a recombinant vector or other transferred DNA, including the original progeny of the original transfected cell.

  As used herein, “replicon” means any genetic element, such as a plasmid, chromosome, or virus, that behaves as an independent unit of polynucleotide replication in a cell. That is, the replicon can replicate under its own control.

  A “vector” is a replicon added so that another polynucleotide segment can be replicated and / or expressed.

  The term “control sequence” refers to a polynucleotide sequence necessary to effect the expression of a coding sequence to which they are linked. The characteristics of the control sequence vary depending on the host organism. In prokaryotic cells, control sequences generally include a promoter, ribosome binding site and terminator, and in eukaryotic cells, control sequences generally include a promoter, terminator, and optionally an enhancer. Thus, the term “control sequence” is intended to include at least all components whose presence is necessary for expression, and may also include additional components that are advantageous if present, such as leader sequences.

  “Operably coupled” refers to a situation in which the components are in a relationship where they can function as expected. Thus, for example, a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence occurs under conditions compatible with the control sequences.

  The term “open reading frame” or “ORF” refers to a region of a polynucleotide sequence that encodes a polypeptide. This region may provide part of the coding sequence or the entire coding sequence.

  A “coding sequence” is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation initiation codon at the 5 'end and a translation termination codon at the 3' end. Coding sequences include but are not limited to mRNA, cDNA, and recombinant polynucleotide sequences.

  The term “immunologically identifiable with / as” refers to the presence of epitopes and polypeptides that are present and unique in a particular polypeptide, usually an HGBV protein. Immunological identity can be determined by antibody binding and / or competition for binding. Such methods are known to those skilled in the art and are also described herein. The uniqueness of an epitope can be determined by computer searching for polynucleotide sequences encoding the epitope in a known data bank, such as GenBank, or by comparing the amino acid sequence with other known proteins.

  As used herein, “epitope” means an antigenic determinant of a polypeptide. Perhaps an epitope may contain 3 amino acids in a spatial arrangement that is unique to that epitope. Usually an epitope consists of at least 5 such amino acids, and more usually consists of at least 8-10 amino acids. Methods for investigating the spatial arrangement are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

  A polypeptide is "immunologically reactive" to an antibody when it binds to the antibody because it recognizes a particular epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, particularly antibody binding rate, and / or binding competition using a known polypeptide containing an epitope for the antibody as a competitor. Methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.

  As used herein, the term “immunogenic polypeptide comprising an HGBV epitope” refers to a native HGBV polypeptide or fragment thereof, as well as other means, such as chemical synthesis or in a recombinant microorganism. It means a polypeptide produced by expression.

  The term “transformation” refers to the insertion of a foreign polynucleotide into a host cell, regardless of the method of insertion. For example, direct uptake, transduction or f-mating is included. The foreign polynucleotide can be maintained as a non-integrating vector, such as a plasmid, or can be integrated into the host genome.

  “Treatment” refers to prophylaxis and / or treatment.

  As used herein, the term “individual” refers to animals, particularly those of mammalian species, including but not limited to farm animals, sport animals, primates and humans, although the term in particular includes tamarins. And human.

  As used herein, “positive strand” (or “+”) refers to a nucleic acid comprising a sequence encoding a polypeptide. “Negative strand” (or “−”) indicates a nucleic acid comprising a sequence complementary to the sequence of the “positive” strand.

  A “positive strand genome” of a virus indicates that the genome, whether RNA or DNA, is single stranded and encodes a viral polypeptide.

  “Test sample” refers to a component of an individual that is the source of an analyte (eg, an antibody or antigen of interest). Such ingredients are known in the art. Test samples include biological samples that can be tested by the method of the present invention, including human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph, and respiratory, gastrointestinal and urine. Various exocrine secretions of the genital tract, tears, saliva, milk, leukocytes, myeloma and the like, as well as biological fluids such as cell culture supernatants, fixed tissue samples, fixed cell samples.

  “Purified HGBV” refers to a preparation of HGBV isolated from cellular components to which the virus is normally associated, and other types of viruses that may be present in infected tissues. Methods for isolating viruses are known to those skilled in the art and include, for example, centrifugation and affinity chromatography.

  “PNA” refers to a “peptide nucleic acid analog” that can be used in an assay such as determining the presence of a target, eg, a target. PNA is an electrically neutral substance for RNA targets or DNA. For example, PNA probes that are used in assays instead of DNA probes offer advantages that are not obtained when using DNA probes. Such advantages include manufacturability, large scale labeling, reproducibility, stability, low sensitivity to changes in ionic strength, and resistance to enzymatic degradation present in methods using DNA or RNA. PNA can be labeled with signal generating compounds such as fluorescein, radionuclides, chemiluminescent compounds and the like. Thus, PNA can be used in the method instead of DNA or RNA. Although the present specification describes an assay using DNA, it is also within the normal practice to change the assay reagents appropriately and use PNA instead of RNA or DNA if necessary.

(General use)
Once a particular recombinant protein, synthetic peptide, or purified viral polypeptide of the invention has been produced, the recombinant or synthetic peptide can be used to detect the presence of an antigen or antibody against HGBV as described herein. A unique assay can be developed. Such compositions can also be used to develop monoclonal and / or polyclonal antibodies using specific recombinant proteins or synthetic peptides that specifically bind to the desired immunological epitope of HGBV. It is also conceivable to develop vaccines according to methods known in the art using at least one polynucleotide of the invention.

  The reagents used in the assay are test kits that include one or more containers, eg, vials or bottles, each containing a reagent such as a monoclonal antibody or mixture of monoclonal antibodies or a (recombinant or synthetic) polypeptide used in the assay. It can be provided in the form of Other components, such as buffers and controls known to those skilled in the art, may be included in such test kits.

  “Solid phases” (“solid supports”) are known to those skilled in the art and include reaction tray well walls, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles (eg latex particles), sheep (Or other animal) red blood cells, Duracytes, and the like. The “solid phase” is not limiting and can be selected by one skilled in the art. Latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, microtiter well walls, glass or silicon chips, sheep (or other suitable animal) red blood cells, and Duracytes are all suitable examples. is there. Suitable methods for immobilizing the peptide on the solid phase include ionic, hydrophobic, and covalent interactions. As used herein, “solid phase” refers to any material that is insoluble or can be rendered insoluble by subsequent reactions. The solid phase can be selected for its inherent ability to attract and immobilize the capture agent. Alternatively, the solid phase may hold an auxiliary receptor that has the ability to attract and immobilize the capture agent. Auxiliary receptors can include a charged material having a charge opposite to that of the capture agent itself or the charged material bound to the capture agent. Alternatively, the receptor molecule can be any specific binding member that is immobilized (attached) on a solid phase and has the ability to immobilize the capture agent by a specific binding reaction. The capture agent can be indirectly bound to the solid phase material by the receptor molecule before or during the assay. The solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, test tubes with glass or silicon surfaces, microtiter wells, sheets, beads, microparticles, chips, sheep (or other suitable animal) red blood cells, Duracytes and other configurations known to those skilled in the art may be used.

  It is contemplated that the solid phase may be composed of any suitable porous material having sufficient porosity that the detection antibody can access and appropriate surface affinity to bind the antigen, and is within the scope of the present invention. . In general, a microporous structure is preferable, but a material having a gel structure in a hydrated state can also be used. Such useful solid supports include natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives such as agar, agarose, cross-linked alginic acid, substituted cross-linked guar gum, especially cellulose esters with nitric acid and carboxylic acids, mixed cellulose esters, cellulose Ethers; natural polymers containing nitrogen, such as proteins and derivatives including cross-linked or modified gelatin; natural hydrocarbon polymers such as latex and rubber; synthetic polymers that can be made to have a suitable porous structure, such as polyethylene, polypropylene, polystyrene, Polyvinyl chloride, vinyl polymers including polyvinyl acetate and partially hydrolyzed derivatives thereof, polyacrylamide, polymethacrylate, copolymers and terpolymers of the above polycondensates such as polyester, polyamido , And other polymers such as polyurethane or polyepoxides; porous inorganic materials such as alkaline earth metals and magnesium sulfates or carbonates (eg barium sulfate, calcium sulfate, calcium carbonate), alkali and alkaline earth metals, aluminum and Magnesium silicates; as well as oxides or hydrates of aluminum or silicon, such as clay, alumina, talc, kaolin, zeolite, silica gel or glass (these materials can be used as fillers with the above polymer materials ); As well as mixtures or copolymers thereof, such as graft copolymers obtained by initiating polymerization of synthetic polymers over existing natural polymers. All of these materials can be used in any suitable shape, such as a film, sheet, or plate, or can be coated, adhered, or laminated to a suitable inert carrier such as paper, glass, plastic film, or fiber. .

  The porous structure of nitrocellulose exhibits excellent absorbency and adsorption for a wide range of reagents including monoclonal antibodies. Nylon has similar properties and is still suitable. The porous solid support as described above is considered to be in the form of a sheet having a thickness of about 0.01 to 0.05 mm, preferably about 0.1 mm. The pore size can vary over a wide range, but is preferably about 0.025 to 15 microns, particularly about 0.15 to 15 microns. The surface of such a support can be activated by a chemical treatment that causes a covalent bond between the antigen or antibody and the support. In general, an antigen or antibody is irreversibly bound by adsorbing on a porous material by a less well understood hydrophobic force. Suitable solid supports are also described in US Patent Application No. 227,272.

  An “indicator” includes a “signal generating compound” (label) that can generate or generate a measurable signal that can be detected by external means, bound (attached) to a specific binding member of HGBV. As used herein, a “specific binding member” is a specific binding pair, that is, a member of two molecules in which one molecule specifically binds to a second molecule through chemical or physical means. means. In addition to being an antibody member of a specific binding pair for HGBV, the indicator may be a hapten-anti-hapten system such as biotin or anti-biotin, avidin or biotin, carbohydrate or lectin, complementary nucleotide sequence, effector molecule or receptor molecule, enzyme cofactor And enzymes, enzyme inhibitors or enzymes. An immunoreactive specific binding member can bind to HGBV in a sandwich assay, to a capture agent in a competitive assay, or to any specific binding member in an indirect assay, an antibody, antigen, or antibody / antigen complex. It can be.

  Various possible “signal generating compounds” (labels) include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetane, acridinium, phenanthridinium and luminol, radioactive elements Direct visible labels. Examples of the enzyme include alkaline phosphatase, horseradish peroxidase, β-galactosidase and the like. The selection of a particular label is not limiting, and the label can generate a signal by itself or together with one or more other substances.

  The present invention provides assays that use specific binding members. As used herein, a “specific binding member” is a specific binding pair, ie a member of two different molecules in which one molecule specifically binds to a second molecule through chemical or physical means. It is. Thus, in addition to antigen and antibody specific binding pairs in general immunoassays, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector molecules and reporter molecules, cofactors and enzymes, Enzyme inhibitors and enzymes may be mentioned. Specific binding pairs may further include members that are analogs of the original specific binding pair, eg, analyte analogs. Immunoreactive specific binding members include those formed by recombinant DNA molecules and include antigens, antigen fragments, monoclonal and polyclonal antibodies and antibody fragments, and complexes thereof. The term “hapten” as used herein refers to a partial antigen or non-protein binding member that can bind to an antibody but cannot induce antibody formation unless bound to a carrier protein.

  As used herein, an “analyte” is a substance to be detected that may be present in a test sample. The analyte can be a substance against which a natural specific binding member (eg, an antibody) is present or for which a specific binding member can be prepared. That is, an analyte is a substance that can bind to one or more specific binding members in an assay. “Analyte” includes any antigenic substance, hapten, antibody, and combinations thereof. As a member of a specific binding pair, the analyte can be detected by a natural specific binding partner (pair), for example, the measurement of vitamin B12 uses an intrinsic factor protein as a member of a specific binding pair, and folic acid A folate binding protein is used to measure and a lectin is used as a member of a specific binding pair to measure carbohydrate. Analytes include proteins, peptides, amino acids, nucleotide targets and the like.

  Other embodiments using a variety of other solid phases are also contemplated and are within the scope of the present invention. For example, described in co-pending US Patent Application No. 150,278 and US Patent Application No. 375,029 (European Patent Application Publication No. 0406473) corresponding to European Patent Application No. 0326100 The ion capture method of immobilizing an immobilizable reaction complex using a negatively charged polymer can be used in accordance with the present invention to perform a fast solution-phase immunochemical reaction. The immobilizable immune complex is separated from the rest of the reaction mixture by ionic interaction between a negatively charged polyanion / immune complex and a pretreated positively charged porous matrix, and published as an EPO patent application. Various signal generation systems specified in the literature are used, including those described in chemiluminescent signal measurements such as those described in co-pending US Patent Application No. 921,979 corresponding to No. 0,273,115. Can be detected.

  The method of the invention can also be adapted for use in systems using microparticle technology, including automated and semi-automated systems in which the solid phase consists of (magnetic or non-magnetic) microparticles. Such systems include pending US patent applications Nos. 425,651 and 425,643 corresponding to EPO Patent Application Publication Nos. 0 425 633 and 0 424 634, respectively. The thing of description is mentioned.

  Using a scanning probe microscope (SPM) for immunoassays, the monoclonal antibodies of the invention can be readily adapted. In scanning probe microscopes, particularly atomic force microscopes, a capture phase, for example at least one monoclonal antibody of the present invention, is attached to a solid phase and is present on the surface of the solid phase using a scanning probe microscope. The resulting antigen / antibody complex is detected. Using a scanning tunneling microscope eliminates the need for labels that must normally be used in many immunoreactor systems to detect antigen / antibody complexes. Such a system is described in pending US Patent Application No. 662,147. SPM can be used in many ways to monitor specific binding reactions. In one embodiment, one member of a specific binding partner (analyte-specific substance that is a monoclonal antibody of the invention) is attached to a surface suitable for scanning. The analyte-specific substance can be attached by adsorbing it on a test piece made of a solid phase of a plastic or metal surface according to a method known to those skilled in the art. Alternatively, a specific binding partner (analyte-specific substance) can be covalently bound to a test piece made of a derivatized plastic, metal, silicon, or glass solid phase. Covalent binding methods are known to those skilled in the art and include various means for irreversibly binding a specific binding partner to a test strip. If the specimen is silicon or glass, the surface needs to be activated before attaching the specific binding partner. Triethoxyaminopropylsilane (available from Sigma Chemical Co., St. Louis, MO), triethoxyvinylsilane (Aldrich Chemical Co., Milwaukee, WI) and (3-mercapto-propyl) -trimethoxysilane (Sigma Chemical Co) , St. Louis, MO) can be used to introduce reactive groups such as amino, vinyl and thiol, respectively. Such an activated surface can be used to bind the binding partner directly (in the case of amino or thiol) or the activated surface can be further coupled with glutaraldehyde, bis (succinimidyl) suberate, SPPD9 (succinimidyl 3 -[2-pyridyldithio] propionate), SMCC (succinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate), SIAB (succinimidyl [4-iodoacetyl] aminobenzoate) and SMPB (succinimidyl 4- [1 It can also be reacted with a linker such as -maleimidophenyl] butyrate) to separate the binding partner from the surface. The vinyl group can be oxidized to provide a covalent means, or can be used as an anchor to polymerize various polymers such as polyacrylic acid that can provide multiple points of attachment to specific binding partners. it can. The amino surface can also be reacted with oxidized dextrans of various molecular weights to give hydrophilic linkers of various sizes and binding capabilities. Examples of oxidizable dextran include: Dextran T-40 (molecular weight 40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons (all of which are available from Pharmacia), or Ficoll (molecular weight 70,000 daltons) (available from Sigma Chemical Co., St Louis, Mo.). In addition, using the methods and chemicals described in pending US Patent Application No. 150,278 filed on Jan. 29, 1988 and 375,029 filed on Jul. 7, 1989, a polymer electrolyte was used. Interactions can also be used to immobilize specific binding partners on the surface of the test strip. A preferred attachment method is by covalent bonding. After attaching the specific binding member, the surface may be further treated with serum, protein, or other blocking agent to minimize non-specific binding. To verify that the surface is suitable for the assay, it may be scanned at the point of manufacture or at the point of use. The scanning method shall not change the specific binding characteristics of the test strip.

  A variety of other assay formats may be used, including “sandwich” immunoassays and probe assays. For example, the monoclonal antibodies of the invention can be used in various assay systems to determine the presence, if any, of HGBV protein in a test sample. Fragments of such monoclonal antibodies given may be used. For example, in a rapid assay format, a solid phase coated polyclonal or monoclonal anti-HGBV antibody or fragment thereof or a combination of these antibodies is contacted with a test sample that may contain HGBV protein to form a mixture. This mixture is incubated for a time and under conditions sufficient to form an antigen / antibody complex. An indicator comprising a monoclonal or polyclonal antibody that specifically binds to the HGBV region, or a fragment thereof, or a combination of these, to which a signal generating compound is attached is contacted with the antigen / antibody complex to form a second mixture. . This second mixture is incubated for a time and under conditions sufficient to form an antibody / antigen / antibody complex. The presence of HGBV antigen present in the test sample and captured on the solid phase, if any, is determined by detecting a measurable signal generated by the signal generating compound. The amount of HGBV antigen present in the test sample is proportional to the signal produced.

  Alternatively, a monoclonal or polyclonal antibody that specifically binds to HGBV antigen, to which a polyclonal or monoclonal anti-HGBV antibody or fragment thereof or a combination of these antibodies or a combination of these antibodies, a test sample, and a signal-generating compound are attached to a solid support Alternatively, a mixture is formed by contacting with an indicator comprising a fragment thereof or a combination of these antibodies. This mixture is incubated for a time and under conditions sufficient to form an antibody / antigen / antibody complex. The presence of HGBV protein present in the test sample and captured on the solid phase, if any, is determined by detecting a measurable signal generated by the signal generating compound. The amount of HGBV protein present in the test sample is proportional to the signal produced.

  In yet another assay format, one or a combination of two or more of the monoclonal antibodies of the invention can be used as a competitive probe to detect antibodies against HGBV protein. For example, the HGBV protein can be coated on the solid phase alone or in combination. A test sample suspected of containing an antibody against the HGBV antigen is then combined with the test sample and the indicator and the solid phase, or the indicator and the solid phase, together with an indicator comprising a signal generating compound and at least one monoclonal antibody of the present invention. Incubating for a time and under conditions sufficient to form an antigen / antibody complex. A decrease in the binding of the monoclonal antibody to the solid phase can be quantitatively measured. If a decrease in signal can be measured compared to a signal generated from a test sample that has been confirmed to be non-A, non-B, non-C, non-D, non-E hepatitis negative, anti-HGBV antibody is present in the test sample. It can be seen that it exists.

  In yet another detection method, each of the monoclonal or polyclonal antibodies of the invention can be used to detect HGBV antigen in fixed tissue pieces and fixed cells by immunohistochemical analysis. Such antibodies can be directly labeled (fluorescein, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) or labeled using a second labeled anti-species antibody (using various labels exemplified herein). Cytochemical analysis that tracks the history of the disease is also within the scope of the present invention.

  In addition, the monoclonal antibody is bound to a matrix similar to CNBr-activated Sepharose and used for affinity purification of specific HGBV proteins from cell cultures or biological tissues such as blood and liver, recombinant or natural viral HGBV antigens and proteins Can also be purified.

  The monoclonal antibodies of the present invention can also be used to generate chimeric antibodies for therapeutic purposes and other similar uses.

  Monoclonal antibodies or fragments thereof can be provided individually to detect HGBV antigens. The combinations of monoclonal antibodies (and fragments thereof) provided herein are different binding specificities, each in a mixture or “cocktail” of at least one anti-HGBV antibody of the invention and antibodies against other HGBV regions. They can be used together as ingredients having properties. That is, the cocktail may comprise a monoclonal antibody of the invention against the HGBV protein and other monoclonal antibodies against other antigenic determinants of the HGBV genome.

  Polyclonal antibodies or fragments thereof that can be used in such an assay format should specifically bind to the specific HGBV region or other HGBV protein used in the assay. The polyclonal antibodies used are preferably derived from mammals, and human, goat, rabbit or sheep anti-HGBV polyclonal antibodies can be used. Most preferably, the polyclonal antibody is a rabbit polyclonal anti-HGBV antibody. The polyclonal antibody used in the assay can be used alone or as a cocktail of polyclonal antibodies. Because the cocktail used in the assay format consists of either monoclonal or polyclonal antibodies with different HGBV specificities, they are useful for the diagnosis, evaluation and prevention of HGBV infection and for studying the differentiation and specificity of HGBV proteins It will be.

  It is contemplated that by using synthetic, recombinant or natural peptides common to all HGBV viruses, the HGBV family of viruses can be detectable in the assay and are within the scope of the present invention. It is also within the scope of the present invention that various synthetic, recombinant or natural peptides identifying various epitopes from HGBV-A, HGBV-B, HGBV-C, and even other HGBV viruses can be used in the assay format. is there. In the latter case, they may be coated onto one solid phase, or each peptide may be coated onto a separate solid phase, such as a microparticle, and combined to form a mixture of peptides that can be used later in the assay. It may be formed. Variations on such assay formats are known to those skilled in the art and are described below.

  In another assay format, the presence of antibodies and / or antigens against HGBV can be detected in a simultaneous assay as follows. The test sample is a first analyte capture agent containing a first binding member specific to the first analyte attached to the solid phase and a second analyte for the second analyte attached to the second solid phase. Simultaneous contact with a second analyte capture agent comprising one binding member thereby forming a mixture. This mixture is incubated for a time and under conditions sufficient to form a capture agent / first analyte complex and a capture agent / second analyte complex. The complex formed in this manner is specific to an indicator containing a member of a binding pair specific for the first analyte labeled with the signal generating compound and a second analyte labeled with the signal generating compound. Contact with an indicator comprising a member of a suitable binding pair to form a second mixture. This second mixture is incubated for a time and under conditions sufficient to form a capture agent / first analyte / indicator complex and a capture agent / second analyte / indicator complex. The presence of one or more analytes was generated in association with a complex formed on one or both solid phases as an indication of the presence of one or more analytes in the test sample. Determined by detecting signal. In this assay format, a protein derived from a human expression system can be used as well as a monoclonal antibody produced from a protein derived from a mammalian expression system as described herein. Such an assay system is described in detail in co-pending U.S. patent application 07 / 574,821, corresponding to European patent application publication 0 473 065, entitled "Simultaneous Assay for Detecting One More More Analyzes". .

In yet another assay format, recombinant proteins and / or synthetic peptides can be used to detect the presence of anti-HGBV in a test sample. For example, the test sample is incubated with a solid phase to which at least one recombinant protein or synthetic peptide is attached. These are reacted for a time and under conditions sufficient to form an antigen / antibody complex. After incubation, the antigen / antibody complex is detected. Depending on the assay system chosen, indicators may be used to facilitate detection. In another assay format, a test sample is contacted with a solid phase to which a recombinant protein or synthetic peptide produced as described herein is attached, and the protein preferably labeled with an indicator. Contact with a specific monoclonal or polyclonal antibody. After incubation for a time and under conditions sufficient for antibody / antigen complexes to form, the solid phase is separated from the free phase and the label as an indicator of the presence of the HGBV antibody is detected in the solid phase or free phase. Other assay formats using the proteins of the invention are also contemplated. They include contacting a test sample with a solid phase to which at least one antigen from a first source is attached, and the solid phase and test sample for a time sufficient to form an antigen / antibody complex and Incubating under conditions and then contacting the solid phase with a labeled antigen derived from a second source different from the first source. For example, using a recombinant protein derived from a first source such as E. coli as a capture antigen on a solid phase, a test sample is added to the solid phase thus prepared and a different source (ie E using the recombinant proteins derived from non .Coli) as part of the indicator. Similarly, a recombinant antigen on the solid phase can be combined with a synthetic peptide in the indicator phase. Also contemplated are assay formats that use an antigen specific for HGBV from a first source as the capture antigen and an antigen specific for HGBV from a different second source. Various combinations of recombinant antigens, as well as the use of synthetic peptides, purified viral proteins, etc. are within the scope of the present invention. Such assays and others are described in US Pat. No. 5,254,458 in the name of the Applicant, the contents of which are hereby incorporated by reference.

  In other assay systems (polyclonal, monoclonal or natural) antibodies that specifically bind to HGBV viral particles or subviral particles containing the viral genome (or fragments thereof) by contact of specific antibodies with viral proteins (such as peptides) Is used. The captured particles can then be analyzed by methods such as LCR or PCR to determine if the viral genome is present in the test sample. Test samples that can be analyzed according to the method include blood, liver, sputum, urine, stool, saliva, and the like. The advantage of using such an antigen capture amplification method is that specific antibodies can be used to separate the viral genome in the test sample from other molecules. Such a method is described in pending US patent application Ser. No. 08 / 141,429.

  Although the present invention has been described for the use of a solid phase, it is contemplated that reagents such as antibodies, proteins and peptides of the present invention can be used in non-solid phase assay systems. Such assay systems are known to those skilled in the art and are intended to be within the scope of the present invention.

(Materials and methods)
(General method)
Unless otherwise stated, the practice of the present invention uses conventional techniques and methods that are conventional in the fields of molecular biology, microbiology, recombinant DNA and immunology. Such techniques are explained fully in the literature. For example, J. et al. Sambrook et al., Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N .; Y. (1989); N. Glover, DNA Cloning, Volumes I and II (1985); J. et al. Edited by Gait , Oligonucleotide Synthesis , (1984); D. Edited by Hames et al., Nucleic Acid Hybridization , (1984); D. Edited by Hames et al., Transcription and Translation , (1984); I. Edited by Freshney, Animal Cell Culture , (1986); Immobilized Cells and Enzymes , IRL Press (1986); Perbal, A Practical Guide to Molecular Cloning , (1984); Series, Methods in Enzymology , Academic Press, Inc. , Orlando, Florida; H. Edited by Miller et al., Gene Transfer Vectors for Mammalian Cells , Cold Spring Harbor Laboratory, Cold Spring Harbor, N .; Y. (1987); Wu et al., Methods in Enzymology, Vol. 154 and 155; edited by Mayer et al., Immunological Methods In Cell and Molecular Biology , Academic Press, London (1987); Scopes, Protein Purification , Pr. Y. And D. Edited by Weir et al., Handbook Of Experimental Immunology , Vol. I-IV (1986); See Litisisyn et al., Science 259: 946-951 (1993).

  The reagents and methods of the present invention are highly relevant in plasma, serum or liver homogenates of HGBV-infected individuals that are tamarin or human isolated by improved representative difference analysis as described herein. This can be realized by providing a group of nucleotide sequences. This nucleotide sequence group does not hybridize to human or tamarin genomic DNA from an uninfected individual, exists only in the liver (or liver homogenate), plasma or serum of an HGBV infected individual, and does not exist in GenBank. Therefore, it is not derived from human or tamarin. Furthermore, the sequence group does not show significant coincidence at the nucleic acid level with sequences contained within the HAV, HBV, HCV, HDV and HEV genomes, and the coincidence level of translation products is too low to be considered significant. Infectious serum, plasma or liver homogenate from HGBV infected humans contains such polynucleotide sequences, whereas serum, plasma or liver homogenate from uninfected humans does not contain such sequences. Northern blot analysis of infected livers containing some of these polynucleotide sequences reveals that they are derived from large RNA transcripts similar in size to the viral genome. Serum, plasma or liver homogenate from HGBV-infected humans contains antibodies that bind to this polypeptide, whereas serum, plasma or liver homogenates from uninfected humans do not contain antibodies to this polypeptide, such antibodies are Induced in individuals after A, non-B, non-C, non-D and non-E infections. Based on these, the sequence is considered to be a viral sequence that causes or is associated with non-A, non-B, non-C, non-D and non-E hepatitis.

  By using this group of nucleic acid sequences, non-A, non-B, non-C, non-D, non-E hepatitis caused by HGBV infection can be diagnosed, blood donors, donated blood, blood products and individuals can be infected. DNA probes and polypeptides useful in screening can be constructed. For example, the sequence is useful, for example, as a hybridization probe or PCR primer for detecting the presence of a viral genome in the serum of a subject suspected of carrying the virus, or screening for the presence of virus in donated blood A DNA oligomer of about 8-10 or more nucleotides useful for the synthesis can be synthesized. The nucleic acid sequences can design and produce HGBV-specific polypeptides that are useful as diagnostic reagents for the presence of antibodies produced upon infection with HGBV. It is also possible to detect viral antigens in infected individuals and blood using antibodies against purified polypeptides derived from the nucleic acid sequences. Such nucleic acid sequences make it possible to design and produce polypeptides that can be used as vaccines against HGBV and for the production of antibodies, so that they can be used for disease prevention and / or treatment of HGBV-infected individuals. Can do.

  Such a group of nucleic acid sequences allows further characterization of the HGBV genome. Using polynucleotide probes derived from such sequences, genomic or cDNA libraries can be screened for other overlapping nucleic acid sequences and further used to obtain overlapping sequences. If the genome is segmented but the segments do not lack a common sequence, the entire genome sequence can be obtained using this method. However, if the genome is segmented, repeat the RDA cloning process as described to modify as described below, or repeat the λ-gt11 serological screening process as described below to isolate the clones described below, Alternatively, other segments of the genome can be obtained by isolating the genome from purified HGBV particles.

  CDNA sequences and polypeptides derived from the above sequences and antibodies against such polypeptides are also useful for the isolation and identification of HGBV causative agents. For example, antibodies against HGBV epitopes contained in polypeptides derived from such nucleic acid sequences can be used in affinity chromatography-based methods to isolate viruses. Alternatively, antibodies can be used to identify viral particles isolated by other methods. Viral antigens and genomic material in the isolated viral particles can also be further characterized.

  Non-A, non-B, non-C, non-D, non-E induced by HGBV by further sequencing the HGBV genome, further characterizing the HGBV antigen, and information obtained by characterizing the genome Other probes and polypeptides and antibodies can be designed and synthesized that can be used for the diagnosis, prevention and treatment of hepatitis B and for screening infected blood and blood related products.

  Once a probe for HGBV is obtained, including antigens, antibodies and polynucleotides derived from the genome from which such nucleic acid sequences are derived, a tissue culture system particularly used to characterize the biological properties of HGBV. Development becomes possible. Once this is known, it would be possible to develop new therapeutic methods based on antiviral compounds that preferentially inhibit HGBV replication or its infection.

One method used to identify and isolate the etiological agent of HGBV is N. Lisitsyn et al., Science . 259: 946-951 (1993), the GB material was cloned / isolated by a modification of a known method known as reproducibility analysis (RDA). This method clones DNA differences between two complex mammalian genomes based on the principle of subtractive hybridization. In summary, this method genusally distinguishes two test genomes from a “tester” (including the relevant target sequence) and a “driver” (corresponding to normal DNA). Lisitsyn et al.'S description of RDA is limited to the identification and cloning of DNA differences between similar complex DNA backgrounds. These differences can be attributed to any large DNA virus (eg, ≧ 25,000 DNA base pairs) present in a particular cell line, blood, plasma or tissue sample and absent in an uninfected cell line, blood, plasma or tissue sample. May be included. Since the previous literature indicates that HGBV appears to be a small virus containing a DNA or RNA genome of ≦ 10,000 bases, the RDA protocol was modified to detect small viruses. The main steps of the method are described below and shown in FIG.

  In summary, in Step 1, total nucleic acids (DNA and RNA) are isolated using a commercial kit. RDA requires that the samples be highly consistent. Ideally, tester and driver nucleic acid samples should be obtained from the same source (animal, human, etc.). If highly related, non-identical materials may be used for the tester and driver nucleic acid source. Double-stranded DNA is generated from the total nucleic acid by randomly initiated reverse transcription of RNA followed by randomly initiated DNA synthesis. This treatment converts single-stranded RNA virus and single-stranded DNA virus into double-stranded DNA molecules that can be used for RDA. Assuming that the unknown virus has one of the DNA or RNA genomes, double-stranded DNA can be generated using DNA-only or RNA-only extraction procedures as described in the literature.

  Stage 2 amplifies the tester and driver nucleic acids to produce large amounts of material (ie tester and driver amplicons) corresponding to pre-inoculation and total nucleic acid extracted from infectious plasma sources. This is achieved by cleaving the double stranded DNA prepared as described above with a restriction endonuclease having a 4 bp recognition site (eg Sau3AI). The DNA fragment is ligated to the oligonucleotide adapter (set # 1). The DNA fragment is end-filled and PCR amplified. After PCR amplification, the oligonucleotide adapter (set # 1) is removed by restriction endonuclease digestion (eg, Sau3A I) to liberate large amounts of tester and driver nucleic acids for use in subsequent subtractive hybridization methods.

  The purpose of the stage 3 experiment is to accumulate DNA unique to the tester genome. This is achieved by combining subtractive hybridization and dynamic accumulation as a single step. In summary, an oligonucleotide adapter set (# 2 or # 3) is ligated to the 5 'end of the tester amplicon. Tester amplicon and excess driver amplicon are mixed, denatured and hybridized for 20 hours. Large amounts of sequences that are conserved in common between tester and driver DNA anneal during this time. Furthermore, the sequence unique to the tester apricon reanneals. However, since the hybridization time is limited, some single-stranded tester and driver DNA remain.

  In step 4, the 3 'end of the reannealed tester and driver DNA is filled with a thermostable DNA polymerase at high temperature as described in the literature. The tester-specific reannealed sequence includes adapters linked on both strands of the annealed sequence. Thus, the 3 'end filling of these molecules produces sequences complementary to the PCR primers on the two DNA strands. Therefore, these DNA species are exponentially amplified when subjected to PCR. In contrast, a relatively large amount of hybrid molecules that contain sequences conserved in common between the tester and the driver amplicon (ie, one strand is derived from the tester amplicon and one strand is derived from the driver amplicon. ) Is linearly amplified by PCR. This is because it contains an adapter sequence in which only the strands (derived from the tester amplicon) are linked, and the 3 'end filling only produces a sequence complementary to the PCR primer on the strand derived from the driver adapter.

  In step 5, the corresponding double-stranded DNA is quantitatively increased using PCR for 10 amplification cycles. As described in Step 4, the reannealed tester sequence is exponentially amplified, and the conserved sequence in common between the tester and driver anneal is linearly amplified.

  In step 6, residual single stranded DNA is removed by single stranded DNA nuclease digestion using jaenalinuclease as described in the literature.

  In step 7, the double-stranded DNA remaining after nuclease digestion is further PCR amplified for 15 to 25 cycles.

  Finally, in step 8, these DNA products are cleaved with a restriction endonuclease to remove the oligonucleotide adapter. These DNA products are then either subjected to the next amplification (starting at step # 3 using an oligonucleotide adapter that was not used in the previous cycle of RDA) or cloned into an appropriate plasmid vector for further analysis. .

  The RDA method is a variation of the reproducibility difference analysis known to those skilled in the art. The method was modified to isolate virus clones from pre-inoculation and infectious serum sources. These variations are described below with respect to the preparation of tester and driver DNA amplicons. First of all, the starting material was total nucleic acid extracted from infectious pre-inoculated biological blood samples obtained from tamarin rather than double stranded DNA obtained from genomic DNA of mammalian cells as previously reported. . Other biological samples (eg, organs, tissues, bile, stool or urine) may be used as a nucleic acid source to generate testers and driver amplicons. Second, the amount of starting nucleic acid is substantially less than described in the literature. Third, a restriction endonuclease with a 4 bp recognition site rather than 6 bp was used. This is substantially different from the prior art. According to the teachings of Lisitsyn et al., RDA is effective because the production of amplicons (ie, reproductions) reduces the degree of hybridization of the hybridized (ie, subtracted) DNA.

  In the prior art, the genome was fractionated using a restriction enzyme having a 6 bp recognition site. These restriction endonucleases cleave approximately every 4000 bp. On the other hand, under the PCR conditions described in the prior art, the amplification size of the sequence is ≦ 1500 bp. Thus, subsequent PCR amplification of a complex species (eg, genome) of a DNA fractionated with a restriction enzyme that recognizes a 6 bp sequence results in an amplicon containing a DNA fraction of size ≦ 1500 bp after restriction endonuclease digestion. . It has been reported that this reduction in DNA complexation (estimated to be 10-50 times) is necessary for the RDA hybridization step to be performed effectively. If the degree of complexity does not decrease, the unique sequences in the tester cannot effectively hybridize during the deduction phase, and therefore these unique sequences are not exponentially amplified during the subsequent PCR step of RDA.

  As the complexity of nucleic acid sequences undergoing RDA decreases, it becomes difficult to use RDA to isolate relatively small viruses. The probability that there are two 6 bp recognition sites within 1.5 kb of each other is so low that small (≦ 10 kb) viruses may not be isolated (Table 1).

  In contrast, the present inventors have found that RDA is useful for cloning small viruses if the RDA test DNA is fractionated using a restriction endonuclease having a higher cleavage frequency. As shown in Table 1, an amplicon based on a 4 bp recognition site enzyme almost certainly contains several fragments from any small virus, such as a restriction endonuclease with a 4 bp recognition site fragment DNA about every 250 bp. . On the other hand, almost all of the DNA species are PCR amplified after digestion with the 4 bp recognition restriction endonuclease at ≦ 1500 bp, so the amplicon appears to be as complex as the nucleic acid source from which it was produced. Since the relative viral sequence copy number is expected to be greater than any specific or endogenous sequence copy number, the unique viral sequence present in the tester amplicon forms a double-stranded molecule during the hybridization step (step 3 above). It should be possible. Therefore, these sequences are amplified exponentially as described above. Use a restriction endonuclease that recognizes overlapping 6 bp sequences (eg BstYI or HincII) and cleaves about every 1000 bp, or recognizes 6 bp sequences, as the relative viral sequence copy number approaches background or endogenous nucleic acid sequence copy number It is inferred that several restriction endonucleases can be used simultaneously to fractionate DNA prior to amplification by PCR. In this way, the complexity of amplicons undergoing RDA can be moderately reduced while minimizing the risk of excluding viral sequences from tester amplicons. The usefulness of this method is demonstrated by the cloning of HGBV sequences from infectious tamarin plasma as described herein.

Immunoscreening to identify HGBV immunoreactive epitopes Immunoscreening as described below was also an additional means of identifying HGBV sequences. Viral particles are isolated using individual serum, plasma or liver homogenates or pools thereof from individuals who meet the criteria within the parameters below with acute or chronic HGBV infection. Nucleic acids isolated from these particles are used as templates in the construction of genomic libraries and / or cDNA libraries for viral genomes. The procedure used to isolate putative HGBV particles and construct a genomic and / or cDNA library with λ-gt11 or similar systems known to those skilled in the art is described below. λ-gt11 is a vector developed to specifically express the inserted cDNA as a fusion polypeptide with β-galactosidase and to screen a large number of recombinant phages with a specific antiserum against a defined antigen. A coding epitope capable of specifically binding a λ-gt11 cDNA library generated from a cDNA pool containing cDNA to serum from non-A, non-B, non-C, non-D and non-E hepatitis history individuals Screen for V. Hunyh et al. Glover, DNA Cloning Techniques; A Practical Approach , IRL Press, Oxford, England, pp. 49-78 (1985). About 10 ⁶ to 10 ⁷ phage are screened to identify and purify positive phage, and then tested for specificity to bind to serum from each individual previously infected with HGBV material. Phages that selectively bind to serum or plasma that are derived from patients that meet the following criteria and are not present in patients that do not meet these criteria are suitable for subsequent testing. By using the method of isolating overlapping nucleic acid sequences, clones containing additional upstream and downstream HGBV sequences are obtained. The nucleotide sequence of the HGBV nucleic acid sequence encoded within the isolated clone is analyzed to determine whether the composite sequence contains one long continuous ORF.

  Sequences recovered from HGBV sequences as described herein (and their complements) and sequences or any portion thereof may be prepared using synthetic methods or similar as described herein. The method may be used to prepare synthetic methods by combining partial sequence recovery. This description relates to a method capable of isolating genomic or cDNA sequences corresponding to the complete HGBV genome. However, other methods for isolating these sequences will be apparent to those skilled in the art and are considered to be within the scope of the present invention.

Replicates (clone 2, 4, 10, 16, 18, 23, and 50) from the strain deposited HGBV nucleic acid sequence library are located at 12301 Parklawn Drive, Rockville, Maryland 20852, dated February 10, 1994, based on the Budapest Treaty. At the American Type Culture Collection, and will be stored for 30 years from the date of deposit, 5 years from the latest sample order, or the longest of any of the lifetimes of US patents. The above deposited strains and all other deposited materials described herein are presented for convenience only and are not necessary to practice the invention in light of the teachings herein. The HGBV cDNA sequences in all deposited materials are part of this specification as reference material. The plasmid was prepared as follows. T.A. C. C. The deposit number was deposited. Clone 2 is A. T.A. C. C. Deposit No. 69556, Clone 4 is A. T.A. C. C. Deposit No. 69557, clone 10 is A. T.A. C. C. Deposit No. 69558, clone 16 is A. T.A. C. C. Deposit No. 69559, Clone 18 is A. T.A. C. C. Deposit No. 69560, clone 23 is A. T.A. C. C. Deposit No. 69561, clone 50 is A. T.A. C. C. Deposit number 69562 has been assigned.

  Replicates from the HGBV nucleic acid sequence library (clones 11, 13, 48 and 119) were deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852, dated April 29, 1994, under the Budapest Treaty. , 30 years from the date of deposit, 5 years from the latest sample order, or the longest of any US patent lifetime. The above deposited strains and all other deposited materials described herein are presented for convenience only and are not necessary to practice the invention in light of the teachings herein. The HGBV cDNA sequences in all deposited materials are part of this specification as reference material. The plasmid was prepared as follows. T. T. et al. C. C. The deposit number was deposited. Clone 11 is A. T. T. et al. C. C. Deposit No. 69613, Clone 13 is A. T. T. et al. C. C. Deposit No. 69611, Clone 48 is A. T. T. et al. C. C. Deposit No. 69610, clone 119 is A. T. T. et al. C. C. Deposit number 69612 has been assigned.

  Additional strains (clone 4-B1.1, 66-3A1.49, 70-3A1.37 and 78-1C1.17) from the HGBV nucleic acid sequence library were 12301 dated July 28, 1994 under the Budapest Treaty. Deposited with the American Type Culture Collection located at Parklawn Drive, Rockville, Maryland 20852, and will be stored for 30 years from the date of deposit, 5 years from the latest sample order, or the longest of any US patent lifetime . The above deposited strains and all other deposited materials described herein are presented for convenience only and are not necessary to practice the invention in light of the teachings herein. The HGBV cDNA sequences in all deposited materials are part of this specification as reference material. The plasmid was prepared as follows. T. T. et al. C. C. The deposit number was given. Clone 4-B1.1 is A.I. T. T. et al. C. C. Deposit No. 69666, clone 66-3A1.49 T. T. et al. C. C. Deposit No. 69665, clone 70-3A1.37 is A. T. T. et al. C. C. Deposit No. 69664, clone 78-1C1.17 is A. T. T. et al. C. C. Deposit number 69663 has been assigned.

  Clone pHGBV-C Clone # 1 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, dated November 8, 1994, based on the Budapest Treaty. From the longest of any of the five years to date or the duration of a US patent. The above deposited strains and all other deposited materials described herein are presented for convenience only and are not necessary to practice the invention in light of the teachings herein. pHGBV-C clone # 1 is T. T. et al. C. C. Deposit number 69971 has been assigned. The HGBV cDNA sequences in all deposited materials are part of this specification as reference material.

Preparation of viral polypeptides and fragments The availability of nucleic acid sequences allows construction of expression vectors that encode the antigenic active region of a polypeptide encoded by either chain. These antigenic active regions include, for example, envelope (coat) or core, in addition to non-structural regions of the virus, including, for example, polynucleotide binding proteins, polynucleotide polymerases, and other viral proteins necessary for viral particle replication and / or assembly. It can be derived from the structural region of the virus containing the antigen. Fragments encoding the desired polypeptide are obtained from genomic or cDNA clones by conventional restriction digestion or synthesis methods, such as β-galactosidase (β-gal), superoxide dismutase (SOD) or CMP-KDO synthetase (CKS). Ligated into a vector that may contain part of the fusion sequence. Methods and vectors useful for the production of polypeptides containing SOD fusion sequences are described in published EPO 0196056, published Oct. 1, 1986, and are useful for the production of polypeptides containing CKS fusion sequences and The vector is described in published EPO 03131961, dated September 13, 1989. Any desired portion of the nucleic acid sequence containing an open reading frame in either direction strand can be obtained as a recombinant protein such as a mature or fusion protein, or a polypeptide encoded by the HGBV genome or cDNA can be provided by chemical synthesis You can also

  The nucleic acid sequence encoding the desired polypeptide is linked to an appropriate expression vector for any suitable host, regardless of whether it is in fused or mature form, and whether it contains a signal sequence that allows secretion. can do. In the art, both eukaryotic and prokaryotic host systems can be used to form recombinant proteins, and here are some of these hosts. The polypeptide is then isolated from lysed cells or medium and purified to the extent necessary for the intended use. Purification can be carried out by methods known to those skilled in the art, and in particular, salting out, ion exchange resin chromatography, affinity chromatography, centrifugation and the like can be used. Such polypeptides can be used as diagnostic agents or in passive immunotherapy. In addition, antibodies against these polypeptides are useful for isolating and identifying HGBV particles. HGBV antigens can also be isolated from HGBV particles. These viral particles can be propagated in tissue culture or HGBV infected cells in infected individuals.

Antigenic polypeptide preparation and solid phase binding The antigenic region or fragment of a polypeptide is relatively small, usually about 8-10 amino acids in length or less. Even a fragment of about 5 amino acids can characterize the antigenic region. These segments can correspond to regions of the HGBV antigen. By using the HGBV genome or cDNA sequence as a basis, a nucleic acid sequence encoding a short segment of an HGBV polypeptide can be recombinantly expressed as a fusion protein or an isolated polypeptide. These short amino acid sequences can also be obtained by chemical synthesis. Small chemically synthesized polypeptides can be bound to a suitable carrier molecule if the resulting synthetic polypeptide is properly positioned to provide the proper epitope and is too small to be antigenic. Methods of conjugation are known to those skilled in the art and non-limiting examples include N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) is used. Polypeptides without sulfhydryl groups can be modified by adding cysteine residues. These substances create a disulfide bond between the substance and a peptide cysteine residue on one protein, and an amide bond via an ε-amino or other free amino group on lysine in the other protein To do. Various such disulfide / amide forming materials are known. Other bifunctional coupling agents form thioesters rather than disulfide bonds. Many of these thioether formers are commercially available and known to those skilled in the art. The carboxy group can be activated by conjugation with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid sodium salt. Any carrier can be used provided that it does not itself induce the production of antibodies harmful to the host. Suitable carriers may include proteins, polysaccharides (eg latex functionalized sepharose, agarose, cellulose, cellulose beads), polymeric amino acids (eg polyglutamic acid, polylysine, amino acid copolymers) and inactive virus particles. Examples of protein substrates can include serum albumin, mussel hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin and other proteins known to those skilled in the art.

Preparation of hybrid particle immunogens containing HGBV epitopes The immunogenicity of HGBV epitopes can also be enhanced by preparing them in a mammalian or yeast system fused or bound to a particle-forming protein (eg as in the HBV surface antigen). . A construct in which the HGBV epitope is directly linked to the sequence encoding the particle-forming protein produces an immunogenic hybrid to the HGBV epitope. Furthermore, all vectors containing epitopes specific for the prepared HGBV have different degrees of immunogenicity. Particles constructed from particle-forming proteins that contain HGBV sequences are immunogenic to HGBV and HBV.

Hepatitis B surface antigen S. cerevisiae and mammalian cells are confirmed to form and bind to particles, and the formation of these particles has been reported to enhance the immunogenicity of the monomer subunits. P. Valenzuela et al., Nature 298: 334 (1982); Valenzuela et al. Edited by Millman et al., Hepatitis B , Plenum Press, pp. 225-236 (1984). The construct may comprise an immunodominant epitope of HBsAg. Such constructs have been reported to be expressible in yeast and hybrids containing heterologous viral sequences for yeast expression are disclosed. See for example EPO 174,444 and EPO 174,261. It has also been reported that these constructs can be expressed in mammalian cells such as Chinese hamster ovary (CHO) cells. See Michelle et al., International Symposium on Viral Hepatitis , 1984. In HGBV, the part of the sequence encoding the particle-forming protein can be replaced with a codon encoding the HGBV epitope. This substitution eliminates regions that are not necessary to mediate aggregation of units to form immunogenic particles in yeast or mammals, thus eliminating additional HGBV antigenic sites from competition with HGBV epitopes. be able to.

Vaccine production A vaccine can be produced from one or more immunogenic polypeptides or nucleic acids derived from the HGBV nucleic acid sequence or the HGBV genome corresponding to the nucleic acid sequence. The vaccine may comprise a recombinant polypeptide comprising an epitope of HGBV. These polypeptides may be expressed in bacterial, yeast or mammalian cells or may be isolated from viral preparations. It is also anticipated that various structural proteins may contain epitopes of HGBV that induce protective anti-HGBV antibodies. Thus, synthetic peptides can also be used in the production of these vaccines. Thus, a polypeptide comprising at least one epitope of HGBV can be used alone or in combination in an HGBV vaccine. Furthermore, it is expected that nonstructural proteins and structural proteins may provide protection against viral pathogenicity even if they do not produce neutralizing antibodies.

  In view of the above, a multivalent antibody against HGBV may comprise one or more structural proteins and / or one or more nonstructural proteins. These vaccines can be composed, for example, of recombinant HGBV polypeptides and / or polypeptides and / or synthetic peptides isolated from viral particles. These immunogenic epitopes can be used in combination, i.e., as a mixture of recombinant proteins, synthetic peptides and / or polypeptides isolated from viral particles, and these vaccines can be used at the same or different times. Can be administered. Furthermore, it may be possible to use inactivated HGBV in the vaccine. Such inactivation may prepare a virus lysate or other means known to those skilled in the art to cause inactivation of hepatitis-like viruses, such as treatment with organic solvents or surfactants, or formalin You may implement by a process. The present invention also discloses an attenuated HGBV strain preparation. Some proteins in HGBV can cross-react with other known viruses, so there is a shared epitope between HGBV and other viruses, resulting in protective antibodies against one or more of the diseases caused by these pathogens It is expected to be. It is expected that multipurpose vaccines can be designed based on this belief.

  The preparation of vaccines comprising at least one immunogenic peptide as an active ingredient is known to those skilled in the art. Typically, such vaccines are prepared as injections as solutions or suspensions. Solid forms suitable for dissolving or suspending in liquid prior to injection can also be produced. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients are often mixed with pharmaceutically acceptable excipients that are compatible with the active ingredients. Non-limiting examples of suitable excipients include water, saline, dextrose, glycerol, ethanol, and the like, and various amounts of these excipients may be used in combination. Vaccines can also contain minor amounts of auxiliary substances such as wetting agents, emulsifiers, pH buffering agents and / or adjuvants that enhance the efficacy of the vaccine. For example, such adjuvants include aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11687). , Also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 ′, 2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine (CGP 19835A) , Also known as MTP-PE) and RIBI (MPL + TDM + CWS) 2% squalene / Tween-80® emulsion. The efficacy of an adjuvant can be determined by measuring the amount of antibody to an immunogenic polypeptide comprising an HGBV antigen sequence that results from administration of a polypeptide in a vaccine that is also composed of various adjuvants.

  Vaccines are usually administered by intravenous or intramuscular injection. Other formulations suitable for other modes of administration include suppositories, and in some cases oral formulations. In the case of suppositories, non-limiting examples of conventional binders and carriers include polyalkylene glycols or triglycerides. Such suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations contain commonly used excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, and are about 10% to about 95%, preferably about 25% to about 70% active ingredient. May be contained.

  Proteins used in vaccines can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the peptide) and formed with inorganic acids (eg hydrochloric acid or phosphoric acid) or organic acids (acetic acid, succinic acid, tartaric acid, maleic acid). Alternatively, other salts known to those skilled in the art can be used. Salts formed with free carboxyl groups are inorganic bases (eg sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide (III) etc.) and organic bases (eg isopropylamine, trimethylamine, 2-ethyl). Aminoethanol, histidine, procaine) and other bases known to those skilled in the art.

  The vaccine is administered in a manner compatible with the dosage form and in a prophylactic and / or therapeutically effective amount. The dosage is generally about 5 μg to about 250 μg of antigen at a time, depending on the patient being administered, the ability of the patient's immune system to synthesize antibodies and the degree of protection desired. The exact amount of active ingredient required to administer will also depend on the judgment of the prescribing physician and will vary from patient to patient. The vaccine may be administered on a single dose schedule or on a multiple dose schedule. In the multiple administration, after the primary vaccination process is performed in 1 to 10 times, the secondary administration is performed after a period necessary for maintaining and / or strengthening the immune response, for example, 1 to 4 months later. If necessary, administer several months later. The dosage regimen is determined at least in part by the individual's needs and is at the discretion of the prescribing physician. It is anticipated that vaccines containing immunogenic HGBV antigens can be administered in combination with other immunoregulatory agents such as immunoglobulins.

Production of Antibodies Against HGBV Epitopes Polyclonal or monoclonal antibodies are produced using immunogenic peptides prepared as described herein. In producing a polyclonal antibody, a selected mammal (eg, mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide having at least one HGBV epitope. Serum from the immunized animal is collected after an appropriate incubation time and processed according to known procedures. When serum containing polyclonal antibodies against HGBV epitopes contains antibodies against other antigens, the polyclonal antibodies can be purified, for example, by immunoaffinity chromatography. Methods for producing and processing polyclonal antibodies are known to those skilled in the art and are described in particular in Mayer and Walker, Immunochemical Methods In Cell and Molecular Biology , Academic Press, London (1987). Polyclonal antibodies can also be obtained from mammals previously infected with HGBV. One example of a method for purifying antibodies against HGBV epitopes from sera of individuals infected with HGBV using affinity chromatography is described herein.

Monoclonal antibodies against the HGBV epitope can also be produced by those skilled in the art. General methods for producing such antibodies are well known and are described, for example, in Kohler and Milstein, Nature 256: 494 (1975). G. R. Hurrel, Monoclonal Hybridoma Antibodies: Techniques and Applications , CRC Press Inc. , Boco Raton, FL (1982). T.A. Mimms et al., Virology 176: 604-619 (1990). Immortal antibody-producing cell lines can be created by cell fusion, and can also be created by other methods such as direct transformation of B lymphocytes with oncogene DNA or transfection with Epstein-Barr virus. Similarly, M.M. Schreier et al., Hybridoma Techniques, Scopes (1980) Protein Purification, Principles and Practice, 2nd ed., Springer-Verlag, New York (1984); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (1981); Kennet et al., Monoclonal Antibodies (1980). Examples of the use and techniques of monoclonal antibodies are disclosed in US Patent Applications Nos. 748,292, 748,563, 610,175, 648,473, 648,477 and 648,475.

Monoclonal and polyclonal antibodies against HGBV epitopes thus developed are useful for diagnostic and prognostic applications, and neutralizing antibodies are useful for passive immunotherapy. Monoclonal antibodies can be used in particular to produce anti-idiotype antibodies. These anti-idiotype antibodies are immunoglobulins that have an “internal picture” of the antigen of the infectious agent for which protection is desired. For example, A.I. Nisonoff et al . , Clin. Immunol. Immunopath . 21: 397-406 (1981) and Driesman et al., J. MoI. Infect. Dis . 151: 761 (1985). Methods for inducing such idiotypic antibodies are known to those skilled in the art and are described, for example, by Grych et al., Nature 316: 74 (1985); MacNamara et al., Science 226: 1325 (1984); and Uytdehaag et al . Immunol . 134: 1225 (1985). These anti-idiotype antibodies may also be useful in the treatment of HGBV infection and elucidation of the immunogenic region of HGBV antigen.

Diagnostic oligonucleotide probes and kits Using a predetermined portion of the isolated HGBV nucleic acid sequence as a substrate, hybridize with the HGBV genome, identify viral agents, further characterize the viral genome, and virus in affected individuals Oligomers of about 8 nucleotides or more that are useful for detection can be produced by excision or synthesis. Natural or derived probes for HGBV polynucleotides are of a length that allows detection of a single viral sequence by hybridization. While 6-8 nucleotides can be of a processable length, a sequence of 10-12 nucleotides is preferred and a sequence of about 20 nucleotides may be most preferred. These sequences are preferably derived from regions lacking heterogeneity. These probes can be produced using routine standard methods, including automated oligonucleotide synthesis methods. The complement of any single part of the HGBV genome will be satisfactory. Full complementarity may be unnecessary as the fragment length increases, but is desirable for use as a probe.

  When used as a diagnostic reagent, a test sample to be analyzed, such as blood or serum, may be processed, eg, extracting nucleic acids contained within the sample. The nucleic acid obtained from the sample may be subjected to gel electrophoresis or other size separation techniques, or the nucleic acid sample may be dot blotted without size separation. The probe is then labeled. Suitable labels and methods for attaching the label to the probe are known in the art and include radioactive labels, biotin, fluorescent and chemiluminescent probes that are incorporated by nick translation or kinase reaction. There is no limitation. Many examples of these labels are disclosed herein. The nucleic acid extracted from the sample is then treated with a labeled probe under appropriately stringent hybridization conditions.

  The probe can be perfectly complementary to the HGBV genome. Therefore, usually a high stringency condition is desirable to avoid false positives. However, high stringency conditions should only be used if the probe is complementary to a region of the HGBV genome lacking heterogeneity. The stringency of hybridization is determined by several factors during the washing procedure (eg, temperature, ionic strength, time and formamide concentration). For example, J. et al. See Sambrook (supra). Hybridization can be performed by several different techniques. Amplification can be performed by, for example, ligase chain reaction (LCR), polymerase chain reaction (PCR), Q-β replicase, NASBA, and the like.

HGBV genomic sequence, for example, at a relatively low level of about ¹⁰ 2 to 10 ³ sequences / ml, is considered to be present in the serum of infected individuals. At this level, it may be necessary to use amplification techniques such as ligase chain reaction or polymerase chain reaction in hybridization assays. Such techniques are well known in the art. For example, the “Biobridge” system uses terminal deoxynucleotide transferase to add unmodified 3′-poly-dT-tails to nucleic acid probes (Enzo Biochem. Corp.). A probe with a poly-dT-tail is hybridized to the target nucleotide sequence and then hybridized to biotin-modified poly-A. Furthermore, a DNA hybridization assay in which an analyte is annealed to a single-stranded DNA probe complementary to an enzyme-labeled oligonucleotide and the resulting tailed double strand is hybridized to the enzyme-labeled oligonucleotide is disclosed in European Patent No. No. 124221. European Patent No. 204510 has a probe having an analyte DNA having a tail (eg, poly-dT-tail) and a sequence (eg, poly-A sequence) that hybridizes to the tail of the probe, and a plurality of labeled strands. A DNA hybridization assay in which an amplifier strand that can bind to is contacted is described. This technique may consist of first amplifying the target HGBV sequence in the serum to about 10 ⁶ sequences / ml. This can be performed according to the method described in Saiki et al., Nature 324: 163 (1986). The amplified sequence can then be detected using hybridization assays as are known in the art. The probe can be packaged in a diagnostic kit containing a probe nucleic acid sequence that can be labeled. Alternatively, the labeling component may be included in the kit without labeling the probe. The kit may further include other appropriately packaged reagents and materials necessary or desirable for a particular hybridization protocol, such as standards and instructions for performing the assay.

Other known amplification methods that can be used in the present invention include the so-called “teach” of PNAS USA 87: 1874-1878 (1990) and discussed in Nature : 350 (No. 6313): 91-92 (1991). NASBA "or" 3SR "technology and Q-beta replicase are included, but are not limited to these.

  Fluorescence in situ hybridization ("FISH") can also be performed using the reagents described herein. In situ hybridization is the collection of uninjured tissues, cells or chromosomes by nucleic acid hybridization methods to demonstrate the existence of special genetic information and the specific location of the information in individual cells. Become. This does not require homogenization of the cells and extraction of the target sequence, thus revealing the exact location and distribution of the sequences in the cell population. In situ hybridization can identify problematic sequences that are concentrated in the cell, and can further identify the type and fraction of cells in the heterogeneous population of cells that contain the sequence. DNA and RNA can be detected with the same assay reagent. PNA can be used in the FISH method to detect the target without amplification. If an increase in signal is desired, multiple fluorophores can be used to increase the signal and increase the sensitivity of the method. Various FISH methods are known including one-step methods using multiple oligonucleotides or conventional multi-step methods. It is within the scope of the present invention that these types of methods can be automated by various means (eg, flow cytometry and image analysis).

Immunoassays and diagnostic kits Polypeptides that immunoreact with sera containing HGBV antibodies and complexes thereof, and antibodies against HGBV-specific epitopes in these polypeptides are both present in the presence of HGBV antibodies or viruses and / or in biological test samples. Useful in immunoassays to detect the presence of viral antigens. These immunoassay designs are often subject to variations, and these variations are well known in the art. These variations are described herein. The immunoassay is derived from a single viral antigen (eg, a clone containing HGBV nucleic acid sequences, or a composite nucleic acid sequence derived from HGBV nucleic acid sequences in these clones, or the HGBV genome that is the source of the nucleic acid sequences in these clones. Polypeptide). Alternatively, the immunoassay may use a combination of viral antigens derived from these sources. Immunoassays can use, for example, monoclonal antibodies against the same viral antigen or polyclonal antibodies against different viral antigens. Assays include, but are not limited to, those based on competitive assays, direct reaction assays or sandwich type assays. The assay may use a solid phase or may be performed by immunoprecipitation or any other method that does not use a solid phase. Assays that use labels as signal-generating compounds and examples of these labels are described herein. The signal further uses biotin and avidin, enzyme labels or biotin anti-biotin systems as described in pending US patent applications 608,849, 070,647, 418,981 and 687,785. And may be amplified. Recombinant polypeptides comprising epitopes derived from the immunodominant region of HGBV can be useful for detecting viral antibodies in biological test samples of infected individuals. It is also believed that antibodies can be useful in distinguishing between acute and non-acute infections. Package appropriate materials (eg, HGBV epitopes or polypeptides of the invention comprising antibodies to HGBV epitopes) in appropriate containers along with other reagents and materials necessary to perform the assay and appropriate assay instructions Thus, a kit suitable for immunodiagnosis containing an appropriate reagent is produced.

  Assay formats can be designed that use the recombinant proteins detailed herein. Although we have described CKS proteins, other expression systems such as superoxide dismutase (SOD) in the present invention can be used in various ways, such as fusion proteins, such as antigens for immunoassays, It is also considered that an immunogen for antibody production can be produced. In an assay format for detecting the presence of antibodies to a specific analyte (eg, an infectious agent such as a virus) in a human test sample, the human test sample is coated with at least one recombinant protein (polypeptide). Incubate in contact with the solid phase. If an antibody is present in the test sample, this antibody forms a complex with the antigen polypeptide and is immobilized on the solid phase. After formation of the complex, the solid phase is washed to remove unbound substances and reagents. The complex is reacted with the indicator reagent and incubated under the time and conditions for formation of the second complex. The generated signal is detected to determine the presence of antibodies to the CKS recombinant polypeptide in the test sample. A signal generation higher than the cut-off value indicates that antibodies to the analyte are present in the test sample. With multiple indicator reagents such as enzymes, the amount of antibody present is proportional to the signal produced. Depending on the type of test sample, the test sample may be diluted with an appropriate buffer reagent, concentrated, or contacted with a solid phase without manipulation (“neat”). For example, it is usually preferable to test a prediluted serum or plasma sample or concentrate a sample such as urine to determine the presence of antibody and / or the amount of antibody present.

  Furthermore, the aforementioned assay format, which uses CKS fusion proteins against various antigenic epitopes of the viral genome of the infectious agent under study to test for the presence of antibodies to specific infectious agents, uses two or more recombinant proteins. be able to. Thus, using a recombinant polypeptide comprising an epitope within a specific viral antigen region and an epitope derived from another antigenic region derived from the viral genome, rather than using a polypeptide derived from one epitope. It would be preferable to provide an assay with increased sensitivity and possibly high specificity. Such an assay can be used as a confirmatory assay. In this particular assay format, a known amount of test sample is applied to a known amount of at least one species coated with at least one recombinant protein for a time and under conditions sufficient to form a recombinant protein / antibody complex. Contact with the solid phase. The complex is contacted with a known amount of a suitable indicator reagent under suitable conditions for the time to cause the reaction. The generated signal is compared to a negative test sample to determine the presence of antibodies to the analyte in the test sample. When using certain solid phases, such as microparticles, each recombinant protein used in the assay is attached to an individual microparticle or a mixture of the microparticles produced by combining various coated microparticles. It can be optimized for the assay. Variations of the aforementioned assay formats include various analyte CKS recombinant proteins (eg, coated on the same or different solid phases) attached to the same or different solid phases to detect the presence of antibodies to each analyte. A CKS recombinant protein specific to one antigenic region of the infectious agent) together with a CKS recombinant protein specific to one antigenic region of a different infectious agent, Detecting the presence.

  As another assay format, CKS recombinant proteins containing antigenic epitopes are useful in competitive assays such as neutralization assays. To perform a neutralization assay, a recombinant polypeptide exhibiting an epitope of the antigenic region of an infectious agent such as a virus is solubilized and mixed with a sample diluent to give a final of 0.5-50.0 μg / ml. Concentrate. A known amount of test sample (preferably 10 μl) diluted as necessary is added to the reaction well, followed by 400 μl of test diluent containing the recombinant polypeptide. If desired, the mixture may be preincubated for about 15 minutes to 2 hours. The solid phase coated with the CKS recombinant protein described herein is then added to the reaction well and incubated at about 40 ° C. for 1 hour. After washing, a known amount of an indicator reagent, eg, 200 μl of peroxidase-labeled goat anti-human IgG in binding diluent, is added and incubated at 40 ° C. for 1 hour. After washing, when using an enzyme conjugate as described above, an enzyme substrate (eg, OPD substrate) is added and incubated at room temperature for 30 minutes. Stop the reaction by adding a stop agent such as 1N sulfuric acid to the reaction well. Read absorbance at 492 nm. In a test sample containing an antibody against a specific polypeptide, signal generation due to competitive binding between the peptide and the antibody in solution is reduced. The percentage of competitive binding can be calculated by comparing the absorbance value of the sample in the presence of the recombinant polypeptide to the absorbance value of the sample assayed in the absence of the same dilution of the recombinant polypeptide. Thus, the difference in signal that occurs between the sample in the presence of the recombinant protein and the sample in the absence of the recombinant protein is a measure used to determine the presence or absence of the antibody.

  As another assay format, a recombinant protein can be used in an immunodot blot assay system. The immunodot blot assay system uses a panel of purified recombinant polypeptides juxtaposed on a nitrocellulose solid support. The produced solid phase carrier is brought into contact with the sample, and a specific antibody (specific binding element) against the recombinant protein (other specific binding element) is captured to form a specific binding element pair. The captured antibody is detected by reacting with an indicator reagent. Preferably, the conjugate specific reaction is quantified using a reflectance optics assembly in the instrument described in US patent application Ser. No. 07 / 227,408 filed Aug. 2, 1988. Related U.S. patent applications 07 / 227,586 and 07 / 227,590 (both filed on August 2, 1988) are further assigned by the applicant and are hereby incorporated by reference. Similar to US Pat. No. 5,075,077 (US patent application Ser. No. 07 / 227,272, filed Aug. 2, 1988), specific methods and apparatus useful for performing immunodot assays are described. Briefly, nitrocellulose-based test cartridges are treated with a number of antigen polypeptides. Each polypeptide is contained within a specific reaction zone on the test cartridge. After all the antigen polypeptide has been placed on the nitrocellulose, it blocks excess binding sites on the nitrocellulose. The test cartridge is then contacted with the test sample so that each antigen polypeptide in each reaction zone reacts if the test sample contains the appropriate antibody. After the reaction, the test cartridge is washed and any antigen-antibody reaction is identified using well-known appropriate reagents. The entire process can be automated as described in the patents and patent applications cited herein. The foregoing application relating to methods and apparatus for performing immunodot blot assays is incorporated herein by reference.

  The CKS fusion protein can be used in an assay using the first and second solid phase carriers described later for detecting an antibody against the specific antigen of the analyte in the test sample. In this assay format, a first aliquot of a test sample is coated with an analyte-specific CKS recombinant protein for a time and under conditions sufficient to form a recombinant protein / analyte antibody complex. Contact with the prepared first solid phase carrier. The complex is then contacted with an indicator reagent specific for the recombinant antigen. An indicator reagent is detected to determine the presence of antibodies to the recombinant protein in the test sample. A second aliquot of the test sample is then coated with a CKS recombinant protein specific for the second antibody for a time and under conditions sufficient to form a recombinant protein / second antibody complex. The presence of different antigenic determinants of the same analyte is examined by contacting with a second solid phase carrier. The complex is contacted with a second indicator reagent specific for the antibody of the complex. A signal is detected to determine the presence of the antibody in the test sample. The presence of antibodies to one or both analyte recombinant proteins indicates the presence of anti-analyte in the test sample. It is also believed that the solid support can be tested simultaneously.

  The use of haptens is well known in the art. It is believed that haptens can be used in assays with CKS fusion proteins to enhance assay performance.

Further characterization of HGBV genomes, virions and viral antigens using probes HGBV nucleic acid sequences can be used to obtain further information about the sequence of the HGBV genome and the identification and isolation of HGBV agents. Therefore, this knowledge is considered to be useful for analysis of the characteristics of HGBV (for example, the type of HGBV genome, the structure of virus particles, and the type of antigen constituting HGBV). With this information, additional polynucleotide probes, polypeptides derived from the HGBV genome, and antibodies to HGBV epitopes can be obtained. They are useful for the diagnosis and / or treatment of non-A, non-B, non-C, non-D and non-E hepatitis caused by HGBV.

  The nucleic acid sequence information is useful for designing probes or PCR primers to isolate additional nucleic acid sequences derived from non-deterministic regions of the HGBV genome. For example, using a PCR primer or a labeled probe comprising a sequence of 8 or more nucleotides, preferably 20 or more nucleotides, derived from a region near the 5 ′ end or 3 ′ end of the HGBV nucleic acid sequence family, It may be isolated from an HGBV genomic or cDNA library or directly from viral nucleic acid. Then another overlap that does not necessarily overlap with the nucleic acid sequence in the clone, using said sequence that further overlaps with the HGBV nucleic acid sequence but further comprises a sequence derived from a region of the genome that is not the source of the HGBV nucleic acid sequence Probes for identifying fragments may be synthesized. If the HGBV genome is segmented but lacking a common sequence, it is possible to determine the sequence of the entire viral genome using techniques for isolating overlapping nucleic acid sequences derived from the viral genome. Alternatively, genomic segments can be characterized from viral genomes isolated from purified HGBV particles. Methods for purifying HGBV particles and detecting the particles during the purification procedure are described herein. Procedures for isolating polynucleotide genomes from viral particles are well known in the art. The isolated genomic segment can then be cloned and sequenced. Therefore, it is possible to clone and sequence the HGBV genome regardless of its type.

  Methods for constructing HGBV genomic or cDNA libraries are known in the art, and vectors useful for this are also known in the art. These vectors include λ-gt11, λ-gt10, and the like. The isolated polynucleotide may be digested with appropriate restriction enzymes and the HGBV-derived nucleic acid sequence detected by the HGBV genome or cDNA-derived probe isolated from the clone and sequenced.

  Sequence information derived from these overlapping HGBV nucleic acid sequences is useful for determining homology and heterogeneity areas within the viral genome. This may indicate the presence of various genomic strains and / or defective particle populations. This is also useful for the design of hybridization probes for detecting HGBV or HGBV antigens or HGBV nucleic acids in biological samples during the isolation of HGBV using the techniques described herein. Duplicate nucleic acid sequences may be used to produce expression vectors for polypeptides derived from the HGBV genome. An antigen containing an epitope believed to be unique to HGBV is encoded within the nucleic acid sequence family. That is, antibodies against these antigens are not present in individuals infected with HAV, HBV, HCV and HEV, and the homology between GenBank genomic sequences and these nucleic acid sequences and the source polynucleotide sequence is It is considered small. Thus, antibodies against antigens encoded by HGBV nucleic acid sequences may be used to identify non-A, non-B, non-C, non-D and non-E particles isolated from infected individuals. Furthermore, they are useful for the isolation of HGBV material.

  HGBV particles can be produced, for example, by size discrimination based techniques (eg sedimentation or exclusion), or density based techniques (eg density gradient ultracentrifugation, or precipitation with substances such as polyethylene glycol (PEG), or anions Or the sera or cell culture of infected individuals by any method known in the art, including cation exchange materials, materials that bind by hydrophobic interactions, and chromatography on various materials such as affinity columns). Can be isolated from Presence of HGBV during the isolation procedure by performing hybridization analysis of the extracted genome with a probe derived from the HGBV nucleic acid sequence or by immunoassay using as an probe an antibody against the HGBV antigen encoded within the HGBV nucleic acid sequence family Can be detected. The antibody may be polyclonal or monoclonal and it may be desirable to purify the antibody prior to use in an immunoassay. The antibody against HGBV antigen immobilized on a solid phase is useful for isolation of HGBV by immunoaffinity chromatography. Immunoaffinity chromatography methods are well known in the art and include methods of immobilizing antibodies on a solid phase to retain immunoselective activity. These methods include adsorption and covalent bonding. A spacer group may be included in the bifunctional coupling agent so that the antigen binding site of the antibody remains accessible.

  During the purification procedure, the presence of HGBV may be detected and / or verified by nucleic acid hybridization or PCR using the HGBV genome or cDNA sequence family and polynucleotides derived from overlapping HGBV nucleic acid sequences as probes or primers. . Fractions are processed under conditions that cause destruction of the viral particles, for example using detergent in the presence of a chelating agent, and the presence of viral nucleic acid is determined by hybridization techniques or PCR. Whether the individual (preferably tamarin) is infected with the isolated viral particles and then the non-A, non-B, non-C, non-D and non-E hepatitis symptoms described herein are due to infection By examining whether it is possible, another confirmation can be obtained that the isolated particles are HGBV infection inducers.

  The properties of the aforementioned virus particles obtained from the purified preparation can then be further analyzed. Once the genomic nucleic acid is purified, it can be tested to determine its sensitivity to RNAse or DNAseI. Based on these tests, HGBV may be examined as an RNA genome or a DNA genome. The number of chains and the cyclicity or acyclicity can be examined by methods known in the art, for example by visualization by electron microscopy, migration by density gradient and sedimentation characteristics. From the hybridization of the HGBV genome, the negative or positive strandedness of the purified nucleic acid can be examined. Furthermore, if the genomic material is RNA, the purified nucleic acid can be cloned and sequenced by known techniques (eg, reverse transcriptase). The entire genome can then be sequenced with or without segmentation using nucleic acids derived from viral particles.

  Determination of a polypeptide containing a conserved sequence is useful for selecting probes that bind to the HGBV genome, which leads to isolation of the probe. Furthermore, the conserved sequences may be used together with sequences derived from HGBV nucleic acid sequences to design primers for use in systems that amplify genomic sequences. Furthermore, the structure of HGBV may be examined to isolate its components. For example, the form and dimensions can be examined by electron microscopy. Determine whether the antigen is present in the major viral component or in the minor viral component, and use the antibody against the specific antigen encoded in the isolated nucleic acid sequence as a probe to detect the specific viral polypeptide antigen [e.g. Envelope (coat) antigens or internal antigens (eg, nucleic acid binding proteins or core antigens)] or polynucleotide polymerases can also be identified and located. This information can be useful for diagnostic and therapeutic applications. For example, it may be preferable to include an external antigen in the vaccine preparation, or perhaps the multivalent vaccine may be a polypeptide derived from a genome that encodes a structural protein, and a polypeptide derived from another part of the genome (eg, a nonstructural polypeptide Peptide).

Cell culture systems and animal model systems for HGBV replication Generally, cells or cell lines suitable for culturing HGBV include monkey kidney cells (eg MK2 and VERO), porcine kidney cell lines (eg PS), hamster infant kidney cell lines ( BHK), mouse macrophage cell lines (eg P388D1, MK1 and Mm1), human macrophage cell lines (eg U-937), human peripheral blood leukocytes, human adherent monocytes, hepatocytes or hepatocyte cell lines (eg HUH7 and HepG2) Embryonic cells or embryonic cells (eg chicken embryo fibroblasts), or invertebrates, preferably cell lines derived from insects (eg Drosophila cell lines), more preferably cell lines derived from arthropods (eg mosquito cell lines) Or a tick cell line). It is also possible to culture primary hepatocytes and then infect them with HGBV. Alternatively, hepatocyte cultures can be derived from the liver of an infected individual (human or tamarin). The latter case is an example of a cell line that has been in vivo infected and passaged in vitro . Furthermore, cell lines derived from hepatocyte cultures can be obtained using various immortalization methods. For example, primary liver cultures (before and after enrichment of the hepatocyte population) can be fused with various cells to maintain stability. Furthermore, the culture may be infected with a transforming virus or transfected with a transforming gene to produce a permanent or semi-permanent cell line. Furthermore, cells in the liver culture may be fused to a defined cell line such as PehG2. Cell fusion methods are well known to those skilled in the art and include, inter alia, the use of fusion agents such as PEG and Sendai virus.

It is believed that cell line HGBV infection can be achieved by techniques such as incubating cells with a viral preparation under conditions that allow viral entry into the cells. It may also be possible to transfect cells with the isolated viral polynucleotide to obtain a viral preparation. Methods for transfecting tissue culture cells are known in the art and include, but are not limited to, techniques using electroporation and precipitation with DEAE-dextran or calcium phosphate. Virus is replicated and propagated in vitro by transfection with cloned HGBV genome or cDNA. In addition to cultured cells, animal model systems may be used for virus replication. Thus, HGBV replication can occur in chimpanzees, as well as, for example, marmoset and infant mice.

Screening of HGBV antiviral agents Since cell culture systems and animal model systems for HGBV can be used , screening of antiviral agents that inhibit HGBV replication, particularly preferably substances that enable cell proliferation while inhibiting viral replication is also possible It becomes. These screening methods are well known in the art. In general, animal model systems with various concentrations of antiviral agents in cell culture systems that support viral replication for their effects on the prevention of viral replication, followed by inhibition of infectivity or viral pathogenicity, and low levels of toxicity Inspect at. The methods and compositions for detection of HGBV antigens and HGBV polynucleotides described herein are useful for screening antiviral agents. This is because they are probably a more sensitive alternative for detecting the effect of antiviral agents on viral replication than the cell plaque assay or ID ₅₀ assay. For example, the HGBV polynucleotide probes described herein may be used to quantify the amount of viral nucleic acid produced in a cell culture. This can be done by hybridizing or competitively hybridizing infected cell nucleic acid with a labeled HGBV polynucleotide probe. In addition, anti-HGBV antibodies may be used to identify and quantify HGBV antigens in cell cultures by the immunoassay described herein. Furthermore, since it may be desirable to quantify HGBV antigen in infected cell cultures by competition assays, the polypeptides encoded within the HGBV nucleic acid sequences described herein are useful in these assays. In general, a recombinant HGBV polypeptide derived from an HGBV genome or cDNA is labeled, and inhibition of binding of the labeled polypeptide to the HGBV polypeptide by an antigen produced in a cell culture system is monitored. These methods are particularly useful when HGBV can replicate in cell lines without causing cell death.

Preparation of HGBV attenuated strains Using tissue culture systems and / or animal model systems as described herein, it may be possible to isolate HGBV attenuated strains. These attenuated strains are useful as vaccines or for the isolation of viral antigens. Attenuated strains can be isolated after multiple passages in cell culture and / or animal models. Detection of attenuated strains in infected cells or individuals can be accomplished according to the following methods known in the art, including the use of antibodies as probes for one or more epitopes encoded by HGBV, Alternatively, the use of a polynucleotide comprising a HGBV sequence of at least about 8 nucleotides in length as a probe can be included. In addition or alternatively, attenuated strains may be constructed using the genomic information of HGBV described herein and using recombinant techniques. Usually, not viral replication, but a region of the genome that encodes a polypeptide associated with virulence is deleted. Once the genome is constructed, epitopes that produce neutralizing antibodies against HGBV can be expressed. The denatured genome can then be used to transform cells that allow HGBV replication and the cells can be grown under conditions that allow viral replication. The attenuated HGBV strain is useful not only for vaccines but also as a source for commercial production of viral antigens. This is because the treatment of these viruses does not require so much safeguards against employees involved in virus production and / or production of virus products.

Extract host and expression control sequence genomes from viruses, create and probe genomic libraries, sequence clones, construct expression vectors, transform cells, perform immunoassays, and in culture General techniques used to grow cells are known in the art and are encompassed by the present invention.

When using appropriate control sequences that are compatible with the designated host, both prokaryotic and eukaryotic host cells can be used for the expression of the desired coding sequence. Prokaryotic hosts include E. coli . E. coli is most often used. Prokaryotic expression control sequences optionally include a promoter containing an operator moiety and a ribosome binding site. Transfer vectors that are compatible with prokaryotic hosts are typically derived from plasmid pBR322 containing an operon conferring ampicillin resistance and tetracycline resistance, and various pUC vectors containing sequences conferring antibiotic resistance markers. Using these markers, better transformed cells can be obtained by selection. Commonly used prokaryotic control sequences are reported by β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 198: 1056 [1977]), (Goeddel et al., Nucleic Acid Res 8: 4057 [1980]. A hybrid Tac promoter (De Boer et al., Proc ) derived from tryptophan promoter system, λ-inducible P1 promoter and N gene ribosome binding site (Shimatake et al., Nature 292: 128 [1981]), and trp and lac UV5 promoter sequences Natl.Acad.Sci.USA 292: 128 [1983]). The aforementioned system is particularly suitable for E.I. Other prokaryotic hosts such as Bacillus strains or Pseudomonas strains may be used with corresponding control sequences if desired, although compatible with E. coli .

Eukaryotic hosts include cultured yeast and mammalian cells. Saccharomyces cerevisiae and Saccharomyces carlsbergensis are the most commonly used yeast hosts and convenient fungal hosts. Yeast-compatible vectors carry markers that make auxotrophic mutants prototrophic or confer heavy metal resistance to wild-type strains, allowing the selection of good transformed cells. Yeast compatible vectors may be 2 micron replicating origins (Broach et al., Meth. Enz. 101; 307 [1983]), a combination of CEN3 and ARS1, or other means for performing replication (eg, suitable into the host cell genome). Sequences that result in the incorporation of small fragments). Regulatory sequences for yeast vectors are known in the art and include a glycolytic enzyme synthesis promoter including a promoter for 3-phosphophycerate kinase. For example, Hess et al . Adv. Enzyme Reg . 7: 149 (1968), Holland et al., Biochemistry 17: 4900 (1978) and Hitzeman J. et al . Biol. Chem . 255: 2073 (1980). Holland, J.M. Biol. Chem . A terminator such as that derived from the enolase gene reported in 256: 1385 (1981) may also be included. Particularly useful control systems include the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase (ADH) regulatable promoter, a terminator derived from GAPDH, and yeast α-factor if secretion is desired. It is considered that the system contains the derived leader sequence. Furthermore, the operably linked transcriptional regulatory region and transcription initiation region may be such that they are not innately related in wild type organisms.

  Mammalian cell lines that can be used as hosts for expression are known in the art and include a number of immortalized cell lines available from the American Type Culture Collection. These include HeLa cells, Chinese hamster ovary (CHO) cells, hamster infant kidney (BHK) cells and the like. Promoters suitable for mammalian cells are also known in the art, such as Simian virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV), cytomegalovirus (CMV). Viral promoters as derived from are included. Mammalian cells may further require terminator sequences and poly A addition sequences. Enhancer sequences that increase expression may be included, and sequences that induce gene amplification may be desired. These sequences are known in the art. Vectors suitable for mammalian cell replication ensure the incorporation of viral replicons or appropriate sequences encoding non-A, non-B, non-C, non-D, non-E epitopes into the host genome. Can be included. An example of a mammalian expression system for HCV is described in US patent application Ser. No. 07 / 830,024, filed Jan. 31, 1992.

Transformation Transformation is possible by known methods for introducing polynucleotides into host cells, including transduction of host cells by a virus that has incorporated the polynucleotide into the virus, and direct uptake of the polynucleotide. The transformation procedure selected will depend on the host to be transformed. Bacterial transformation by direct uptake generally uses treatment with calcium chloride or rubidium chloride. Cohen, Proc. Natl. Acad. Sci. USA 69: 2110 (1972). Graft transformation by direct uptake may be performed using the calcium phosphate precipitation method of Graham et al., Virology 52: 526 (1978) or variations thereof.

Vector Construction Vector construction uses methods known in the art. In general, site-specific DNA cleavage is performed by treatment with an appropriate restriction enzyme under conditions generally defined by commercial enzyme manufacturers. Typically, about 1 microgram (μg) of plasmid or DNA sequence is cleaved by incubating at 37 ° C. for 1-2 hours with 1-10 units of enzyme in about 20 μl of buffer solution. After incubation with the restriction enzyme, the protein is removed by phenol / chloroform extraction and precipitated with ethanol to recover the DNA. The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis by methods known to those skilled in the art.

Present in the mixture in the presence of a suitable deoxynucleotide triphosphates (dNTPs), E. The cohesive end cleavage fragment may be made blunt end using E. coli DNA polymerase 1 (Klenow). Treatment with S1 nuclease may be used to hydrolyze the single stranded DNA portion.

T4 DNA ligase and ATP are used to bind under standard buffer and temperature conditions. Sticky end bonding requires less ATP or ligase than blunt end bonding. When vector fragments are used as part of a ligation mixture, vector fragments are often treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove 5'-phosphate and prevent vector recombination. Alternatively, restriction enzyme digestion of undesired fragments can be used to prevent binding. The binding mixture is treated with E.I. It is transformed into a suitable cloning host such as E. coli or a good transformed cell selected by methods including antibiotic resistance and then screened for the correct construction.

Construction of the desired DNA sequence Synthetic oligonucleotides may be produced using an automated oligonucleotide synthesizer as described by Warner, DNA 3: 401 (1984). If desired, the synthetic strand can be labeled with ³² P by treatment with polynucleotide kinase in the presence of ³² P-ATP using standard reaction conditions. A DNA sequence (eg, a DNA sequence isolated from a genomic or cDNA library) is obtained using known methods [eg, Zoller, Nucleic Acid Res . 10: 6487 (1982)]. Briefly, the DNA to be modified is packaged in a phage as a single stranded sequence and is complementary to the DNA portion to be modified, using a synthetic oligonucleotide as a primer that contains the desired modification in its own sequence. Convert to double-stranded DNA with DNA polymerase. Plaques are obtained by agar plating cultures of transformed bacteria containing replication regions for each strand of phage. Theoretically, 50% of the new plaques contain phage with the mutated sequence and the remaining 50% have the original sequence. The plaque replica is hybridized to the labeled synthetic probe under temperature and conditions suitable for hybridization with the correct strand, but not the unmodified sequence. The sequence identified by hybridization is recovered and cloned.

Hybridization with probes Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 73: 3961 (1975) may be used to probe HGBV genomic or DNA libraries. Briefly, the DNA to be probed is immobilized on a nitrocellulose filter, denatured, 0-50% formamide, 0.75M NaCl, 75 mM sodium citrate, 0.02% (w / v) each of bovine serum. Prehybridize with buffer containing albumin (BSA), polyvinylpyrrolidone and Ficoll, 50 mM sodium phosphate (pH 6.5), 0.1% SDS and 100 μg / ml carrier denatured DNA. The percentage of formamide in the buffer and the time / temperature conditions of the prehybridization and subsequent hybridization steps depend on the stringency required. Oligomeric probes, which may have low stringency conditions, are generally used with a low rate of formide, a lower temperature, and a longer hybridization time. Probes containing 30 or 40 or more nucleotides, such as those derived from cDNA or genomic sequences, generally use higher temperatures such as about 40-42 ° C. and a higher percentage of formamide, for example 50%. After prehybridization, ³² P-labeled oligonucleotide probe is added to the buffer and the filter is incubated in this mixture under hybridization conditions. After washing, the treated filter is subjected to autoradiography to indicate the position of the hybridized probe. The DNA at the corresponding position on the original agar plate is used as the source of the desired DNA.

Construction Validation and Sequencing In standard vector construction, the ligation mixture is E. coli. E. coli strain XL-1 Blue or other suitable host is transformed and good transformed cells are selected by antibiotic resistance or other markers. Then, usually Clewell et al . Bacteriol . 110: 667 (1972) followed by chloramphenicol amplification followed by Clewell et al . , Proc. Natl. Acad. Sci. USA 62: 1159 (1969) to produce a plasmid derived from transformed cells. DNA is isolated and analyzed, usually by restriction enzyme analysis and / or sequencing. Messing et al., Nucleic Acid Res . 9: 309 (1981), Sanger et al . , Proc. Natl. Acad. Sci. USA 74: 5463 (1977), or by the method described in Maxam et al., Methods in Enzymology 65: 499 (1980). The problem of band compression that is sometimes observed in the GC rich region is overcome with T-deazoguanosine by the method described in Barr et al., Biotechniques 4: 428 (1986).

Enzyme linked immunosorbent assay An enzyme linked immunosorbent assay (ELISA) can be used to measure antigen concentration or antibody concentration. This method relies on the binding of an enzyme label to an antigen or antibody and uses the bound enzyme activity (generated signal) as a quantitative label (measurable generated signal). Methods for using enzymes as labels, and examples of such enzyme labels are described herein.

Generation of HGBV nucleic acid sequences Sources of non-A, non-B, non-C, non-D, non-E materials are humans infected with HGBV virus that meet the clinical and experimental criteria described herein Or individual plasma, serum or liver homogenates from tamarins or pools thereof. Alternatively, tamarin can be experimentally infected with blood of other individuals suffering from non-A, non-B, non-C, non-E hepatitis that meet the criteria described below. Multiple pools of individual plasma, serum or liver homogenate samples containing high levels of alanine transferase activity can be prepared. This activity is due to hepatic damage due to HGBV infection. The virus TID (Tamarin Infectivity) calculated in one of our experiments was 4 × 10 ⁵ cells / ml or more (see Example 2 below).

  For example, a nucleic acid library derived from plasma, serum or liver homogenate that is preferably high titer, but not necessarily, is prepared by the following method. First, virus particles are isolated from plasma, serum or liver homogenate. The aliquot is then diluted in a buffer solution (eg, a buffer solution containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl). For example, debris is removed by centrifugation at 15,000 × g and 20 ° C. for 20 minutes. Centrifugation is then performed under appropriate conditions that can be routinely determined by one skilled in the art to pellet the viral particles in the resulting supernatant. To release the viral genome, the pellet is subjected to SDS suspension [eg suspension containing 1% SDS, 120 mM EDTA, 10 mM Tris-HCl (pH 7.5) and 2 mg / ml proteinase K] The particles are then suspended in an aliquot of and then incubated under appropriate conditions, eg, at 45 ° C. for 90 minutes to break the particles. For example, 0.8 μg MS2 bacteriophage RNA is added as a carrier, and the mixture is added to phenol: chloroform [0.5 M Tris-HCl (pH 7.5), 0.1% (v / v) β-mercaptoethanol, 0.1 Nucleic acids are isolated by extraction four times with a 1: 1 mixture of phenol saturated with% (w / v) hydroxyquinolone and then twice with chloroform. The aqueous phase is concentrated, for example with 1-butanol, and then precipitated overnight at −20 ° C. with 2.5 volumes of absolute ethanol. For example, the nucleic acid is recovered by centrifugation at 40,000 rpm and 4 ° C. for 90 minutes in a Beckman SW41 rotor, dissolved in water, treated with 0.05% (v / v) diethylpyrocarbonate, and autoclaved. To do.

The nucleic acid obtained by the above procedure is denatured with, for example, 17.5 mM CH ₃ HgOH, and then cDNA is synthesized using this denatured nucleic acid as a template. For example, phages can be synthesized using the method described in Huynh (1985, supra). Cloning into the EcoRI site of λ-gt11. However, the random primer is replaced with oligo (dT) 12-18 during the synthesis of the first nucleic acid strand by reverse transcriptase (see Taylor et al., [1976]). The resulting double-stranded nucleic acid sequence is fractionated, for example, by sizing on a Sepharose CL-4B column. Elution material with an average size of about 400, 300, 200 and 100 base pairs is pooled into the genome pool. A λ-gt11 cDNA library is created from the cDNA in at least one pool. Alternatively, when the etiological agent is a DNA virus, genomic DNA cloning methods are useful and are known to those skilled in the art.

Serum, plasma or liver from a non-A-type, non-B-type, non-C-type, non-E-type hepatitis individual (according to the criteria described below) is stored in the λ-gt11 genomic library thus prepared. Screen for epitopes that can specifically bind to the homogenate. Using the method of Huyng et al. (Supra), about 10 ⁴ to 10 ⁷ phage are screened with serum, plasma or liver homogenate. Bound human antibodies can be detected with sheep anti-human Ig antiserum radiolabeled with ¹²⁵ I or other suitable reporter molecules (eg, HRPO, alkaline phosphatase, etc.). Positive phage are identified and purified. The phage is then tested for specificity of binding with the serum of a predetermined number of humans previously infected with HGBV substance using the same method. Ideally, the phage encodes a polypeptide that reacts with all or most of the serum, plasma or liver homogenate to be tested and is described herein for HGBV substances and hepatitis A, B, C, D, and E. It does not react with serum, plasma or liver homogenate from individuals who are determined to be “negative” by the criteria described in the document. A polypeptide that is immunologically specifically recognized by serum, plasma, or liver homogenate from a patient identified as non-A, non-B, non-C, non-D, non-E according to these procedures A clone encoding can be isolated.

  The invention is illustrated by the examples below. These examples are illustrative and do not limit the scope of the invention.

(Example)
The examples described herein describe in detail how to find viruses in the HGBV group. The examples are described in chronological order so as to trace the discovery of HGBV-A, HGBV-B and HGBV-C viruses in the HGBV group. In general, transmission and infectivity studies were first conducted. These studies described herein and subsequent studies demonstrated the existence of two HCV-like viruses, GB-A and GB-B, as HGBV. In addition, HGBV-C was discovered by subsequent experiments using degenerative primers, also described in detail herein. Prevalence of the virus group in humans demonstrated by serologic studies, viral characterization of the virus group, relevance to other viruses in that particular genus and correlation of HGBV-A, HGBV-B and HGBV-C Teach sex.

Example 1
Propagation of HGBV Experimental protocol . Sixteen tamarins (Saguinus labiatsu) were obtained from LEMSIP (Laboratory for Experimental Medicine and Surgery in Primates, Tuxedo, New York) for transmission and infectivity studies. All animals were maintained and monitored with LEMSIP according to the protocol approved by LEMSIP (Note: one animal died spontaneously and the other sick animal was euthanized before the start of the infectivity experiment). Serum liver enzymes alanine transaminase (ALT), γ-glutamyltransferase (GGT) and isocitrate dehydrogenase (ICD) for 2-3 weeks, based on serum samples obtained once a week or once every two weeks Liver enzyme values were determined. Prior to inoculation, a minimum of 8 serum liver enzyme values were obtained for each animal. A cut-off value (CO) was determined for each animal based on the mean liver enzyme value + 3.75 times the standard deviation. Liver enzyme values above the cut-off value were abnormal and the liver was considered damaged. Several tamarins were inoculated as described below and monitored for changes in ALT, GGT and ICD serum levels. Thereafter, at certain times during the monitoring process, several animals were killed to obtain serum and tissue for subsequent experiments.

B. Animal inoculation (initial experiment) . 11 infectious tamarin GB serum pools (shown as H205 GB pass 11) from serum collected during the early acute phase of hepatitis (19-24 days after inoculation) from 9 tamarins inoculated with HGBV ) Was prepared. The pool has already been described (JL Dienstag et al., Nature 264, supra; E. Tabor et al., J. Med. Virol. 5, supra) and was studied to determine the associated etiological agent. Aliquots of the pool were maintained under liquid nitrogen storage conditions in Abbott Laboratories (North Chicago, IL60064) until used for this experimental study. Other aliquots of HGBV are available from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 under ATCC Deposit Number VR-806.

  On the first day, 4 of the remaining 14 tamarins in the initial group, identification numbers T-1053, T-1048, T-1057 and T-1061 were diluted 1:50 in advance. Pool H205, 11 passages 0.25 ml were inoculated intravenously. These animals were monitored weekly for changes in liver enzymes ALT, GGT and ICD. Table 2 shows liver enzyme data for these four tamarins (T-1053, T-1048, T-1057, and T-1061) before and after inoculation. Figures 1-4 show the ALT and ICD levels of these 4 tamarins before and after inoculation. As the data showed, a significant increase over CO was obtained in ALT, GGT and ICD of 4 tamarins inoculated with a 1:50 dilution of pool H205.

  On the same day (Day 1), one tamarin (T-1047) was intravenously inoculated with 0.25 ml of pooled normal tamarin serum and used as a negative control, and another tamarin (T-1042) Added an additional negative control by intravenous inoculation with pooled normal human serum. Figures 5 and 6 and Table 3 show the ALT and ICD levels before and after inoculation of two control tamarins (T-1047 and T-1042). As the data show, there was no increase in ALT or ICD in the two control tamarins during 8 weeks.

On the same day (first day), one tamarin (T-1044) was intravenously inoculated with 0.2 ml of convalescent serum obtained from the surgeon (GB origin) about 3 weeks after the occurrence of acute hepatitis. This specimen was stored at −20 ° C. [F. Deinhardt et al., J. Exper. Med . 125: 673-688 (1967)]. Another tamarin (T-1034) was inoculated with 0.1 ml of the convalescent serum. As shown in FIG. 7 and FIG. 8 and Table 4, during 11 weeks after inoculation, no increase was observed in the serum liver enzymes of these tamarins. Therefore, these data indicate that no infectious HGBV was detected in the convalescent sera collected from the original patient and stored at -20 ° C, indicating that the patient recovered from infection. And that the virus has been removed from the patient's serum or that the virus titer has been reduced to an undetectable level by storage at -20 ° C.

C. Later experiments . Tamarin T-1053 showed a significant increase in serum liver enzymes one week after inoculation and was retested for liver enzymes 11 days after inoculation. On day 12, it was determined that there was a significant increase in serum liver enzymes and the animals were killed on that day. Plasma, liver and spleen tissue samples were obtained for subsequent experiments. Plasma derived from T-1053 was used as the starting material for the RDA procedure described in Example 3 below, and liver tissue was used in Example 8 below.

  Tamarins T-1048, T-1057 and T-1061 were monitored for serum liver enzymes. All the animals were found to have elevated liver enzyme levels within 2 weeks after inoculation. These increased values were seen after 6 weeks after inoculation. All three tamarins were found to have decreased serum liver enzyme levels below CO by 84 days after inoculation. On day 97 after inoculation, these three tamarins (T-1048, T-1057, and T-1061) were treated with 0.10 ml of neat plasma derived from tamarin T-1053 (provided to be infectious, Example 2). ) And re-challengeed to determine whether hepatitis, indicated as an increase in serum liver enzymes, could be re-induced. The data is shown in Table 2, FIG. 1, FIG. 3 and FIG. As the data show, the serum liver enzyme levels of the two tamarins (T-1057 and T-1061) remained below CO at 3 weeks after inoculation. One tamarin (T-1048) showed a gradual increase in serum liver enzyme levels in the 2 weeks immediately following revaccination. It was estimated that the gradual rise in T-1048 was due to reinfection of liver tissue with HGBV or not fully recovering from the initial inoculation of H205.

(Example 2)
Infectivity experiment Experimental protocol . Reference readings for 4 tamarins were obtained as described in Example 1 (A). The baseline serum liver enzyme (ALT, GGT and ICD) values were briefly determined for each animal prior to inoculation. A cut-off value (CO) was determined for each animal based on the mean liver enzyme value + 3.75 times the standard deviation. Liver enzymes above the cut-off value were abnormal and considered liver damaged.

B. Tamarin inoculation . Plasma derived from tamarin T-1053 (see Example 1 [C]) killed 12 days after inoculation was used as an inoculum for subsequent experiments. On the first day, one tamarin (T-1055) was inoculated intravenously with 0.25 ml of neat plasma of T-1053. On the same day, two tamarins (T-1038 and T-1051) were serially diluted to 10 ⁻⁴ (T-1038) or 10 ⁻⁵ (T-1051) in pooled normal tamarin plasma. 0.25 ml of -1053 plasma was inoculated intravenously. On the same day, Tamarin T-1049 was filtered through a series of filters with reduced pore sizes (0.8 μm, 0.45 μm, 0.22 μm and 0.10 μm) and diluted to 10 ⁻⁴ in pooled normal tamarin plasma. 0.25 ml of T-1053 plasma was inoculated intravenously.

  All tamarins (T-1055, T-1038, T-1051 and T-1049) were monitored weekly as described in Example 1 for changes in serum liver enzymes ALT, GGT and ICD. Table 5 shows the serum liver enzyme data before and after inoculation for these 4 tamarins. FIG. 9 shows ALT and ICD values before and after inoculation with T-1055. Referring to FIG. 9, it can be seen that serum liver enzymes ALT and ICD have risen above CD. The tamarin was killed 12 days after inoculation. Figures 10 and 11 show the serum levels before and after inoculation in ALT and ICD for tamarin T-1051 and T-1038, respectively. Referring to FIG. 10 and FIG. 11, it can be seen that the serum liver enzymes ALT and ICD of both animals increased until 11 days after the inoculation. T-1038 was killed 14 days after inoculation. Table 5 and FIG. 12 show the data obtained for T-1049. As can be seen from Table 5 and FIG. 12, an increase in serum liver enzyme exceeding CO was observed at T-1049 within 11 days after inoculation.

Filtration experiments performed on T-1049 show that HGBV can pass through a 0.10 μm filter, suggesting that HGBV can be viral and have a diameter of less than 0.1 μm. . In addition, infectious titration experiments performed on T-1038 show that T-1053 serum contains at least 4 × 10 ⁵ tamarin infections per ml.

  In order to demonstrate the transmission of a single HGBV pathogen, inoculum consisting of T-1057 serum collected in tamarin T-1044 7 days after H205 inoculation and diluted 1: 500 in normal tamarin serum. 25 ml was inoculated. From 14 to 63 days after inoculation, a gradual increase in the ALT level exceeding the cutoff (ie, an increase in the range of 82 to 106) was observed.

  Tamarin T-1047 and T-1056 were sequentially inoculated with 0.25 ml of T-1044 serum collected 14 days after inoculation and diluted 1: 2 in normal tamarin serum. First, on the 42nd day after inoculation, T-1047 and T-1056 showed an increase in the ALT level exceeding the cut-off, but returned to the normal level on the 64th and 91st day after the inoculation, respectively. Tamarin T-1058 was inoculated with 0.25 ml of T-1057 neat serum collected 22 days after challenge using T-1053 serum. There was no increase in ALT levels during 112 days after inoculation.

Example 3
Representative difference analysis (subtraction hybridization)
A. Production of double-stranded DNA for amplicons Collected 12 days after inoculation of H205 serum (see Examples 1C and 2B) using the procedure described in the above materials and methods herein and referenced in FIG. Tester amplicons were synthesized from total nucleic acids derived from the tamarin T-1053 infectious plasma (see Example 1A). Driver amplicons were synthesized from pre-inoculation plasma of tamarin T-1053 pooled between 17-30 days prior to inoculation. Briefly, both plasmas were filtered through a 0.1 μm filter as described in Example 2B. Subsequently, 50 μl of filtered plasma was extracted using a commercially available kit [United States Biochemical (USB), Cleveland, OH, cat. # 73750] and 10 μg of yeast tRNA as a carrier. This nucleic acid was randomly started and reverse transcribed, and then DNA was synthesized using a commercially available kit. Briefly, an 80 μl reverse transcription reaction was performed using random hexamers using a Perkin Elmer's (Norwalk, CT) RNA PCR kit (cat. # N808-0017) as directed by the manufacturer. Incubated for 10 minutes at 42 ° C and then for 2 hours at 42 ° C. The reaction was then stopped and the cDNA / RNA duplexes were denatured by incubating at 99 ° C. for 2 minutes. The reaction material contains 10 μl of 10 × RP buffer (100 mM NaCl, 420 mM Tris (pH 8.0), 50 mM DTT, 100 μg / ml BSA), 250 pmol of random hexamer and 13 units of Sequenase (total 20 μl). Supplemented with registered version 2.0 polymerase (USB, cat. # 70775). The reaction material was incubated at 20 ° C. for 10 minutes and then at 37 ° C. for 2 hours. After extraction with phenol: chloroform and ethanol precipitation, the double-stranded DNA products of these reactions were combined with 4 units of restriction endonuclease Sau3AI (New England Biolabs [30] in a 30 μl reaction material volume as directed by the manufacturer. NEB], cat. # 169L) for 30 minutes.

B. Amplicon synthetic Sau3AI digested DNA was extracted and precipitated as described above. In a 30 μl reaction material volume, the entire Sau3AI digestion product was buffered in 1 × T4 DNA ligase buffer (NEB) by placing the reaction material in a dry heating block at 50-55 ° C. and incubating for 1 hour at 4 ° C. 465 pmol R Bgl24 (SEQ ID NO: 1) and 465 pmol R Bgl 12 (SEQ ID NO: 2) were annealed. 400 units of T4 DNA ligase (NEB, cat. # 202S) was added to ligate the annealed product. After incubation at 16 ° C. for 14 hours, small scale PCR was performed. 10 μl of ligation reaction material was added to 60 μl H ₂ O, 20 μl 5 × PCR buffer (335 mM Tris (pH 8.8), 80 mM [NH ₄ ] ₂ SO ₄ , 20 mM MgCl ₂ , 0.5 μg / ml. Bovine serum albumin and 50 mM 2-mercaptoethanol), 8 μl of 4 mM dNTP strain, 2 μl (124 pmol) R Bgl 24 (SEQ ID NO: 3) and 3.75 units of Ampli Taq® DNA polymerase (Perkin Elmer, cat. # N808-1012). PCR amplification was performed in a GeneAmp® 9600 thermocycler (Perkin Elmer). Samples were incubated at 72 ° C. for 5 minutes to fill the 5 ′ protruding ends of the ligated adapters. Samples were amplified in 25-30 cycles (1 minute at 95 ° C and 3 minutes at 72 ° C), then extended for 10 minutes at 72 ° C. After confirming that the amplicon was successfully synthesized by agarose gel (ie, a smear of PCR product in the range of about 100 bp to over 1500 bp), large scale amplification of the tester and driver amplicon was performed. Forty 100 μl PCRs and eight 100 μl PCRs were set up as described above for driver and tester synthesis, respectively. 2 μl small scale PCR product per 100 μl reaction material served as a template for large scale amplicon production. Thermocycling as described above was performed to perform an additional 15-20 cycles of amplification. The PCR reaction material for driver and tester DNA was then extracted twice with phenol / chloroform, precipitated with isopropanol, washed with 70% ethanol and digested with Sau3AI to cleave the adapter. The tester amplicon was further purified on a low melting agarose gel. Briefly, 10 μg of tester amplicon DNA was run on a 2% SeaPlaque® gel (FMC Bioproducts, Rockland, ME). A 150-1500 base pair fragment was excised from the gel and the gel slice was thawed at 72 ° C. for 20 minutes with 3 ml H ₂ O, 400 μl 0.5 M MOPS and 400 μl NaCl. DNA was recovered from the melted gel slices using Qiagen-tip 20 (Qiagen, Inc., Chatsworth, Calif.) As directed by the manufacturer.

C. Hybridization and selective amplification of amplicons Approximately 2 μl of purified tester DNA amplicons were ligated to NBgl 24 (SEQ ID NO: 3) and NBgl 12 (SEQ ID NO: 4) as described above. For initial subtractive hybridization, tester amplicons (0.5 μg) and driver amplicons (20 μg) linked to the NBgl primer set were mixed, extracted with phenol / chloroform and precipitated with ethanol. The DNA was resuspended in 4 μl EE × 3 buffer (30 mM EPPS, pH 8.0 [Sigma, St. Louis, MO], 3 mM EDTA at 20 ° C.), and 35 μl mineral oil was overlaid. After heat denaturation (3 min at 99 ° C.), 1 μl of 5M NaCl was added to the denatured DNA, and the DNA was hybridized at 67 ° C. for 20 hours. The aqueous phase was transferred to a new tube and 8 μl of tRNA (5 mg / ml) was added to the sample, followed by 390 μl of TE [10 mM Tris (pH 8.0) and 1 mM EDTA]. 80 μl of hybridized DNA solution was added to 480 μl H ₂ O, 160 μl 5 × PCR buffer (above), 64 μl 4 mM dNTPs and 6 μl AmpliTaq® polymerase (30 units). The solution was incubated at 72 ° C. for 5 minutes to fill the 5 ′ overhang formed by the ligated NBgl 24 primer. N Bgl 24 (SEQ ID NO: 3, 1.24 nmol in ₂₀ μl H ₂ O) was added and the reaction material was divided into aliquots (100 μl / tube) and 10 cycles of amplification were performed as described above. The reaction material was pooled, extracted twice with phenol / chloroform, precipitated with isopropanol, washed with 70% ethanol and resuspended in 40 μl H ₂ O. Single-stranded DNA was removed with Yaenalinuclease (MBN). Briefly, 20 μl of amplified DNA was digested with 20 units of MBN (NEB) in 40 μl reaction material as described by the vendor. 160 μl of 50 mM Tris (pH 8.8) was added to the MBN digest. The enzyme was heat inactivated at 99 ° C. for 5 minutes. 80 μl of MBN digested DNA was PCR amplified for an additional 15 cycles as described above. The reaction material was again pooled, extracted twice with phenol / chloroform, precipitated with isopropanol, washed with 70% ethanol, and resuspended in H ₂ O. The amplified DNA (3-5 μl) was then digested with Sau3AI, extracted and precipitated as described above. The final DNA pellet was resuspended in 100 μl TE.

D. Subsequent hybridization / amplification step 100 ng of DNA from the previous hybridization / selective amplification material was ligated to the J Bgl primer set (SEQ ID NO: 5 and SEQ ID NO: 6) as described above. This DNA (50 ng) was mixed with 20 μg driver amplicon and the hybridization and amplification procedure was repeated as described above, except that the extended temperature during thermocycling was 70 ° C. and the NBgl primer set The final amplification step (after NBN digestion) was 25 cycles instead of 72 ° C for (SEQ ID NO: 3 and SEQ ID NO: 4). Next, 100 ng of the second hybridization-amplification product was ligated to the NBgl primer set (SEQ ID NO: 3 and SEQ ID NO: 4), 200 pg of this material was mixed with 20 μg of driver amplicon, and the third time as described above. Hybridization / amplification was performed, but the final amplification was 25 cycles.

  A 2% agarose gel of the product obtained from representative difference analysis (RDA) performed before HGBV inoculation and on acute T-1053 plasma is shown in FIG. Referring to FIG. 14, lane 1 contains 150 ng HaeIII digested Phi-X174 DNA marker (NEB) with the appropriate size (bp) DNA fragment. The complexity of the driver amplicon (lane 2) and tester amplicon (lane 3) is demonstrated by smears of DNA products found in the samples. This complexity is dramatically reduced when the tester sequence undergoes the first (lane 4), second (lane 5) or third (lane 6) hybridization / selective amplification.

E. Difference Product Cloning The difference product was cloned into the BamHI site of pBluescriptIIKS + (Stratagene, La Jolla, CA, cat. # 212207) as follows. 0.5 μg of pBluescript II was digested with BamHI (10 units, NEB) and 5 ′ dephosphorylated using calf intestinal phosphatase (10 units, NEB) as directed by the vendor. The plasmid was extracted with phenol: chloroform, precipitated with ethanol, washed with 70% ethanol, resuspended in 10 μl H ₂ O (final concentration: approximately 50 ng pBluescript II per μl). The four largest bands from the second hybridization / amplification product were excised from a 2% low melting point agarose gel as described above. Using a Takara DNA ligation reaction kit (Takara Biochemical, Berkeley, Calif.), 4 μl of melted (72 ° C., 5 min) gel slices were ligated to 50 ng BamHI-cut, dephosphorylated pBluescript II in 50 μl reaction material. After 3.5 hours of incubation at 16 ° C., 8 μl of ligation reaction material was used as directed by the vendor . E. coli competent XL-1 Blue cells (Stratagene) were transformed. The transformation mixture was placed on LB plates supplemented with ampicillin (150 μg / ml) and incubated overnight at 37 ° C. The resulting colonies were grown in liquid culture and the miniprep plasmid DNA was analyzed as described in the art to confirm the presence of the cloning product.

In addition to the cloning of the four largest products obtained from the second hybridization / growth stage, the entire population of products obtained from the third hybridization / growth stage was cloned into pBluescript II. 50 ng of pBluescript II vector (synthesized as described above) was ligated to 10 ng of the third hybridization / growth product in 50 μl of reaction material as described above. After 2 hours of incubation at 16 ° C., 10 μl of the ligation product is used . E. coli competent XL-1 Blue cells were transformed. Sixty colonies derived from the obtained transformation products were grown, miniprep DNA was synthesized, and analyzed by methods known in the art as described above. Restriction endonuclease digestion and dot blot hybridization were used to identify unique clones.

Example 4
Immunoisolation of cDNA clones encoding the antigenic region of the HGBV genome Preparation of concentrated virus as a source of cloning material In addition to the method shown in Example 3, the following separation procedure was used to separate the HGBBV genome. Three tamarins (T-1055, T-1038 and T-1049) were inoculated with serum prepared from tamarin T-1053 as described in Example 2. Referring to Table 5, by the 11th day after inoculation, an increase in liver enzyme values was observed in all three tamarins. Tamarin T-1055 was killed 12 days after inoculation, and Tamarin T-1038 and T-1049 were killed 14 days after inoculation. About 3-4 ml of serum derived from each of these three tamarins was pooled to obtain a total volume of about 11.3 ml. Pooled serum was clarified by centrifugation at 10,000 xg for 15 minutes at 15 ° C. This was then continuously passed through 0.8, 0.45, 0.2 and 0.1 μm syringe filters. The filtered material was then passed through a SW41-Ti rotor at 41,000C for 4 minutes at 41,000 rpm with 0.3 ml CsCl cushion [density: 10 mM Tris, 150 mM NaCl, 1 mM EDTA (pH 8.0). 1.6 g / ml] and concentrated by centrifugation. About 0.6 ml of CsCl layer was removed after centrifugation and divided into three 0.2 ml aliquots and stored at -70 ° C.

Tamarin T-1034 was then inoculated with 0.25 ml of a ^10-6 dilution of this pelleted material (prepared in normal tamarin serum). First, an increase in the ALT liver enzyme level was observed in T-1034 at 2 weeks after inoculation, and the value continued to increase for 7 weeks, and finally normalized by 10 weeks after inoculation (FIG. 30, Example). 14). This experiment showed the infectivity of material concentrated from pooled tamarin serum. Since the material was shown to have a relatively high titer, this virus enrichment source was used as a nucleic acid source for construction of a cDNA library, as described below.

B. Construction of cDNA library RNA was extracted from an aliquot (0.2 ml) of concentrated virus (above) using a commercially available RNA extraction kit (Stratagene, La Jolla, Calif.) according to the manufacturer's instructions. Prior to the final precipitation step, the sample was divided into four equal aliquots and then precipitated in the presence of 5 μg / ml yeast tRNA. Only one of these aliquots was used for cDNA synthesis. Others were stored at -80 ° C. As directed by the manufacturer, phosphorylated and blunt terminated double stranded cDNA was synthesized from RNA using a commercially available kit (Stratagene, La Jolla, CA). The double stranded linker / primer was then ligated to the cDNA ends (sense strand, SEQ ID NO: 7) in a 10 μl reaction material volume using the T4 DNA ligase kit (Stratagene, La, Jolla, Calif.) As directed by the manufacturer. An antisense strand, SEQ ID NO: 8). This gave all cDNAs as a mixture with identical 5 ′ and 3 ′ ends containing NotI and EcoRI restriction enzyme recognition sites [G. Reyes and J. Kim, Mol. Cell. Probes 5: 473-481. (1991); A. Akowitz and L. Manuelidis, Gene 81: 295-306 (1989); and G. Inchauspe et al., Viral Hepatitis and Liver Disease , FB Hollinger et al., Eds., Pages 382-387 (1991)]. The linker / primer sense strand oligonucleotide was then used as a primer in the PCR reaction so that the entire cDNA was amplified independent of their sequence. This procedure amplified the trace cDNA present in the total cDNA population to a level where it could be efficiently cloned, thereby forming a cDNA library representing sequences in the starting material.

  PCR was performed on 1 μl aliquots of the ligation material in the presence of sense strand oligonucleotide primers in a PE-9600 thermocycler using the GeneAmp PCR kit (Perkin-Elmer) as directed by the manufacturer (final) Concentration: 1 μM; reaction material volume: 50 μl). 30 cycles of PCR were performed as follows: denaturation at 94 ° C. for 0.5 min, annealing at 55 ° C. for 0.5 min and extension at 72 ° C. for 1.5 min. A 1 μl aliquot of the resulting product was then reamplified as described above. The final PCR reaction product was then extracted once with an equal volume of phenol-chloroform (1: 1, v / v), once with an equal volume of chloroform, then sodium acetate (final concentration, 0.3 M) and 2 .5 volumes of absolute ethanol was added and precipitated on dry ice for 10 minutes. The resulting DNA pellet was resuspended in water and digested with the restriction enzyme EcoRI (New England Biolabs) as directed by the manufacturer. The digested cDNA was then purified from the reaction mixture using DNA binding resin (Prep-a-Gene, BioRad Laboratories) as directed by the manufacturer and eluted in 20 μl of distilled water.

  cDNA (8 μl) was ligated to 3 μg of lambda gt11 vector DNA arm (Stratagen, La Jolla, Calif.) in 30 μl reaction material volume at 4 ° C. for 1-5 days. 11 μl of ligation material was packaged into phage heads using GigaPackIII Gold package extract (Stratagene, supra) as directed by the manufacturer. The resulting library contained a total of about 1.73 million members (PFU) with a recombination frequency of 89.3% and an average insert size of about 350 base pairs.

C. Immunoscreening of recombinant GB cDNA library The antiserum used for the immunoscreening of the cDNA library was taken from tamarin that showed an increase in serum liver enzyme levels after inoculation. Two separate antiserum pools were used for immunoscreening. The first pool contains sera from two animals (T-1048 and T-1051; see Example 1, Table 2, and Example 2, Table 5, respectively), and the second pool is It contained serum from one animal (T-1034; see FIG. 30, Example 14). The specific sera used are shown in Table 6.

  At the time these samples were selected for use in immunoscreening the cDNA library, these samples were tested for their immunoreactivity with 1.4 or 1.7 recombinant CKS proteins (Example 13). It wasn't. Thus, the results presented herein were obtained without regard to any information regarding the presence or absence of HGBV antibodies against these recombinant proteins in the antisera used.

The procedure used for immunosegregation of recombinant phage was based on the method described by Young and Davis (RA Young and RW Davis, PNAS 80: 1194-1198 (1983)) with the following changes: there were. Two immunoscreening experiments were performed, one using antiserum pooled from T-1048 and T-1051 and the other using antiserum from T-1034. In either case, the antibody and E. coli. In order to reduce non-specific interactions with E. coli proteins, the primary antiserum is pre- pre-adsorbed on the E. coli extract. In the first experiment, 1.29 million recombinant phages were immunoscreened using the T-1048 / T-1051 antiserum pool. In the second experiment, 300,000 recombinant phages were immunoscreened with T-1034 antiserum. Recombinant phage libraries were obtained from E. coli. E. coli strand Y1090 _r- was placed on a lawn and grown at 37 ° C for 3.5 hours. The plate was then covered with a nylon filter saturated with IPTG (10 mM) and incubated at 42 ° C. for 3.5 hours. The filters were then blocked for 1 hour at 22 ° C. in Tris-saline buffer containing 1% BSA, 1% gelatin and 3% Tween-20 (“Blocking Buffer”). The filter was then incubated in primary antiserum (1: 100 dilution in blocking buffer) at 4 ° C. for 16 hours. The primary antiserum was then removed and set aside for the next plaque purification, and the filter was washed 4 times in Tris-saline containing 0.1% Tween-20. The filters were then incubated for 60 minutes at 22 ° C. in blocking buffer containing 125-I-labeled (or alkaline-phosphatase-conjugated) goat anti-human IgG (available from Jackson ImmunoResearch, West Grove, Pa.) As above Washed and then exposed to X-ray film (or developed according to established procedures as in J. Sambrook et al., Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989). Five immunopositive phages (4-3BI, 48-1A1, 66-3A1, 70-3A1, 78-1C1) were isolated from this library, and then 3 infected tamarins (T -1048, T-1051, T-1034) were tested for binding specificity to antisera. These recombinants encoded polypeptides that reacted with convalescent sera from each of the three infected tamarins but did not react with pre-inoculated sera (data not shown).

In order to prove the specificity of the immunological reactivity of the polypeptide encoded by the recombinant phage, a primer located at the 5 ′ position (SEQ ID NO: 9) and a primer located at the 3 ′ position relative to the EcoRI cloning site Each cDNA was recovered from the lambda phage genome by PCR using (SEQ ID NO: 10). The PCR product was then digested with EcoRI and E. coli as described in Example 13 . The plasmid was then ligated to the E. coli expression plasmid pJO201. By inserting the cDNA into the EcoRI site of pJO201, the translation reading frame of this cDNA was maintained as it was in the lambda phage clone. The subclones in the pJO201 expression vector were 4-3B1.1, 48-1A1.1, 66-3A1.49, 70-3A1.37 and 78-1C1.17. E. coli prepared from cultures expressing these cDNAs including convalescent sera from Tamarin T-1034, T-1048 and T-1051 (1: 100 dilution) . Immunoblot analysis of E. coli lysates (as in Example 13) showed specific immunological reactivity with the expected size protein for each CKS fusion protein (data not shown). The DNA sequence of each of the cDNAs was determined and these clones were found to have almost 100% sequence identity with the sequence of HGBV-B virus (SEQ ID NO: 11). The sequence of the 4-3B1.1 insert (SEQ ID NO: 12 and 13) was not determined in its entirety, but the sequencing portion was the sequence portion in HGBV-B (SEQ ID NO: 11) from base pair 6834-7458. With 99.5% sequence identity. This region of the HGBV-B (SEQ ID NO: 11) sequence showing identity with the sequence obtained from clone 4-3B1.1 has been translated into the +1 reading frame and is shown in the sequence listing as SEQ ID NO: 14. The sequence of the 48-1A1.1 insert (SEQ ID NO: 15) exhibits 100% sequence identity with a portion of the sequence of HGBV-B (SEQ ID NO: 11, see Example 9) from base pair 4523-4752. Show. The DNA sequence corresponding to SEQ ID NO: 15 is translated into a +1 reading frame and is shown in the sequence list as SEQ ID NO: 16. The sequence of the 66-3A1.49 insert (SEQ ID NO: 17) shows substantially 100% sequence identity with the sequence of clone 48-1A1.1, and therefore does not show protein translation in the sequence list. The sequence of the 70-3A1.37 insert (SEQ ID NO: 18) shows 100% sequence identity with the portion of the sequence of HGBV-B (SEQ ID NO: 11) from base pair 6450-6732, provided that HGBV-B Three base pairs corresponding to bases 6630-6632 of the sequence (SEQ ID NO: 11) are applied. The DNA sequence corresponding to SEQ ID NO: 18 is translated into a +2 reading frame and is shown in the sequence listing as SEQ ID NO: 19. The sequence of the 78-1C1.17 insert (SEQ ID NO: 20) shows 100% sequence identity with the sequence of clone 70-3A1.37, and therefore no protein translation is shown in the sequence listing. These data indicate that a cDNA clone isolated from a lambda gt11 cDNA library is derived from the genome of an HGBV pathogen and the library encodes a polypeptide that is immunologically specifically recognized by sera from GB infected tamarins. It is shown that. Clones 48-1A1.1 (“Clone 48”) 4-3B1.1, 66-3A1.49, 70-3A1.37 and 78-1C1.17 have been deposited with the American Type Culture Collection.

(Example 5)
DNA sequence analysis of HGBV clone The unique clone obtained in Example 3 was sequenced using the dideoxynucleotide chain termination method (Sanger et al., Supra) in kit form [Sequenase® version 2.0, USB]. These sequences do not overlap, clone 4 (SEQ ID NO: 21), clone 2 (SEQ ID NO: 22), clone 10 (SEQ ID NO: 23), clone 11 (SEQ ID NO: 24), clone 13 (SEQ ID NO: 25) , Clone 16 (SEQ ID NO: 26), clone 18 (SEQ ID NO: 27), clone 23 (SEQ ID NO: 28), clone 50 (SEQ ID NO: 29) and clone 119 (SEQ ID NO: 30). Clones 4, 2, 10, 11, 13, 16, 18, 23, 50 and 119 T.A. C. C. Has been deposited. Clone 2 is A. T.A. C. C. Accession No. 69556, clone 4 is A. T.A. C. C. Accession No. 69557, clone 10 is A. T.A. C. C. Accession No. 69558, clone 16 is A. T.A. C. C. Accession No. 69559, clone 18 is A. T.A. C. C. Accession No. 69560, clone 23 is A.E. T.A. C. C. Accession No. 69561, clone 50 is A. T.A. C. C. Accession No. 69562, clone 11 is A. T.A. C. C. Accession No. 69613, clone 13 is A. T.A. C. C. Accession No. 69611, and clone 119 are A.I. T.A. C. C. Accession number 69612 was given.

Sequences were searched in the GenBank database using the BLASTN algorithm (Altschul et al., J. Mol. Biol . 215: 403-410 [1990]). None of these sequences were found in GenBank, indicating that these sequences have not yet been characterized in the literature. The DNA sequence is translated into six possible open reading frames and the sequence list (SEQ ID NO: 21 is SEQ ID NO: 31-36, SEQ ID NO: 22 is SEQ ID NO: 37-42, SEQ ID NO: 23 is SEQ ID NO: 43-48, SEQ ID NO: 26 is shown in SEQ ID NO: 49-54, SEQ ID NO: 27 is translated into SEQ ID NO: 55-60, SEQ ID NO: 28 is translated into SEQ ID NO: 61-66, and SEQ ID NO: 29 is translated into SEQ ID NO: 67-72) Yes. SEQ ID NO: 24 is contained in SEQ ID NO: 73 (described in Example 9) and translated into SEQ ID NOs: 74-79. SEQ ID NOs: 25-30 are included in SEQ ID NO: 80 (described in Example 9) and are translated into SEQ ID NOs: 81-86. Using the BLASTX algorithm (Gish et al., Nature Genetics 3: 266-272 [1993]), translated sequences were used to search the SWISS-PROT database. Again, these sequences were not found in SWISS-PROT, indicating that these sequences have not yet been characterized in the literature.

Searching for homologues using the BLASTN, BLASTX, and FASTdb algorithms reveals some, if any, sequence similarity to the hepatitis C virus (Table 7 below). In particular, clones 4 (SEQ ID NO: 35), 10 (SEQ ID NO: 44), 11 (residues 1 to 166 of GB-4, frame 3 [SEQ ID NO: 76]), 16 (SEQ ID NO: 50), 23 (SEQ ID NO: 65) ), 50 (SEQ ID NOs: 70 and 72) and 119 (residues 912 to 988 of GB-A, frame 3 [SEQ ID NO: 83]) are translated into 24.1-45 for various HCV isolates at the amino acid level. There is a homology in the range of 1%. Of particular importance is that the translation of clone 10 (SEQ ID NO: 44) showed slight homology to the putative RNA-dependent RNA polymerase of HCV. Comparison of the conserved amino acids present in the putative RNA-dependent RNA polymerase of other positive strand viruses (Jiang et al., PNAS 90: 10539-10543 [1993]) with the putative amino acid translator of clone 10 (SEQ ID NO: 44) It was revealed that conserved amino acid residues of other RNA-dependent RNA polymerases are also conserved in clone 10 (SEQ ID NO: 44). This includes the canonical GDD (Gly-Asp-Asp) signature sequence of RNA-dependent RNA polymerase. Therefore, clone 10 (SEQ ID NO: 44) appears to encode a viral RNA-dependent RNA polymerase. Surprisingly, only clone 10 (SEQ ID NO: 44) showed sequence homology with HCV at the nucleotide level when using the BLASTN algorithm. Clones 4 (SEQ ID NO: 21), 16 (SEQ ID NO: 26), 23 (SEQ ID NO: 28) and 50 (SEQ ID NO: 29) and 119 (SEQ ID NO: 30), which have low HCV homology at the amino acid level, were found in the GenBank search. , Not detected in BLASTN. In addition, clones 2 (SEQ ID NOs: 37-42), 13 (SEQ ID NOs: 25 and 37-42) and 18 (SEQ ID NOs: 27 and 55-60) were tested against HCV when searched with the above GenBank or SWISS-PROTO. Significant nucleotide or amino acid homology was not shown as follows.

(Example 6)
Exogenous HGBV clones of HGBV clones include normal or HGBV-infected tamarin liver DNA, normal human lymphocyte DNA, yeast DNA or E. coli. It was not detected in E. coli DNA. This was shown for HGBV clones 2 (SEQ ID NO: 22) and 16 (SEQ ID NO: 26) by the Southern method. In addition, all HGBV clones were analyzed by genomic PCR to detect tamarin, human, yeast and E. coli. The exogenous origin of the HGBV sequence for the E. coli genome was confirmed. These data are consistent with the viral nature of the HGBV sequence described in Example 5.

A. Southern blot analysis HGBV-infected tamarin and normal tamarin liver homogenates were pelleted at low speed to obtain tamarin liver nuclei (described below). DNA was extracted from the nuclei using a commercially available kit (USB cat. # 73750) as directed by the vendor. Tamarin DNA was treated with RNase during the extraction process. Human placental DNA (Clontech, Palo Alto, CA), yeast DNA ( Saccharomyces cerevisiae , Clontech) and E. coli . E. coli DNA (Sigma) was obtained from a commercial source.

  Each DNA sample was digested with BamHI (NEB) according to the manufacturer's instructions. Digested DNA (10 μg) and RNA product (0.5 μg each from Example 3B) were electrophoresed on a 1% agarose gel and Hybond-N + nylon as described by Sambrook et al. (Pp. 9.34ff). Membrane (Amersham, Arlington Heights, IL) was blotted with a capillary. The DNA was treated with alkali as directed by the membrane vendor and fixed to the membrane. The membrane was prehybridized in Rapid Hyb solution (Amersham) at 65 ° C. for 30 minutes.

A radiolabeled probe of HGBV sequence was prepared by PCR. 1 × PCR buffer II (Perkin Elmer), 2 mM MgCl ₂ , 20 μM dNTP, 1 μM clone specific sense and antisense primers respectively (SEQ ID NO: 87 and 88 for clone 2; SEQ ID NO: 87 for clone 4) 89 and 90; SEQ ID NO: 91 and 92 for clone 10; SEQ ID NO: 93 and 94 for clone 16; SEQ ID NO: 95 and 96 for clone 18; SEQ ID NO: 97 and 98 for clone 23; For SEQ ID NO: 99 and 100), 1 ng of HGBV clone plasmid (described in Example 3 [E]), 60 μCiα- ³² P-dATP (3000 Ci / mmol) and 1.25 units of AmpliTaq® polymerase ( 50 μl PC using Perkin Elmer) R was formed. The reaction material was incubated at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds for a total of 30 cycles of amplification followed by a final extension at 72 ° C. for 3 minutes. Unincorporated label was removed with a Quick-Spin® G-50 spin column (Boehringer Mannheim, Indianapolis, IN) as directed by the vendor. The probe was denatured (99 ° C., 2 minutes) prior to addition to the prehybridized membrane.

To the prehybridized membrane (2 × 10 ⁶ dpm / ml), a radiolabeled probe was added and the filter was hybridized for 2.5 hours at 65 ° C. as directed by the Rapid Hyb® distributor. . The hybridized membrane was washed with mild stringency (1 × SSC, 0.1% SDS at 65 ° C.) and then exposed to autoradiographic film at −80 ° C. for 72 hours using an intensifying plate. These conditions were devised to detect single copy genes using similar radiolabeled probes.

The results show that clone 2 (SEQ ID NO: 22) and clone 16 (SEQ ID NO: 26) sequences are normal or HGBV-infected tamarin liver-derived DNA (FIGS. 15 and 16, lanes 1B and 3B, respectively), human DNA (FIG. 15). And FIG. 16, lane 1A), yeast DNA (FIGS. 15 and 16, lane 2A) or E. coli. E. coli DNA (FIGS. 15 and 16, lane 3A) was not hybridized. Furthermore, no hybridization was detected with driver amplicon DNA (derived from pre-HGBV inoculated tamarin plasma as described in FIGS. 15 and 16, lane 4A, Example 2.B.). In contrast, tester amplicons (derived from infectious HGBV tamarin plasma using the total nucleic acid extraction and reverse transcription steps described in FIGS. 15 and 16, lane 6A, Example 2.B) and 3 subtractions In the case of the product of / selective amplification (FIGS. 15 and 16, lanes 7A, 8A and 4B for products from the first, second and third subtraction / selective amplification, respectively), a strong hybridization signal is observed It was. These data show that HGBV clones 2 (SEQ ID NO: 22) and 16 (SEQ ID NO: 26) were detected in the nucleic acid sequence amplified from the infectious source, and HGBV clones 2 (SEQ ID NO: 22) and 16 (SEQ ID NO: 26) , Tamarin, human, yeast or E. coli. It is not derived from the E. coli genomic DNA sequence.

B. PCR analysis of the genome to further demonstrate the exogenous nature of the HGBV sequences and support their viral origins, tamarin, human, yeast and E. coli. PCR was performed on genomic DNA derived from E. coli . Normal tamarin kidney and liver tissue-derived DNA was synthesized as described by J. Sambrook et al. Yeast, rhesus monkey kidney and human placental DNA were obtained from Clonotech. E. E. coli DNA was obtained from Sigma.

PCR was performed mainly using GeneAmp® reagent from Perkin-Elmer-Cetus as directed by the vendor. 300 ng of genomic DNA was used for each 100 μl of reaction material. HGBV cloned sequence (SEQ ID NO: 87 and 88 for clone 2; SEQ ID NO: 89 and 90 for clone 4; SEQ ID NO: 91 and 92 for clone 10; SEQ ID NO: 93 and 94 for clone 16; clone PCR primers from SEQ ID NO: 95 and 96 for 18; SEQ ID NO: 97 and 98 for clone 23; SEQ ID NO: 99 and 100 for clone 50) were used at a final concentration of 0.5 μM. PCR was performed for 35 cycles (94 ° C. for 1 minute; 55 ° C. for 1 minute; 72 ° C. for 1 minute), followed by a 7 minute extension cycle at 72 ° C. PCR products are separated by agarose gel electrophoresis, nucleic acid is directly stained with ethidium bromide and then irradiated with ultraviolet light and / or transferred to a nitrocellulose filter by the Southern method and then hybridized to a radiolabeled probe. Visualized. Probes were formed as described in Example 6A. Filters were prehybridized for 3-5 hours in Fast-Pair Hybridization Solution from Digene (Belstville, MD), then 100-200 cpm / cm ² at 42 ° C. for 15-25 hours in Fast-Pair Hybridization Solution. Hybridized. The filter is washed as described in GG Schlauder et al., J. Virol. Methods 37: 189-200 (1992), and Kodak X-Omat-AR film is used at −70 ° C. for 15 to 72 hours using an intensifying plate. Exposed to.

FIG. 17 shows a 1.5% agarose gel stained with ethidium bromide. FIG. 18 shows an autoradiogram by Southern blotting from the same gel after hybridizing to a radiolabeled probe from clone 16 (SEQ ID NO: 26). Although it is possible to detect clone 16 (SEQ ID NO: 26) sequences spiked in normal tamarin liver and kidney DNA with 0.05 genomic equivalents (lanes 17 and 18), clone 16 (SEQ ID NO: 26 ) sequence, consistent with its exogenous by genomic PCR analysis, tamarin (FIGS. 17 and 18, lanes 9 and 10), rhesus (lane 11) or human genomic DNA (lane 12) even during, yeast or E . Also not detected in E. coli DNA (data not shown). Furthermore, primers derived from human dopamine D1 receptor gene, 1000-1019 base pairs (sense primer) and 1533-1552 base pairs (antisense primer) (GenBank accession number: X55760, RK Sunahara et al., Nature 347: 80-83 [1990] ]) Successfully amplified dopamine D1 receptor DNA from primate genomic DNA (Figure 17, lanes 2, 3, 4, and 5, corresponding to tamarin kidney, tamarin liver, rhesus monkey and human DNA), It demonstrates the utility of this method for detecting low copy number (ie, single copy) sequences. Lanes 1 and 8 are H ₂ O controls for the dopamine D1 receptor and 16 clone primers (SEQ ID NOs: 93 and 94), respectively. Lane 6 contains 100 fg of clone 16 (SEQ ID NO: 26) plasmid DNA amplified with dopamine receptor primers. Lanes 14, 15, 16, and 20 contain 1, 3, 10, and 100 fg of clone 16 (SEQ ID NO: 26) plasmid DNA, respectively. Lanes 7 and 19 are markers. Similar results were obtained using PCR primers specific for clones 2, 4, 10, 18, 23 and 50 above (data not shown). Clone 2 (SEQ ID NO: 22), 4 (SEQ ID NO: 21), 10 (SEQ ID NO: 23), 18 (SEQ ID NO: 27), 23 (SEQ ID NO: 28), and 50 (SEQ ID NO: 29) have yielded results at this point. Absent. However, the clone 4 (SEQ ID NO: 21), 10 (SEQ ID NO: 23), 18 (SEQ ID NO: 27) and 50 (SEQ ID NO: 29) sequences are tamarin, human, yeast and E. coli. E. coli DNA (rhesus monkeys not tested) detected that these sequences are exogenous to the genomic DNA source tested and support the viral origin of these sequences. Show.

(Example 7)
Presence of HGBV sequences in tamarin serum Presence of inoculation and presence of HGBV clone sequences in acute T-1053 plasma were examined by PCR. Since the HGBV genome can be DNA or RNA, PCR and RT-PCR were performed. In particular, total nucleic acid was extracted from plasma as described in Example 3 (A). PCR was performed on an equivalent amount of 5 μl of plasma nucleic acid as described in Example 6 (B), mainly using the GeneAmp® RNA PCR kit from Perkin-Elmer-Cetus according to the manufacturer's instructions, Primers at 1 μM concentration during PCR (SEQ ID NO: 87 and 88 for clone 2; SEQ ID NO: 89 and 90 for clone 4; SEQ ID NO: 91 and 92 for clone 10; SEQ ID NO: 93 and 94 for clone 16 RT-PCR was carried out using clone No. 95 and 96; clone 23 SEQ ID No. 97 and 98; clone 50 SEQ ID No. 99 and 100). CDNA synthesis was initiated using random hexamers.

  By staining and hybridization with ethidium bromide of the PCR product, HGBV clone sequences 2 (SEQ ID NO: 22), 4 (SEQ ID NO: 2), 10 (SEQ ID NO: 23), 16 (SEQ ID NO: 23) were obtained in the acute phase T-1053 plasma. 26), 18 (SEQ ID NO: 27), 23 (SEQ ID NO: 28) and 50 (SEQ ID NO: 29) were present but not present in the pre-inoculated T-1053 plasma (data not shown). In addition, the HGBV clone 2 (SEQ ID NO: 22), 4 (SEQ ID NO: 21), 10 (SEQ ID NO: 23), 18 (SEQ ID NO: 27), 23 (SEQ ID NO: 28) and 50 (SEQ ID NO: 29) sequences are Tamarin T Could be detected in H205, an HGBV inoculum injected into -1053 (see Example IB). These results are summarized in Table 8. Note that the HGBV clone sequence was detected only by RT-PCR in acute phase plasma. The fact that HGBV clone sequences were detected in acute plasma by PCR for the first time after the reverse transcription step to convert RNA to cDNA is due to the low homology between some of these clones and HCV isolates, and HGBV The presence of a sequence encoding a conserved amino acid found in the RNA-dependent RNA polymerase of clone 10 (SEQ ID NO: 23; Example 5) suggests that HGBV is an RNA virus.

RT-PCR analysis of a tamarin plasma panel with HGBV clone 16 sequence (SEQ ID NO: 26) was performed to confirm the presence of HGBV clone 16 (SEQ ID NO: 26) in other individuals experimentally infected with HGBV. Tamarin-derived plasma obtained before and after experimental infection with H205 inoculum as described previously (GG Schlauder et al., J. Virological Methods 37: 189-200 [1992]) Nucleic acids were separated from 25 μl. Nucleic acid precipitated with ethanol was resuspended in 3 μl DEPC-treated H ₂ O. CDNA synthesis and PCR were performed using a GeneAmpRNA PCR kit from Perkin-Elmer-Cetus, mainly according to the manufacturer's instructions. CDNA synthesis was initiated using random hexamers. The resulting cDNA was subjected to PCR at a final concentration of 0.5 μM using clone 16 primer (SEQ ID NOs: 93 and 94). PCR was performed for 35 cycles (94 ° C. for 1 minute; 55 ° C. for 1 minute; 72 ° C. for 1 minute) followed by an extended cycle at 72 ° C. for 7 minutes. PCR products are separated by agarose gel electrophoresis and nucleic acids are stained directly with ethidium bromide and then irradiated with UV light and / or after transfer to a nitrocellulose filter by Southern blotting as described in Example 6B. Hybridized to a radiolabeled probe and visualized.

FIG. 19 shows a 1.5% agarose gel stained with ethidium bromide. FIG. 20 shows an autoradiogram by Southern blotting from the same gel after hybridizing to a radiolabeled probe from clone 16 (SEQ ID NO: 26). H ₂ O and human normal serum are shown in lanes 1 and 2. Lanes 3, 19 and 20 are markers. Lanes 4, 8, 12, and 16 are derived from uninfected tamarin serum, and lanes 6, 10, 14, and 18 are derived from infected tamarin serum. These results indicate that HGBV clone 16 sequence (SEQ ID NO: 26) was detected in other individuals infected with HGBV in addition to tamarin T-1053, but not in uninfected individuals. ing. Acute serum from 5 H205 infected animals was tested. Clone 16 sequence (SEQ ID NO: 26) was found in 3 of these animals [lane 10, T-1049, 14 days after inoculation (dpi); lane 14, T-1051, 28 dpi; lane 18, T-1055. , 16 dpi]. Clone 16 sequence (SEQ ID NO: 26) consists of 5 animals (lane 4, T-1048; lane 8, T-1049; lane 12, T-1051; lane 16, T-1055; T-1057 not shown) None of the pre-inoculation sera were detected. These results suggest that clone 16 sequence (SEQ ID NO: 26) may be derived from an infectious HGBV pathogen. The absence of the clone 16 sequence (SEQ ID NO: 26) in two of the five acute phase plasmas (lane 6, T-1048, 28 dpi; T-1057, 14 dpi, not shown) indicates that HGBV in tamarins. This can be explained by the relatively low sensitivity of clone 16RT-PCR combined with the acute degradation nature of the infection (it is assumed that about ≧ 1000 copies of clone 16 sequence (SEQ ID NO: 26) can be detected). Thus, acute phase plasma from two negative animals may contain HGBV titers below the detection level of the RT-PCR assay used. The observation by RT-PCR that these two animals were positive for clone 4 (SEQ ID NO: 21) (Example 14) is the presence of the RNA sequence of one virus (including clone 4) It may reflect the absence of detectable RNA sequences from two viruses (including clone 16).

(Example 8)
Northern blot analysis of HGBV sequences in the liver of infected tamarins
The fact that HGBV clone sequences could be detected by RT-PCR and H205 inoculation in acute tamarin plasma increased the possibility that these sequences originated from the HGBV genome. Yet another RT-PCR test demonstrated the presence of HGBV sequences in liver RNA extracted from H205 infected tamarin T-1053 (data not shown). Therefore, in order to know the size of the HGBV genome, Northern blot analysis with liver RNA of H205 infected and uninfected tamarins was performed. Total RNA was extracted from 1.25 g liver of H205 infected tamarin T-1053 and 1.0 g liver of control uninfected tamarin T-1040 using an RNA isolation kit (Stratagene, La Jolla, CA) according to its recommended usage. . Total RNA (30 μg) was electrophoresed through a 1% agarose gel containing 0.6 M formaldehyde (RM Fourney et al. Focus 10: 5-7, [1988]) and further 20 × SCC (pH 7.0) as described above. Transferred to Hybond-N nylon membrane (Amersham) by capillary action. J. Sambrook et al., Molecular Cloning-A Laboratory Manual , 2nd edition (1989). The RNA was UV-crosslinked to the nylon membrane, and the nylon membrane was baked at 80 ° C. for 60 minutes in a vacuum oven. The blot was prehybridized in 25 ml of a solution containing PIPES 0.05M, sodium phosphate 50 mM, NaCl 100 mM, EDTA 1 mM and SDS 5% at 60 ° C. for 2 hours. GD Virca et al., Biotechniques 8: 370-371 (1990). Prior to hybridization with the radiolabeled DNA probe, the solution was removed and 10 ml of fresh solution was added. The probes used for hybridization were clone 4 (SEQ ID NO: 21; 221 bp), clone 50 (SEQ ID NO: 29; 337 bp) and 2000 bp cDNA corresponding to human β-actin. P. Gunning, et al . Mol. And Cell. Biol . 3: 787-795 (1983). A random primer labeling kit (Stratagene, La Jolla, Calif.) Was used according to the recommended usage, and the probe (50 ng) was radiolabeled in the presence of [α- ³² P] dATP. The specific activity of each probe was about 109 cpm / μg. Blots were hybridized at 60 ° C. for 16 hours, washed as described above (GD Virca et al., Supra) and exposed to Kodak X-Omat-AR film at −80 ° C. A photograph of the obtained autoradiograph is shown in FIG. 21A. Lanes 1, 3 and 5 contain liver RNA from T-1040, and lanes 2, 4 and 6 contain liver RNA from T-1053. Lanes 1 and 2 hybridize with the human β-actin cDNA probe, lanes 3 and 4 hybridize with the clone 4 probe (SEQ ID NO: 21), and lanes 5 and 6 have the clone 50 probe (SEQ ID NO: 29 ). The exposure time was 5 hours at −80 ° C. for Lane 1 and Lane 2, and 56 hours at −80 ° C. from Lane 3 to Lane 6. The position of 28S ribosomal RNA and the position of 18S ribosomal RNA are indicated by arrows. The relative sizes of these ribosomal RNAs are 6333 and 2366 nucleotides, respectively. J. Sambrook et al.

  The clone 4 probe (SEQ ID NO: 21) and clone 50 probe (SEQ ID NO: 29) hybridized to RNA species contained in RNA extracted from the liver of infected tamarin (T-1053) (Figure 21A, lanes 4 and lanes). 6). The size of this hybridizable RNA species was calculated to be about 8300 nucleotides based on relative mobility compared to 28S and 18S ribosomal RNA. Both probes are considered to have hybridized with the same RNA species. Both probes did not hybridize with RNA extracted from the liver of uninfected tamarin (T-1040) (FIG. 21A, lane 3 and lane 5). These results indicate that the clone 4 (SEQ ID NO: 21) and clone 50 (SEQ ID NO: 29) sequences are present in the same 8.3 Kb transcript.

To know the strandedness of the HGBV RNA genome, use a Strand-specific radiolabeled DNA probe using the GeneAmp® PCR kit from Perkin-Elmer according to recommended usage, Prepared by asymmetric PCR. Purify clone 50 DNA (SEQ ID NO: 29) in a separate reaction containing either a strand-specific primer that is negative for clone 50 (SEQ ID NO: 99) or a strand-specific primer that is positive for clone 50 (SEQ ID NO: 100). And used as a template to a final concentration of 1 μM. This reaction mixture contained {α ³² P-dATP] (Amersham; 3000 Ci / mmol) instead of dATP normally contained in such reaction mixture. After the 30-line amplification of the template, non-incorporated [α ³² P-dATP] was removed using a Quick-Spin® Sephadex G50 spin column (Boehringer-Mannheim, Indianapolis, IN) according to recommended usage. Hybridization of a radiolabeled probe to a DNA dot blot containing a 10-fold diluted solution of double-stranded clone 50 DNA (SEQ ID NO: 29) confirmed that both probes had approximately the same sensitivity (data Is not shown). A radiolabeled probe was hybridized with an RNA blot containing 30 μg total of liver RNA extracted from uninfected tamarin T-1040 and liver RNA extracted from infected tamarin T-1053 as described above. A photograph of the obtained autoradiograph is shown in FIG. 21B. Lanes 1 and 3 contain liver RNA from T-1040, and lanes 2 and 4 contain liver RNA from T-1053. Lanes 1 and 2 hybridize with a strand probe positive for clone 50 (ie, the positive strand was radiolabeled and the negative strand was detected: SEQ ID NO: 100), and lanes 3 and 4 were negative for clone 50 Hybridization with a simple strand probe (ie, the negative strand was radiolabeled and the positive strand was detected: SEQ ID NO: 99). The blot was exposed at -80 ° C for 18 hours. The position of 28S ribosomal RNA and the position of 18S ribosomal RNA are indicated by arrows.

  As shown in FIG. 21B, positive and negative strand probes for clone 50 (SEQ ID NOs: 100 and 99, respectively) hybridized to the approximately 8.3 kilobase RNA species extracted from the liver of infected tamarin T-1053 (FIG. 21B). 21B, lanes 2 and 4) did not hybridize with RNA extracted from the liver of uninfected tamarin T-1040 (FIG. 21B, lanes 1 and 3). This was consistent with the northern blot results obtained using clone 4 (SEQ ID NO: 21) and clone 50 (SEQ ID NO: 29) double-stranded probes shown above. A stronger signal was obtained using a strand probe negative for clone 50 (SEQ ID NO: 99) (Figure 21B, lane 4 vs. lane 2), indicating that the dominant RNA species present in the liver of infected tamarins are positive ( That is, it was suggested to be a coding) chain.

Example 9
Extension of HGBV clone sequence Generation of HGBV sequences Each of the clones obtained as described in Example 3 and sequenced as in Example 5 was considered to have been obtained from a separate region of the HGBV genome. Therefore, several PCR walking experiments were performed to obtain sequences from other regions of the HGBV genome that remained between the already identified clones and to confirm the sequence of the RNA clone. .

Total nucleic acids were extracted from 50 μl aliquots of infected T-1053 plasma as described in Example 3 (A). Briefly, the precipitated nucleic acid was resuspended in 10 μl of DEPC-treated H ₂ O. Standard RT-PCR was performed with the GeneAmp® RNA PCR kit (Perkin-Elmer) according to recommended usage. Briefly, PCR was performed on the cDNA product of a random sensitized reverse transcription reaction of extracted nucleic acid with 2 mM MgCl ₂ and 1 μM primer. The reaction was subjected to 35 cycles of denaturation-annealing-extension (94 ° C., 30 seconds; 55 ° C., 30 seconds; 72 ° C., 2 minutes) followed by extension at 72 ° C. for 3 minutes. The reaction was kept at 4 ° C. prior to agarose gel analysis. These products were cloned into the pT7 Blue T-vector plasmid (Novagen) by conventional techniques. Table 9 shows the results obtained when the above reaction was performed.

A modified version of the PCR transfer procedure described by Sorensen et al. (J. Virol, 67: 7118-7124 [1993]) was used to obtain additional HGBV sequences. Briefly, total nucleic acid was extracted from infected tamarin T-1053 plasma and reverse transcribed. The resulting cDNA was amplified in a 50 μl PCR reaction (PCR 1) as in the Sorensen et al. (Supra) procedure except that MgCl _{2 2} mM was used. These reactions were subjected to 35 denaturation-annealing-extensions (94 ° C., 30 seconds; 55 ° C., 30 seconds; 72 ° C., 2 minutes), followed by extension at 72 ° C. for 3 minutes. The biotinylated product was isolated using streptavidin-coated paramagnetic beads (Promega) as described by Sorensen et al. (Supra). As described by Sorensen et al., A “nested” PCR (PCR 2) was performed on a total of 20-35 denaturation-annealing-extension and purified products with streptavidin. The resulting products and the PCR primers used to generate them are shown in Table 10.

  These products were isolated from low melting point agarose gels by conventional techniques and cloned into the pT7 Blue T-vector plasmid (Novagen).

As described above, 'in order to obtain a terminal sequences, RNA ligase-mediated 5'5 from a viral genome RNA RACE (rapid amplification of cDNA ends: R apid A mplification of c DNA E nds) was used. Briefly, the 5 ′ AmpliFINDER® RACE kit (Clontech, Palo Alto, Calif.) Was used according to the recommended usage. The source of viral RNA was acute T-1053 plasma extracted as described above. Table 11 shows the oligonucleotides unique to each virus species used for reverse transcription (RT) and the first PCR amplification (PCR 1) and the second PCR amplification (PCR 2). The binding anchor primer and its complementary PCR primer were provided by the manufacturer. PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer) according to recommended usage.

  The product produced by RNA ligase-mediated 5 'RACE was isolated from a low melting point agarose gel as described above and cloned into the pT7 Blue T-vector plasmid (Novagen).

  To obtain different sequences at the 5 'and 3' ends of the HGBV-B sequence (see "Evidence for the existence of two HCV-like flaviviruses in HGBV" below) RNA cyclization experiments (RNA circularization experiment) was performed. (This method is based on the method described in CW Mandl et al. (1991) Biotechniques, Vol 10 (4): 485-486.) (Except for replacing tRNA with 1 μg glycogen during precipitation. Total nucleic acid was purified from 50 μl of T-1057 plasma (14 days after inoculation). Re-dissolve the nucleic acid pellet in 16.3 μl of DEPC-treated water and add 2 μTAP buffer (1 × = 50 mM NaOAc, pH 5.0, 1 mM EDTA, 10 mM 2-mercaptoethanol, 2 mM MATP) 25 μl and tobacco 8.7 μl of acid pyrophosphatase (20 units; Sigma) was added. The mixture was incubated at 37 ° C. for 60 minutes. Samples were extracted with phenol (saturated with water) and further with chloroform and precipitated with NaOAc / EtOH in the presence of glycogen (1 μg). The pellet was dissolved in 83 μl of DEPC water, and 10 μl of 10 × RNA ligase buffer (New England Biolabs, NEB), 2 μl of RNase inhibitor (Perkin Elmer) and 5 μl of T4 RNA ligase (NEB) were added. The mixture was incubated at 4 ° C. for 16 hours. Samples were extracted using phenol and further chloroform as described above and precipitated using NaOAc / EtOH.

  1/10 of the bound RNA was used in a reverse transcriptase (RT) reaction using Superscript RT (GIBCO / BRL) according to its recommended usage and SEQ ID NO: 146 as a primer. Half of the RT reaction mixture was used as described above for PCR1 in the presence of a biotinylated oligonucleotide primer (SEQ ID NO: 146) and a second oligonucleotide primer (SEQ ID NO: 133). The PCR1 product was purified from the reaction mixture using streptavidin-coated magnetic beads as described in Sorensen et al. (Supra). The purified PCR1 product (2 μl out of 30 μl) was used as a template for PCR2. PCR2 using oligonucleotide primers (SEQ ID NO: 147,154) gave a 1200 bp product that was cloned into the pT7 Blue T-vector plasmid and sequenced as follows. By sequence analysis of two independent clones from this experiment, known (although one clone had a sequence of 18 T residues and the other clone had a sequence of 27 T residues) A region overlapping the sequence showed 100% identity, and revealed a new sequence with 270 additional bases.

  The cyclization experiment gave sequences from both the 5 'and 3' ends of the HGBV-B viral genome that were not obtained with the standard 3'-RACE or 5'-RACE procedures. However, it was difficult to find the correct 5'-3 'junction even after another PCR experiment performed using primers designed from the newly obtained sequence. Therefore, in order to better qualify the 5 'end of the HGBV-B RNA genome, primer extension experiments were performed using RNA isolated from T-1053 liver.

As described in Example 7, total cellular RNA was isolated from the liver of T-1053 and the liver of a control (ie, non-infected) animal (T-1040). An antisense oligonucleotide (SEQ ID NO: 155) was end-labeled with γ- ³² P-ATP to a specific activity of about 9.39 × 10 ⁷ CPM / μg using T4 polynucleotide kinase (NEB) (Sambrook et al. ). Primers were annealed to 30 μl of T-1053 liver RNA and T-1040 liver RNA in separate reactions and extended using MMLV reverse transcriptase (Perkin-Elmer) as described above (Sambrook et al.). The product was analyzed on a 6% sequencing gel. A sequence ladder generated from one of the HGBV-B circularized clones using the same primer as that used for primer extension served as a size standard.

  A 176 bp primer extension product was obtained from T-1053. Primer extension using liver RNA extracted from an uninfected animal (T-1040) did not yield such a product, thus indicating a product obtained from the HGBV-B genome. The length of these products obtained indicated that the 5 'end of the genome, such as that present in the liver of infected animals, was located 442 nucleotides upstream from the AUG start codon.

  To confirm the 3 ′ position of the sequence obtained in the cyclization experiment, RT-PCR was performed using primers corresponding to the predicted 3 ′ end (see reaction 1.25 in Table 2). . RT-PCR of infected T-1053 plasma (as described above) using SEQ ID NO: 156 and SEQ ID NO: 157 yielded a 450 bp product. In contrast, RT-PCR using the complement of SEQ ID NO: 157 and SEQ ID NO: 147 did not yield a detectable PCR product (data not shown). These data suggest that the 3 'end of the genome is located 50 nucleotides downstream of the poly T system.

  Cloned products from Table 9, Table 10, Table 11 and the above RNA cyclization experiments were sequenced as previously described in Example 5. Interestingly, it was discovered that the cloned products of reactions 1.4, 1.6, 1.9, 1.10, 1.11 contain only one of the two primer sequences at their ends, which indicates that these products are false It was suggested that it occurred as a result of a random priming event. The sequences detected in products 1.4, 1.6, 1.9, 1.10, 1.11 were associated with clone 4 (SEQ ID NO: 21) and / or clone 50 (SEQ ID NO: 29) by PCR / sequencing experiments. In addition, the sequences obtained from each of the reactions had limited identity with HCV. Thus, these products were considered to contain authentic HGBV sequences, even as a result of incorrect priming at one end of the PCR product. The product obtained from reaction 1.14 was also considered to be the result of an incorrect synthesis start. In this case, the complement of SEQ ID NO: 160 was found at the 5 'end of the product obtained from reaction 1.14 (Figure 22, GB-B). This was unexpected as SEQ ID NO: 160 was obtained from SEQ ID NO: 161 in GB-A. However, the sequence identity between the product from reaction 1.14 and the product from reaction 2.8, together with another PCR / sequencing experiment (data not shown), indicates that reaction 1.14 shows the authentic HGBV sequence. Proven to include. It is clear that the complement of SEQ ID NO: 160 is upstream of SEQ ID NO: 162 and has sufficient identity to serve as a PCR primer for the GB-B sequence.

  The sequences obtained from the products shown in Table 9, Table 10 and Table 11 above and the sequences obtained from the RNA cyclization experiment were contigated using the GCG Package (Version 7) program. ). An outline of this assembled contig is shown in FIG. GB contig A (GB-A) is 9493 bp in length, all of which have been sequenced and are shown in SEQ ID NO: 163. GB-A consists of clone 2 (SEQ ID NO: 22), clone 16 (SEQ ID NO: 26), clone 23 (SEQ ID NO: 28), clone 18 (SEQ ID NO: 27), clone 11 (SEQ ID NO: 24) and clone 10 (SEQ ID NO: 24). 23) and. SEQ ID NO: 163 is translated into three readable frames and is shown in the sequence listing as SEQ ID NOs: 164-392. GB contig B (GB-B) is 9143 bp and is shown in SEQ ID NO: 393. GB-B (SEQ ID NO: 393) includes clone 4 (SEQ ID NO: 21), clone 50 (SEQ ID NO: 29), clone 119 (SEQ ID NO: 30) and clone 13 (SEQ ID NO: 25). SEQ ID NO: 393 is translated into one open reading frame and is shown in the sequence listing as SEQ ID NOs: 396, 397. Each of the UTRs from the 5 'and 3' ends can be translated into six reading frames.

B. Evidence for the presence of two HCV-like viruses in HGBV Evidence for GB-A and GB-B representing two different RNA species
By comparing GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) with HCV-1 (GenBank accession # M67463), GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) It shows that the sequences are all different from HCV-1. A dot plot analysis of the nucleic acid sequences of GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) and HCV-1 was performed using GCGPackage (Version 7). GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) and HCV-1 contain nucleotide sequences that are clearly different from each other using a window size of 21 and a stringency of 14 It was discovered (Figure 23). Accordingly, GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) do not represent different strains or genotypes of HCV, nor do they represent different strains or genotypes. By this analysis, short regions with limited nucleotide identity were identified in the NS3-like and NS5b-like sequences of GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393), as well as NS3 of HCV. Found in the sequence and NS5b sequence. However, since each putative NTP-binding helicase and RNA-dependent RNA polymerase correspond to NS3 and NS5b, which are conserved in all flaviviruses, the nucleotide identity within the region is surprising. Not worth (see below). The 5 'RACE experiment (above) demonstrates that GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) represent separate RNA molecules and that they are not separate regions of the same RNA molecule. , Verified by Northern blot data (described in Example 8). Initially, 5 ′ RACE experiments showed that the 5 ′ ends of GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) were different. Since the RNA molecule has only one 5 'end, GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) represent separate RNA molecules. Second, hepatic RNA of infected tamarins by Northern blotting using clone 4 and clone 50 (both SEQ ID NO: 21, 29, obtained from GB-B [SEQ ID NO: 393], see Example 8). The RNA molecule of 8300 bases detected in it almost coincided with the size of GB-B (SEQ ID NO: 393, 9143 bp). If GB-A and GB-B are each part of the same RNA molecule, there must be a Northern blot product with at least 17,000 bases. These data indicate that GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) are nucleotide sequences of two different RNA molecules that are neither HCV variants nor variants of each other. Indicates.

  According to Northern blot analysis and PCR studies of T-1053, two RNA species corresponding to GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393), respectively, are expressed in the liver of T-1053. It was found that they were not present in equal content. As described above, clones 4 and 50 (SEQ ID NOs: 21 and 29, respectively) obtained from GB-B (SEQ ID NO: 393) are both in the liver of T-1053 (as described in Example 8). Hybridized with the 8.3 kb RNA species present in In contrast, clone 2 (SEQ ID NO: 22), clone 10 (SEQ ID NO: 23), clone 16 (SEQ ID NO: 26) and clone 23 (SEQ ID NO: 163), all obtained from GB-A (SEQ ID NO: 163). 28) did not show any hybridization with T-1053 liver RNA in the same experiment (data not shown). In addition, despite the fact that clone 4 PCR and clone 16 PCR have the same sensitivity using a plasmid template (data not shown), clone 16 PCR is T- Significantly less product was produced on cDNA generated from 1053 liver RNA compared to clone 4 PCR. This was in contrast to the situation found in T-1053 plasma at the time the animals were sacrificed. PCR titration experiments for clone 4 (specific for GB-B (SEQ ID NO: 393)) PCR and clone 6 (specific for GB-A (SEQ ID NO: 163)) PCR on cDNA generated from T-1053 plasma RNA , Suggesting that equal amounts of GB-A (SEQ ID NO: 163) RNA and GB-B (SEQ ID NO: 393) RNA are present in T-1053 plasma (Example 4, E.2). Thus, the T at the time the animal was sacrificed despite the presence of equal amounts of GB-A (SEQ ID NO: 163) RNA and GB-B (SEQ ID NO: 393) RNA in T-1053 plasma. It was considered that a larger amount of GB-B (SEQ ID NO: 393) RNA than that of GB-A (SEQ ID NO: 163) RNA was present in the −1053 liver. These results are further evidence that there are two distinct RNA molecules corresponding to GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) in T-1053 plasma. They suggested that these RNAs are not necessarily present in equal amounts in infected tamarin liver RNA. Therefore, it is unlikely that GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) form separate segments of a single viral genome.

2. Evidence that GB-A (SEQ ID NO: 163) and GB-B (SEQ ID NO: 393) represent the genomes of two different viruses Based on infectivity and PCR studies, GB-A (SEQ ID NO: 163) ) And GB-B (SEQ ID NO: 393) evidence for viral properties. Specifically, Tamarin T-1049 and Tamarin T-1051 inoculated with T-1053 plasma that was filtered (0.1 μm) and diluted to 10 ⁻⁴ or diluted to 10 ⁻⁵ without filtration, Both were positive for both clone 4 (GB-B [SEQ ID NO: 393]) and clone 16 (GB-A [SEQ ID NO: 163]) sequences. Prior to inoculation, both of these animals were negative for clone 4 and clone 16 (Example 4, E.4, E.5). Therefore, the two RNA species contained in the acute phase T-1053 plasma corresponding to GB-A and GB-B are filtered and diluted to give other animals the relevant GB-A (SEQ ID NO: 163). And GB-B (SEQ ID NO: 393) virus could be passaged to maintain the expected properties. The fact that GB-A and GB-B represent RNA molecules from separate virus particles was revealed by PCR investigation of H205 inoculated tamarin. Specifically, all 4 tamarins became positive for clone 4 (GB-B [SEQ ID NO: 393]) by RT-PCR after H205 inoculation. In contrast, only one of the four H205 inoculated tamarins (T-1053) was positive for clone 16 (GB-B [SEQ ID NO: 163]). Example 4, E.2). Therefore, it is assumed that the GB-A (SEQ ID NO: 163) sequence is not really present in T-1048, T-1057, and T-1061, and that the clone 16 PCR result is negative, the infectivity is low. The virus corresponding to the GB-B (SEQ ID NO: 393) sequence (ie, hepatitis GB virus B [HGBV-B]) is independent of the GB-A (SEQ ID NO: 163) sequence. It was thought that it could be inoculated alone. A sample of HGBV-B alone from T-1057 was inoculated twice more (Example 4). In these animals, the GB-A (SEQ ID NO: 163) sequence was not detected by RT-PCR. In addition, it was reported that liver enzyme levels were significantly elevated in these animals (Example 4), indicating that HGBV-B alone can cause hepatitis in tamarins. The GB-A (SEQ ID NO: 163) sequence was confirmed in tamarins where the GB-B (SEQ ID NO: 393) sequence was below the detection limit. Specifically, GB-B only animals (T-1048, T-1057, T-1061) are induced by T-1053 plasma and detected by RT-PCR specific for clone 16. Only -A (SEQ ID NO: 163) developed viremia. Plasma of only GB-A from T-1057 was inoculated once more (Example 4). Therefore, it was considered that the virus corresponding to the GB-A (SEQ ID NO: 163) sequence (hepatitis GB virus A [HGBV-A]) replicates independently regardless of HGBV-B. In the absence of HGBV-B, the inoculation of HGBV-A continues further. To date, it is not known whether HGBV-A causes tamarin hepatitis. However, T-1053-induced tamarin does not have increased liver enzyme levels associated with HGBV-A viremia, and even when subcultured with HGBV-A-only serum from T-1057, liver enzyme levels increased. The absence is in contrast to the liver preference of HGBV-B in tamarin.

  The presence of two viruses in the acute phase T-1053 plasma is thought to be based on the H205 inoculum. Specifically, the data of Example 7 shows that clone 16 (from SEQ ID NO: 26, GB-A [SEQ ID NO: 163]) is in any plasma before inoculation from 7 test tamarins. Indicated that it did not exist. In addition, clones 2, 10, 18, 23 (each SEQ ID NO: 22, 23, 27, 28, all from GB-A [SEQ ID NO: 163]) are also detected in any of the examined HGBV inoculated tamarin plasmas None (Example 7). Similar negative results were obtained when preinoculated tamarin plasma was tested for clone 4 and clone 50 (SEQ ID NO: 21, 29, respectively, all from GB-B [SEQ ID NO: 393]). Therefore, neither HGBV-A nor HGBV-B was present in the plasma of tamarin before inoculation. In contrast, all of the above clones (ie, clones 2, 10, 16, 18, 23 from GB-A [SEQ ID NO: 163] and clones 4, 50 from GB-B [SEQ ID NO: 393]) Detected in H205 inoculum (Table 7). Interestingly, as seen in the cDNA made from T-1053 liver (above), some of the GB-A (SEQ ID NO: 163) in the GB-A (SEQ ID NO: 163) were used when the same cDNA was randomly synthesized from H205. All of these separate PCR targets produced less product than the similar PCR target in GB-B (SEQ ID NO: 393) (data not shown). Therefore, it was concluded that HGBV-A and HGBV-B were present in the initial GB inoculum H205. However, it is considered that H205 contains more HGBV-B than HGBV-A. The relatively low HGBV-A in the H205 inoculum was that only one of the 4 tamarins was positive for HGBV-A after H205 inoculation (Example 4, E.2) The reason is considered.

3. Evidence that HGBV-A and HGBV-B belong to the Flaviviridae family Three frame translation generation of GB-A (SEQ ID NOs: 165-268, 270-384, 386-392) described in Example 5 When the product and GB-B (SEQ ID NO: 397) were searched in the SWISS-PROT database, they showed limited but significant amino acid sequence homology with various strains of HCV. Translation products from GB-A (SEQ ID NO: 164) and GB-B (SEQ ID NO: 393) are directed against nonstructural protein regions of various HCV isolates (ie, NS2, NS3, NS4, NS5). It showed the closest homology. For example, as shown in FIG. 24, conserved residues in the putative NTP-binding helicase domain of Flavivirus (represented by *) (FIG. 24A) and the RNA-dependent RNA polymerase of all viral RNA-dependent RNA polymerases Conserved residues in the domain (represented by *) (FIG. 24B) are similarly represented by HCV-1 NS3 and NS5b (SWISS-PROT accession number p26664), GB-A (SEQ ID NO: 390) and GB-B (SEQ ID NO: 397). ) Was retained in any of the expected translation products. (See Choo et al., PNAS 88: 2451-2455 [1991] and Domier et al., Virology 158: 20-27 [1987].) Thus, both GB-A and GB-B viruses are functional NTP binding. It was thought to encode a helicase (functional NTP-binding helicase) and an RNA-dependent RNA polymerase. However, GB-A (SEQ ID NO: 390) and GB-B (SEQ ID NO: 397) do not have complete amino acid identity between them and / or HCV in other regions of HCV NS3 and NS5b. There is no complete amino acid identity. Specifically, GB-A (SEQ ID NO: 390, residues 1252-1449) virus and HCV-1 (SEQ ID NO: 398) are 47% identical over the 200 residues of NS3 shown in FIG. 24A. Yes, GB-B (SEQ ID NO: 397, residues 1212-1408) virus and HCV-1 (SEQ ID NO: 398) are 55% identical, GB-A (SEQ ID NO: 390, residues 1252-1449) virus and GB -B (SEQ ID NO: 397, residues 1212-1408) The virus was 43.5% identical. In addition, the GB-A (SEQ ID NO: 390, residues 2644-2739) virus and HCV-1 (SEQ ID NO: 398) are 36% identical over the 100 NS3 residues shown in FIG. 24B. , GB-B (SEQ ID NO: 397, residues 2513-2612) virus and HCV-1 (SEQ ID NO: 398) are 41% identical, GB-A (SEQ ID NO: 390, residues 2644-2739) virus and GB- B (SEQ ID NO: 397, residues 2599-2698) The virus was 44% identical. Homology was also relatively low for other putative nonstructural genes of GB-A (SEQ ID NO: 390) and GB-B (SEQ ID NO: 397) when compared to HCV. Even when compared to the various HCV sequences registered with Genbank, the overall level of putative nonstructural protein homology between GB-A and GB-B viruses is GB-A (SEQ ID NO: 164) and GB. Both -B (SEQ ID NO: 393) suggest that they are obtained from two different members of the Flaviviridae family. Flaviviruses contain a single genomic RNA molecule corresponding to one NTP binding helicase domain and one RNA-dependent RNA polymerase domain. The presence of two contigs, each containing a putative RNA helicase domain and a putative RNA-dependent RNA polymerase domain, is consistent with the presence of two HCV-like flaviviruses in acute phase T-1053 plasma .

(Example 10)
PCR
To detect the sequence correlation between HGBV and hepatitis C virus, a PCR-based experiment was performed as follows. 5'- of the HCV genome highly conserved in HCV isolates from various positional sources (Cha, T.-A. et al., J. Clin. Microbiol . 29: 2528-2534 [1991]) PCR primers based on untranslated region (UTR) sequences (JH Han, PNAS 88: 1711-1715 [1991]) were used to detect similar sequences in H205 infected tamarin T-1053 liver RNA. As described in Example 8A, total cellular RNA was extracted from the liver of infected tamarin T-1053 and the liver of uninfected tamarin (T-1040). Using a kit available from Perkin-Elmer, approximately 30 μg of each RNA sample was reverse transcribed and PCR-amplified generally following the recommended usage. An antisense primer (primer 1) containing bases 249-268 of HCV 5′-UTR was used in the reverse transcriptase reaction. Primer 1 and a primer containing HCV 5'-UTR bases 13-46 (primer 2) were used for PCR amplification of the intervening sequence. The conditions for thermocycling are according to Cha et al. In order to increase the detection sensitivity of the HCV 5′-UTR sequence of H205 infected tamarin T-1053, a second amplification reaction using “nested” PCR primers was generated for the PCR reaction. The primer for that was obtained from the sequence between the sequence of the primer 1 and the sequence of the primer 2 in the HCV 5′-UTR. Primer 3 contained sequences from bases 47-69 of HCV 5′-UTR, and primer 4 as an antisense primer contained bases 188-210 of HCV 5′-UTR. In this “nested” PCR reaction, the PCR product from the first PCR reaction (total reaction volume from 100 μl to 2 μl) was used as the DNA template source. The thermocycling conditions were generally the same as above except that the annealing temperature was 55 ° C instead of 60 ° C. The PCR product obtained in the second PCR reaction was analyzed for the desired DNA product using agarose gel electrophoresis and ethidium bromide staining. The size of the intended DNA fragment based on the HCV 5'-UTR sequence (Han et al., Supra) was 253 bp for the product of the first PCR reaction and 163 bp for the product of the nested PCR reaction. It was. A PCR product of the desired size was obtained in a control experiment using 30 μg of total cellular RNA extracted from the liver of an HCV-infected chimpanzee described in Example 8A (data not shown). We show that the experimental procedure can detect 5'-UTR of HCV. However, when either T-1053 liver RNA or T-1040 liver RNA was used, neither desired product could be found on the resulting ethidium bromide stained agarose gel (data not shown) ). Thus, the expected results were not obtained because (i) the 5'-UTR sequence of the above reagent was significantly different from that of HCV, so the oligonucleotide primer used was not efficiently annealed, This suggests that PCR amplification has become impossible, or (ii) that the reagent lacked 5′-UTR. In either case, it is deduced from this result that the nucleotide sequence of the reagent is very different from that of HCV.

Nucleic acids were isolated from chimpanzee plasma pools obtained during the acute phase of experimental infection with HCV as described in Example 7 (G. Schlauder et al., J. Clin. Microbiology 29: 2175-2179 [1991]). RT-PCR was performed as in Example 7 using clone 16 primer (SEQ ID NOs: 93, 94). The expected size bands for these primers were not detected by ethidium bromide staining, nor were they detected after hybridization to a probe specific for clone 16 (data not shown). These results demonstrate the irrelevance of clone 16 sequence (SEQ ID NO: 26) to HCV.

(Example 11)
Reactivity of HGBV-infected sera to other hepatitis viruses.
Obtain serum samples before and after HGBV inoculation using H205 inoculum (T-1048, T-1057, T-1061) or T-1053 inoculum (T-1051) and detect after contact with known hepatitis virus Tests for antibodies that are often performed were performed. Antibodies against hepatitis A virus (using the HAVAB assay available from the applicant), hepatitis B core nucleoprotein (using the Corzyme® test available from the applicant), and hepatitis E The specimens were examined for viruses (HEV) (using the HEV EIA available from the applicant) and hepatitis C virus (HCV) (using the HCV second generation test available from the applicant) . These tests were performed according to the instructions enclosed in the package of each test kit.

  None of the tested tamarins were positive for antibodies against HCV or HEV, either before or after HGBV inoculation (see Table 12). Thus, HGBV infection did not result in detectable antisera against HCV or HEV.

  One of the tamarins (T-1061) was positive for HAV antibody both before and after HGBV inoculation, suggesting that it had previously suffered from HAV ( Table 9, T-1061). However, the other three tamarins (T-1048, T-1057, T-1051) did not show any HAV specific antibodies after HGBV inoculation. Thus, HGBV infection does not produce an anti-HAV response. One of the tamarins (T-1048) was negative for HBV antibody both before and after HGBV inoculation. Two of the tamarins (T-1061, T-1057) were positive before HGBV inoculation. One of the tamarins (T-1051) was positive near the border for HBV antibody before HGBV inoculation, but was negative after inoculation. Based on these data, there is no evidence that infection with HGBV reagent induces an immune response against the HBV nucleus. Overall, these data demonstrated that the HGBV reagent is a unique viral agent and not related to any of the common viral reagents associated with human hepatitis.

(Example 12)
Western blot analysis of HGBV-infected liver As shown in Examples 1 and 2 above, an increase in liver enzyme levels was observed in tamarin inoculated with HGBV. If HGBV is actually a liver tropic virus, it is speculated that viral proteins are produced in infected liver cells and an immune response against these proteins occurs. In this example, a unique protein appears in the liver obtained from HGBV-infected tamarin, which is specifically recognized by Western blot using tamarin serum obtained in the convalescent phase after HGBV infection. The grounds for this should be indicated.

  HGBV-infected tamarin liver and various control tamarin and chimpanzee livers were diced and homogenized in PBS using an Omni-mixer homogenizer (approximately 1 g liver for 5 ml). The resulting suspension was clarified by centrifugation (10,000 × g, 1 hour, 4 ° C.) and ultrafiltration through 5 μm, 0.8 μm and 0.45 μm filters. The clarified homogenate was centrifuged under conditions that resulted in pelleting of all components above 100S. The pellet (100S liver fraction) was dissolved in a small amount of buffer and stored at -70 ° C.

  SDS polyacrylamide gel electrophoresis (PAGE) was performed using standard methods and reagents (Laemmli discontinuous gel). The 100S liver fraction was diluted 1:20 in sample buffer containing SDS and 2-mercaptoethanol and heated to 95 ° C. for 5 minutes. The protein was electrophoresed on either 12% acrylamide or 4 → 15% acrylamide linear gradient gel (7 cm × 8 cm) at 200 volts for 30-45 minutes. Proteins were electro-transferred to nitrocellulose membrane using standard methods and reagents.

  Western blots were generated using standard methods. Briefly, the nitrocellulose membrane is simply rinsed in TBS / Tween and 4 ° C TBS / CS (100 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.18% Tween-20, 4.0% calf serum, pH 8.0) Blocked overnight in. The nitrocellulose membrane was placed in a Multi-screen device and 600 μg of serum was placed in the groove of the device, then maintained at room temperature for 2 hours and incubated at 4 ° C. overnight. The nitrocellulose membrane was removed from the Multi-screen apparatus, and the nitrocellulose membrane was washed three times for 5 minutes each in TBS / Twee (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.0). The nitrocellulose membrane was incubated in 15 ml of goat anti-human: HRPO conjugate (0.2 μg / ml TBS / CS) for 1 hour at room temperature. After washing as above, the nitrocellulose membrane was incubated in TMB enzyme substrate solution, rinsed with water and dried.

  Proteins isolated from T-1053 liver at the time of sacrifice (12 days after GB inoculation) and absorbed and transcribed as described above were obtained at the 5th, 6th, 7th, 9th, or 11th week after GB inoculation. Showed a unique antigenic protein with an apparent molecular weight of about 50-80 kDa upon reaction with T-1057 serum. The band was not present when reacted with T-1057 serum before GB inoculation or 3 weeks after inoculation. When reacted with any of the T-1057 sera, the band did not appear in the lane containing liver protein obtained from uninoculated tamarin (T-1040). In addition, when T-1053 liver protein was reacted with other serum after GB inoculation (T-1048 at 11 weeks after GB inoculation and T-1051 at 8 weeks after GB inoculation), the same size (50 A protein of ~ 80 kDa) appeared, but when pre-inoculation serum obtained from the same animal was reacted with T-1053 liver protein, this same size protein did not appear.

  To examine whether the immunoreactive band is detected in liver tissue from other GB inoculated tamarins or in chimpanzee liver tissue infected with either HCV or HBV Western blot experiments were performed. In each case, react nitrocellulose strips containing liver proteins with serum pools from T-1048 (5, 8, 16 weeks after GB inoculation) and T-1051 (8, 12 weeks after GB inoculation). It was. In the pool, 5 sera were mixed at an equal ratio. 50-80 kDa reactive protein band from GB inoculated tamarins (T-1038, T-1049, T-1055 obtained 14 days after GB inoculation, and T-1053 obtained 12 days after GB inoculation) Detected in all obtained tamarin liver specimens. This immunoreactive band consists of liver samples from T-1040 (non-inoculated), chimpanzee liver samples (CHAS-457 (before HCV inoculation), CHAS-457 (HCV +), CRAIG-454 (HCV +), MUNA-376 (HBV +)) could not be detected.

  Together, these data demonstrated the presence of immunogenic and antigenic proteins with an apparent molecular weight of approximately 50-80 kDa that specifically bind to HGBV-infected tamarin liver. The nature of this HGBV-related protein (ie, whether it encodes a virus or is of host origin) is currently under investigation. Regardless of the origin of the HGBV-related protein, these results are consistent with the fact that HGBV infection induces an antibody response to antigens present in HGBV-infected tamarin liver.

(Example 13)
Expression and detection of immunogenic HGBV-A and HGBV-B polypeptides based on CKS Cloning of HGBV-A and HGBV-B sequences Recombinant proteins were fused to CMP-KDO synthetase (CKS) proteins with cloning vectors pJO200, pJO201, pJO202. Each of these plasmids is derived from plasmid pBR322, but with a modified rack promoter fused to the kdsB gene fragment (encoding the first 239 of the 248 total amino acids that make up the E. coli CKS protein), with a synthetic linker fused to the end of the kdsB gene fragment. This synthetic linker contained multiple restriction sites required for gene insertion, a translation termination signal, and a trpA rho-dependent transcription terminator. Unique restriction sites within this linker region included EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, PstI, SphI, HindIII in the 5 'to 3' direction. Each plasmid allows insertion into any reading frame within the plurality of cloning sites. CKS methods and CKS vectors for protein synthesis are disclosed in Applicant's US Pat. No. 5,124,255, incorporated herein by reference, while the use of CKS fusion proteins in assay formats and test kits is not Applicant's US Application No. 07 / 903,043, which is incorporated herein by reference.

  The HGBV-A and HGBV-B sequences obtained from the walking experiments described in Table 9 and Table 10 (Example 9) are the restriction enzymes (10 units, NEB) shown in Table 13 and Table 14. Was isolated from the appropriate pT7Blue T-vector clone and purified from a 1% low melting point agarose gel as described in Example 3B. Plasmids pJO200, pJO201, and pJO202 were digested with the same restriction enzyme (10 units, NEB) and dephosphorylated with bacterial alkaline phosphatase (GIBCO BRL, Grand Island, NY). Each of the purified HGBV fragments was ligated into digested and dephosphorylated pJO200, pJO201, pJO202 and transformed into E. coli XL1 Blue as described in Example 3B. Standard miniprep analysis confirmed the successful formation of the CKS / HGBV expression vector.

  Two other PCR products were specifically prepared for expression. These two products are represented as 4.1 and 4.2, respectively, and were expected to encode the HGBV-B core region and the HGBV-A core region, respectively (see FIG. 22). As described in Example 9, PCR product 4.1 was prepared using primer core B-s and primer core B-a1 (SEQ ID NOs: 708, 709), and PCR product 4.2 was generated using primer core A- s and primer core 2.2.1 '(SEQ ID NO: 710, 138). The 4.1 sense and antisense primers each had EcoRI and BamHI restriction sites intended to be terminal. The 4.1 PCR product was digested, gel isolated and ligated into pJO200, pJO201, pJO202 as described above. The sense primer for the 4.2 PCR product had an EcoRI restriction site intended to be terminal, but the antisense primer did not have a restriction site. Therefore, the product was cleaved with EcoRI, gel isolated, ligated to pJO200, pJO201, pJO202 already digested with BamHI, end-modified with Klenow fragment of DNA polymerase and dNTP, digested with EcoRI, Dephosphorylated with bacterial alkaline phosphatase as known in the industry.

B. Expression of HGBV-A and HGBV-B sequences Tryptone 32 gm / L, yeast extract 20 gm / L, NaCl 5 gm / L, pH 7.4, ampicillin 100 mg / L, glucose 3 mM E. coli XL1 Blue culture containing the HGBV expression vector was grown at 37 ° C. When this culture reached an OD600 of 1.0-2.0, IPTG was added to a final concentration of 1 mM to induce expression from the modified rack promoter. The culture was grown at 37 ° C. with further shaking for 3 hours and collected. Resuspend cell pellet in SDS / PAGE loading buffer (Tris pH6.8 62.5 mM, SDS 2%, glycerol 10%, 2-mercaptoethanol 5%, bromophenol blue 0.1 mg / ml) to an OD600 of 10. Turbid and boiled for 5 minutes. Prepare an aliquot of the whole cell lysate on a 10% SDS-polyacrylamide gel, color with a solution containing 0.2% Coomassie blue dye in 40% methanol / 10% acetic acid, and until the background is clear Decolorized with 16.5% methanol / 5% acetic acid.

  Whole cell lysates were run on a second 10% SDS-polyacrylamide gel and transferred to nitrocellulose by electrophoresis for immunoblot assays. The nitrocellulose sheet containing the transferred protein is incubated in a blocking solution (5% Carnation non-fat dry milk in Tris buffered saline) for 30 minutes at room temperature and pre-blocked against E. coli cell lysate. Incubated in goat anti-CKS serum for 1 hour at room temperature and diluted 1: 1000 in blocking solution. The nitrocellulose sheet was washed twice with Tris buffered saline (TBS), then incubated with alkaline phosphatase-conjugated rabbit anti-goat IgG for 1 hour at room temperature and diluted 1: 1000 in a blocking solution. The nitrocellulose sheet was washed twice with TBS and developed in TBS containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. The appropriate reading frame for each fragment was determined based on the expression of the correct expected size of the immunoreactive CKS fusion protein and the vector insert binding portion was confirmed by DNA sequencing.

  After determining the appropriate reading frame for each of the fragments, samples from cultures containing the appropriate components were analyzed by SDS-polyacrylamide gel electrophoresis and Western blot. FIG. 25A shows two Coomassie stained 10% SDS-polyacrylamide gels containing the CKS fusion protein whole cell lysate. Lanes 1 and 16 contain molecular weight standards in kilodaltons shown on the left. The order of development on gel 1 (HGBV-A sample) was as follows: lane 2, before clone 1.17 induction: lane 3-15, clone 4.2, clone 1.17, clone 1.8, clone 1.2, clone 1.18 (SEQ ID NO: 390) ), Clone 1.19, clone 1.20, clone 1.21, clone 1.22 (SEQ ID NO: 390), clone 2.12, clone 1.5, clone 1.23, clone 2.18, all 3 hours after induction. The order of development on gel 2 (HGBV-B sample) was as follows: lane 17, clone 4.1 before induction: lane 18-29, clone 4.1, clone 1.15, clone 1.14, clone 2.8, clone 1.13, clone 1.12, Clone 2.1, clone 1.7, clone 1.3, clone 1.4, clone 1.16, clone 2.12 were all 3 hours after induction. These proteins were applied on two separate 10% gels in the same order as above and transferred to nitrocellulose as described above. Samples were analyzed by Western blot using a pool of sera from two convalescent T-1048, T-1051 as follows. The nitrocellulose sheet containing the above sample was incubated in the blocking solution for 30 minutes, E. coli lysate 10%, XL1-Blue / CKS lysate 6 mg / ml, and the convalescent tamarin described in Table 6 (Example 4). Transfer to a sequestration solution containing a 1: 100 dilution of the serum pool. After incubating overnight at room temperature, the nitrocellulose sheet was washed twice in TBS, incubated in HRPO-conjugated goat anti-human IgG for 1 hour at room temperature, and diluted 1: 500 in a blocking solution. The nitrocellulose sheet was washed twice in TBS and developed in TBS containing 2-chloro-1-naphthol 2 mg / ml, hydrogen peroxide 0.02% and methanol 17%. As shown in FIG. 25B, the CKS fusions of the three HGBV-B proteins, clones 1.4, 1.7 and 4.1 showed an immune reaction with the tamarin serum pool. Clone 1.7 contains the sequence encoding the HGBV-B immunogenic region (SEQ ID NO: 610) and clone 1.4 was discovered by immunoscreening of a cDNA library (Example 4) using the same convalescent tamarin serum pool. In addition, it contained sequences (SEQ ID NOs: 12, 13, 18) encoding two HGBV-B immunogenic regions.

  The sample described in the paragraph above was also used as described above, using a 1: 100 dilution of convalescent serum obtained about 3 weeks after the onset of acute hepatitis from the surgeon GB. Analyzed by blot. Tables 13 and 14 show the reactivity of fusion proteins from HGBV-A and HGBV-B to the serum. Only one (2.1) HGBV-B protein showed reactivity to this serum, but this reactivity was very low, whereas the two HGBV-A proteins (1.22 [SEQ ID NO: 390], 2.17) It showed high reactivity to serum. These two HGBV-A proteins are common for 40 amino acids, which may reflect reactivity to one or more epitopes. These two HGBV-A proteins were selected for use in an ELISA assay as described in Example 16. Tamarin infected with the 11th passage GB material (H205 GB pass 11) showed immune responses against several HGBV-B epitopes, but not against HGBV-A epitopes. Interestingly, sera from the first GB source showed significant reactivity against at least one GBV-A epitope. This suggested that HGBV-A may have been the cause of hepatitis in military physician GB.

  Four separate human sera that showed the presence of antibodies to one or more of the CKS / HGBV-A or CKS / HGBV-B fusion proteins by 1.4, 1.7, or 2.17 ELISAS (see Examples 15, 16). Selected for Western blot analysis. Three of these sera (G1-41, G1-14, G1-31) are from the West African “dangerous” population, and the fourth (341C) is the non-A-E hepatitis (Egyptian) specimen (these (See Example 15 for a detailed description of the population). Whole cell lysates from some of the above CKS fusion proteins were applied to another 10% SDS-polyacrylamide gel and transferred to nitrocellulose as described above. Each of these blots was pre-blocked as described above, and one human serum sample diluted 1: 100 in blocking buffer containing 10% E. coli lysate and 6 mg / ml XL1-Blue / CKS lysate. Incubated overnight with one. Blots were washed twice in TBS, reacted with HRPO-conjugated goat anti-human IgG and developed as described above.

  CKS / HGBV-B protein was analyzed using two of the above sera (G1-41, G1-14) and the reactivity is shown in Table 13. In addition to the above three proteins that were reactive against tamarin serum, two additional proteins (1.16, 2.1) were reactive against either of the two human sera. CKS / HGBV-A protein was analyzed with all four human sera and their reactivity is shown in Table 14. In addition to the above two proteins that were reactive against GB serum, another three proteins (1.5, 1.18, 1.19) were reactive against one or more human sera. Two of these proteins (1.5, 1.18) were selected for use in the ELISA assay as described in Example 16. G1-31 serum, which is reactive by Western blot and / or by ELISA against two HGBV-A proteins (1.18, 2.17) and one HGBV-B protein (1.7) (Examples 15, 16), Of particular interest is the C sequence (SEQ ID NO: 673, residues 2274-2640), which is serum isolated therefrom (Example 17).

^ａPCR 生成物は表9 、表10、又は、表13に示した通りである。
^ｂ制限消化は、 pT7Blue T−ベクターからPCR フラグメントを遊離させるために、又は、4.1PCR生成物の直接消化のために使用した。

^a PCR products are as shown in Table 9, Table 10 or Table 13.
^b Restriction digests were used to release PCR fragments from the pT7Blue T-vector or for direct digestion of 4.1 PCR products.

(Example 14)
Epitope mapping of immunoreactive HGBV-A and HGBV-B proteins To confirm the location of the epitope mapping immunogenic region of HGBV-B protein 1.7, overlapping subclones within HGBV-B immunogenic protein 1.7 were isolated from RT-1053 serum as described in Example 7. Performed by PCR. Each PCR primer has an extra 6 bases at the 5 'end to facilitate restriction enzyme digestion, followed by either an EcoRI site (sense primer) or a HindIII site (antisense primer). Followed. In addition, each antisense primer contained a stop codon immediately after the coding region. After digestion, each fragment was cloned into EcoRI / HindIII-digested pJO201 as described in Example 13. CKS fusion protein was expressed as described in Example 13 and analyzed by Western blot using tamarin T-1048 / T-1051 serum. Five overlapping clones, designated 1.7-1 to 1.7-5, were generated. These clones encode a region of 1.7 protein that is in the range of 104 to 110 amino acids in size. Table 15 shows the PCR primers used to generate each clone, the size of the encoded polypeptide, the position within the 1.7 sequence, and the reactivity to tamarin T-1048 / T-1051 serum. Two additional duplicate clones were generated that span the immunogenic region (SEQ ID NO: 678) discovered by immunoscreening of the cDNA library (Example 4). Each of these clones, referred to as 1.7-6 and 1.7-7, encodes a polypeptide of 75 amino acids. Table 15 shows the PCR primers used, the size of the encoded polypeptide, the position within the 1.7 sequence, and the reactivity to tamarin T-1048 / T-1051 serum. Two immunogenic regions were identified in a 1.7 protein length of 507 amino acids. That is, the immunogenic region near the N-terminus was in the range of residues 1-105, and the immunogenic region near the center of the protein was in the range of residues 185-410. Whether there is a single epitope or multiple epitopes within each of these regions requires further consideration.

B. To confirm the location of the epitope mapping immunogenic region of HGBV-B protein 1.4, overlapping subclones within HGBV-B immunogenic protein 1.4 were performed by RT-PCR from T-1053 serum. Each PCR primer has an extra 6 bases at the 5 'end to facilitate restriction enzyme digestion, followed by either an EcoRI site (sense primer) or a BamHI site (antisense primer). Followed. In addition, each antisense primer contained a stop codon immediately after the coding region. After digestion, each fragment was cloned into EcoRI / BamHI-digested pJO201 as described in Example 13. CKS fusion protein was expressed as described in Example 13 and analyzed by Western blot using tamarin T-1048 / T-1051 serum. Four overlapping clones were generated, designated 1.4-1 to 1.4-4. These clones encode a region of the 1.4 protein that ranges in size from 137 to 138 amino acids. Table 15 shows the PCR primers used to generate each clone, the size of the encoded polypeptide, the position within the 1.4 sequence, and the reactivity to tamarin T-1048 / T-1051 serum. Two additional duplicate clones were generated that span the immunogenic region found by immunoscreening of the cDNA library (Example 4). Each of these clones, designated 1.4-5 and 1.4-6, encodes a 75 amino acid long polypeptide. Table 15 shows the PCR primers used, the size of the encoded polypeptide, the position within the 1.4 sequence, and the reactivity to tamarin T-1048 / T-1051 serum. A sequence composed of 265 amino acids including residues 129-393 was confirmed to be an immunogenic region in a 1.4 protein with a length of 522 amino acids. Since two non-contiguous immunogenic clones were identified within the sequence by immunoscreening the library (Example 4), at least two epitopes may be present within the region.

C. Epitope mapping of HGBV-A protein 1.22 (SEQ ID NO: 390) and 2.17
Both HGBV-A proteins 1.22 (SEQ ID NO: 390) and 2.17 (SEQ ID NO: 613) showed immunoreactivity with GB serum by Western blot (Example 13). Since these two proteins overlap by 40 amino acids, the observed immune response may have been caused by one epitope or as a result of the presence of two or more epitopes. The complete 1.22 / 2.17 sequence was 641 amino acids long. In order to find an immunogenic region, overlapping subclones within this region were performed by RT-PCR from T-1053. Each PCR primer has an extra 6 bases on the 5 'end to facilitate restriction enzyme digestion, followed by an EcoRI site (sense primer), or 1.22 / 2.17-2 to 1.22 / Followed by either of the BamHI sites (antisense primers) for 2.17-6. However, since clone 1.22 / 2.17-1 has an internal EcoRI site, the BamHI site was used as the sense primer and the HindIII site was used as the antisense primer. In addition, each antisense primer contained a stop codon immediately after the coding region. After digestion, each fragment was cloned into EcoRI / BamHI-digested (or BamHI / HindIII-digested for 1.22 / 2.17-1) pJO201 as described in Example 13. CKS fusion proteins were expressed as described in Example 13 and analyzed by Western blot using GB serum. This clone encoded a region of 1.22 / 2.17 in the size range of 115-116 amino acids. Table 15 shows the PCR primers used to generate each clone, the size of the encoded polypeptide, the position within the HGBV-A polypeptide sequence, and the reactivity against GB serum. 1.22 / 2.17 In the middle of the protein, the immunogenic region was narrowed to a region 220 amino acids long. This includes a 40 amino acid long overlap region between 1.22 and 2.17, so that the immune response common to the two proteins is due to one epitope shared or due to multiple epitopes It may have been.

(Example 15)
Serological studies of HGBV-B Recombinant protein purification procedure Bacterial cell cultures expressing CKS fusion proteins were frozen and stored at -70 ° C. Bacterial cells from each of the three constructs are thawed and ground with lysozyme and DNase and then sonicated in the presence of phenylmethanesulfonyl fluoride and other protease inhibitors to obtain individual recombinant antigens. A mixture of E. coli proteins was obtained. For each of the three cultures, the insoluble recombinant antigen was concentrated by centrifugation and subjected to a series of washing steps to remove most of the non-recombinant E. coli protein. The composition of the washing solution used in this protocol was distilled water, 5% Triton X-100, and 50 mM Tris (8.5). The resulting precipitate was dissolved in the presence of sodium dodecyl sulfate (SDS). After measuring the protein concentration, 2-mercaptoethanol was added, the mixture was subjected to gel filtration column chromatography using Sephacryl S300 resin, and various proteins were separated and separated according to their sizes. Each fraction was collected and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein separated by this electrophoresis is then stained with Coomassie Brilliant Blue R250, and the molecular weight is about 75 kD (CKS-1.7 / SEQ ID NO: 610), 80 kD (CKS-1.4 / SEQ ID NO: 611), 42 kD. The presence or absence of a protein corresponding to (CKS-4.1 / SEQ ID NO: 612) was examined. Fractions containing the relevant protein were pooled and examined again by SDS-PAGE.

  The immunogenicity and structural integrity of the pool fraction containing the purified antigen was determined by electron transfer to nitrocellulose and immunoblotting according to the method described in Example 13. Since there was no appropriate positive control, recombinant proteins were identified by their reactivity with monoclonal antibodies against the CKS portion of each fusion protein. When CKS-1.7 protein (SEQ ID NO: 610) was examined by Western blotting using an anti-CKS monoclonal antibody to detect a recombinant antigen, a single band was observed at about 75 kD. This result is consistent with the expected size of the CKS-1.7 protein (SEQ ID NO: 610). For CKS-1.4 protein (SEQ ID NO: 611), a quadruple band pattern was detected between 60 kD and 70 kD by anti-CKS monoclonal antibody. These observed bands are shorter than expected for the full-length protein and are probably deleted. When the CKS-4.1 protein (SEQ ID NO: 52) was examined by Western blotting, the anti-CKS monoclonal antibody detected a recombinant antibody as a single band at about 42 kD. This result is consistent with the expected size for the CKS-4.1 protein (SEQ ID NO: 612).

B. Procedure for coating polystyrene beads The above proteins were dialyzed and the antigenicity of these proteins on polystyrene beads was evaluated as described below. Separately, HGBV was used with each of the three purified HGBV recombinant proteins (CKS-1.7 / SEQ ID NO: 610, CKS-1.4 / SEQ ID NO: 611, and CKS-4.1 / SEQ ID NO: 612). An enzyme-linked immunosorbent assay (ELISA) was performed to detect antibodies against. The ELISA performed with these proteins was 1.7 ELISA (using CKS-1.7 (SEQ ID NO: 610) recombinant protein), 1.4 ELISA (using CKS-1.4 (SEQ ID NO: 611) recombinant protein), And 4.1 ELISA (CKS-4.1 (SEQ ID NO: 612) recombinant protein used). In the first study, 1/4 inch polystyrene beads were coated with various concentrations of purified protein (approximately 60 beads / lot), serum from uninoculated tamarin was a negative control, convalescent serum from inoculated tamarin. Was evaluated by ELISA test (described below) using as a positive control. Other controls included a pool of human sera obtained from individuals who were negative for various hepatitis viruses. An additional positive control was a monoclonal antibody against CKS protein to monitor bead coating efficiency. The bead coating conditions were selected so that the ratio of the positive control signal to the negative control signal was the highest and used for the scale assay of the bead coating process. For each of the four ELISA tests, at least two lots of 1000 beads were made and used for serological studies.

  Briefly, to coat polystyrene beads with purified protein, the washed beads were added to a scintillation vial and the beads were immersed in a recombinant antigen-containing buffer (approximately 0.233 ml / bead). A variety of different concentrations were prepared for each recombinant antigen and these were evaluated with a variety of different buffers adjusted to a pH range of 5.0 to 9.5. The vial was then placed on a rotator in a 40 ° C. incubator for 2 hours, after which time the liquid was aspirated and the beads were washed three times with pH 6.8 phosphate buffered saline (PBS). The beads were then treated with 0.1% Triton X-100 for 1 hour at 40 ° C. and washed 3 times with PBS. The beads were then overcoated with 5% bovine serum albumin and incubated at 40 ° C. for 1 hour with stirring. After further washing with PBS, the beads were overcoated with 5% sucrose for 20 minutes at room temperature, and the liquid was removed by suction. Finally, the beads were air dried and used in an ELISA test to detect antibodies against HGBV.

C. ELISA procedure for detecting antibodies against HGBV ELISA was performed in an indirect assay. That is, serum or plasma was diluted with a specimen diluent and reacted with an antigen coated on a solid phase. After the washing step, the beads were reacted with a horseradish peroxidase (HRPO) labeled antibody directed against human immunoglobulin in order to detect tamarin or human antibody bound to the solid phase. Samples that produced a signal that exceeded the truncation value were considered reactive. Additional details regarding the ELISA are provided below.

In the ELISA format, a solid phase coated with an antigen is contacted with tamarin serum previously diluted with a specimen diluent (a buffer solution containing animal serum and a nonionic active agent). In formulating this specimen diluent, the specific antibody binds to the solid phase coated with the antigen while increasing the background signal due to non-specific immunoglobulin binding to the solid phase. Prepared. Specifically, 10 μl of tamarin serum was diluted with 150 μl of a sample diluent, and stirred with rotation. After 10 μl of this pre-diluted sample was added to the wells in the reaction tray, 200 μl of sample diluent and antigen-coated polystyrene beads were added. The reaction tray was incubated in a dynamic incubator (Abbott Laboratories). The incubator was constantly stirred at room temperature. After 1 hour incubation, the liquid was aspirated and the wells containing the beads were washed 3 times with distilled water (5 ml each time). Next, 200 μl of HRPO-labeled goat anti-human immunoglobulin diluted in conjugate diluent (buffer solution containing animal serum and non-ionic active agent) is added to each well and the reaction tray is incubated again for 1 hour as above. did. The liquid was aspirated off and the wells containing the beads were washed 3 times with distilled water as described above. Beads with antigen and bound immunoglobulin was removed from the wells, each bead was placed in a test _{tube, 0.3% o - 0.02% H} 2 O 2 containing 0.1M citrate buffer phenylenediamine dihydrochloride The mixture was reacted with 300 μl of a solution (pH 5.5). After holding at room temperature for 30 minutes, 1N H ₂ SO ₄ was added to stop the reaction. Absorption at 492 nm was read with a spectrophotometer. The resulting color was directly proportional to the amount of antibody present in the test sample.

  Preliminary truncation values were set for each group of specimens to separate specimens that did not contain antibodies against the HGBV epitope and specimens that contained antibodies against the HGBV epitope.

D. Detection of HGBV-derived RNA in the sera of infected individuals Reference has already been made to RT-PCR to correlate the serum response data of 1.7 ELISA and 1.4 ELISA in the presence of HGBV-RNA in tamarin serum or human serum / plasma This was carried out in the same manner as Example 7 of US patent application Ser. No. 08 / 283,314, which was incorporated herein. In practice, oligonucleotides derived from the cloned sequence of HGBV were used. The final concentration of the oligonucleotide for clone 4 (see Example 7) derived from the HGBV-B genome and clone 16 derived from the HGBV-A genome was 0.5 μM.

E. Blood samples were collected from tamarin contained in tamarin serum image LEMSIP on a weekly basis to obtain serum, and liver enzyme levels were examined. Sample serum levels were sent to Abbott Laboratories for further investigation.

1. ELISA results for tamarins (early infectivity study)
Four tamarins (T-1053, T-1048, T-1057, and T-1061) were inoculated with GB serum (referred to as passage number 11 of H205GB). During the first week after inoculation (PI), an increase in liver enzymes was observed with tamarin T-1053. This tamarin was euthanized on PI 12th. Tamarins T-1048, T-1057, and T-1061 showed increased liver enzymes up to 2 weeks after inoculation. This increase continued until PI 8-9 weeks (Figures 2-4) and then returned to pre-inoculation levels. At 14 weeks of PI, these three tamarins were re-inoculated with 0.10 ml of undiluted serum from Tamarin T-1053 (this individual has been found to be infectious-Example 2). Went.

  These three tamarins (Tamarin T-1048, T-1057, and T-1061) were recovered during the recovery phase from CKS-1.7 (SEQ ID NO: 610) recombinant protein, CKS-1.4 (SEQ ID NO: 611). ) Whether or not it has an antibody against the recombinant protein, CKS-4.1 (SEQ ID NO: 612) recombinant protein, was examined by conducting an ELISA (FIGS. 3, 4, and 5) individually. Specific antibodies against each recombinant protein of 1.7 (SEQ ID NO: 610), 1.4 (SEQ ID NO: 611), 4.1 (SEQ ID NO: 612), or 1.5 (SEQ ID NO: 614) Neither was detected from the specimen.

  As shown in FIG. 26, in T-1048 serum, specific antibodies were detected on PI56-84 days by 1.7 and 1.4 ELISA tests, but not on PI97 and 137 days. In addition, T-1048 serum did not detect specific antibodies in the 4.1 ELISA test. As shown in FIG. 27, an antibody against 1.7 protein (SEQ ID NO: 610) was detected in the serum of T-1057 at PI56 and 63 days, but not after PI63 day. Antibody against 4.1 protein (SEQ ID NO: 612) was detected on PI 28-63 days, but not detected on PI 84-97. As described above, each tamarin was re-inoculated with the inoculum H205 on the day of PI97. A specific antibody against the 4.1 protein (SEQ ID NO: 612) was detected on days PI112 and PI126, suggesting a past response to inoculation. No antibody reaction was observed for the 1.4 recombinant protein (SEQ ID NO: 611).

  Specific antibodies against recombinant 1.4 protein (SEQ ID NO: 611) were detected in tamarin T-1061 serum between PI84 days and PI112 days, but not after PI126 days. As shown in FIG. 28, tamarin T-1061 serum was negative at PI 350 days for antibodies against 1.7 protein (SEQ ID NO: 610) and 4.1 protein (SEQ ID NO: 612).

2. Results of PCR for tamarin (study of initial infectivity)
Tamarin T-1048 and T-1057 sera were selected and HGBV-RNA was obtained by RT-PCR using primers obtained from clone 4 described in Example 7 and primers obtained from clone 16 described in Example 7. Examined.

  HGBV-RNA was detected in either serum primer 10 days before inoculation and 17 days before inoculation (T-1048) (FIG. 26), or in serum at days 17, 37, 59 before inoculation, depending on RT-PCR. None of the primers (T-1057) (FIG. 27) was detected. For T-1048, HGBV-RNA was detected using primers from clone 4 for 15 of 17 different sera obtained during PI 7-137 days. HGBV-RNA was not detected by RT-PCR using primers from clone 16 even in any of the 10 sera obtained during the period of PI 7-97 days. When inoculated with T-1053 plasma, 4 out of 5 sera collected during the 8th and 40th days after inoculation were positive for clone 16. As for T-1057, the RT-PCR results were positive in 4 samples collected from PI 7 to 28 as shown in FIG. 27 using the primer from clone 4. The results of RT-PCR performed on each specimen were negative for clone 4 except for day 287, which showed a weak hybridization signal 28 days after PI. All of the 6 specimens of T-1057 obtained from PI 7 days to 97 days were positive for RT-PCR using primers from clone 16. However, sera between 8 and 85 days after inoculation with T-1053 were positive with the use of primers from clone 16.

3. ELISA results for tamarins (titer test / propagation study)
As described in Example 2, 4 tamarins were inoculated with tamarin T-1053 serum. Of these 4 tamarins, 3 were euthanized during the acute phase of the disease (between PI 12 and 14). The RT-PCR results obtained for these three tamarins are described below. Surviving tamarin (T-1051) initially had elevated liver enzyme levels by Day 14 PI, which persisted until at least PI 8 weeks. Tamarin T-1051 specimens were tested in 1.7 ELISA and 1.4 ELISA. The results are shown in FIG. Specific antibodies were not detected in sera collected during the first 41 days after inoculation or in sera before inoculation. However, antibody responses against 1.4 protein (SEQ ID NO: 611) and 1.7 protein (SEQ ID NO: 610) were between PI49 days and PI113 days, and antibody responses against 4.1 protein (SEQ ID NO: 612) were PI28 days. And PI 105 days. Tamarin was euthanized on day 113 of PI.

  Tamarin (T-1034) was previously inoculated with 0.1 ml of potentially infectious serum from a patient (first GB source) recovering from a recently affected hepatitis infection as described in Example 1 and Table 4 Is. No increase in liver enzyme levels was observed at T-1034 for about 10 weeks after inoculation. For this reason, it was determined that tamarin T-1034 could be used in additional studies. Tamarin T-1034 was inoculated with an HGBV preparation. This HGBV preparation was obtained from a serum pool of 3 tamarins (T-1055, T-1038, T-1049) pre-inoculated with tamarin T-1053 serum from Example 4? ? It was prepared according to the description.

  These three tamarins (T-1055, T-1038, T-1049) were inoculated with serum prepared from tamarin T-1053 as described in Example 2. By day 11 PI, all three tamarins had elevated liver enzymes. Tamarin T-1055 was killed on the 12th day of PI. Tamarin T-1038 and Tamarin T-1049 were killed on PI day 14. These tamarin sera were pooled, clarified and filtered. Tamarin T-1034 was inoculated with 0.25 ml of this filtered 10-6 dilution (prepared in healthy tamarin serum).

  An increase in liver enzyme ALT level was first observed at 2 weeks after inoculation with T-1034, maintained an increase for the following 7 weeks, and finally normalized by 10 weeks after inoculation. As shown in FIG. 30, a specific antibody response against the 1.4 (SEQ ID NO: 22) recombinant protein was first detected on PI 49 days and continued to be detected between PI 56 days and 118 days. The antibody response to the 4.1 (SEQ ID NO: 52) recombinant protein was first detected at PI 49 days and continued to be detected between PI 56-77 days. However, it was not detected between days 84 and 118 of PI. The antibody response to the 1.7 (SEQ ID NO: 610) recombinant protein was first detected at PI 56 days and continued to be detected between PI 63-118 days. Tamarin killed on day 118 of PI.

  As described in Example 2, tamarin T-1044 was inoculated with serum from T-1057 7 days after inoculation with H205. This inoculum was only positive for the sequence detected with the clone 4 primer. RT-PCR using the clone 16 primer showed a gradual increase in the ALT value exceeding the cutoff value from the 14th to 63rd days of PI. As described above, a specific antibody response against the 1.7 (SEQ ID NO: 610) recombinant protein was detected between PI 63-84 days. No antibody response to 4.1 (SEQ ID NO: 612) recombinant protein or 1.4 (SEQ ID NO: 611) recombinant protein was detected. Tamarin was killed on PI 161 day.

4). PCR results for tamarins (titer test / propagation study)
Sera collected from T-1049 and T-1055 in the week of 8 weeks prior to inoculation and from T-1038 on the day of inoculation were in clone 16 (SEQ ID NO: 26) and clone 4 (SEQ ID NO: 21). In contrast, RT-PCR was negative. Tamarin T-1049 and Tamarin T-1055 were positive for RT-PCR one week after inoculation against the sequence of clone 4 (SEQ ID NO: 21) (clone 16 PCR was not performed). Prior to the killing date, T-1049 (PI 14 days) and T-1055 (PI 11 days) were positive for RT-PCR against both clone 4 (SEQ ID NO: 21) and clone 16 (SEQ ID NO: 26). Met. Tamarin T-1038 was positive for both primers on the day of killing (PI 14 days).

  As can be seen from FIG. 30, T-1034 was RT-PCR positive for the sequence detected with the clone 4 primer for the first serum sample collected after inoculation (PI 7 days) and remained positive until PI 70 days. The sample collected on PI 112 days was negative. All these samples were negative by RT-PCR with clone 16 primer. Samples taken on days 70 and 101 before inoculation were negative for both primers.

  As can be seen from FIG. 29 for Tamarin T-1051, HGBV-RNA was detected by both primers (from clone 4 and clone 16 above) in serum samples taken 8 weeks prior to inoculation. There wasn't. HGBV-RNA was detected by RT-PCR using primers from clone 4 in 6 sera collected between PI 7-69, but was collected on PI 77, 84, 91, or 105 days It was not detected in serum. In nine samples taken after inoculation, HGBV-RNA was detected by RT-PCR using primers from clone 16.

  As shown in FIG. 7, in the first serum sample collected after inoculation (PI 7 days), T-1044 is positive for RT-PCR against the sequence detected with the clone 4 primer and positive until PI 63 days. It continued to be. Samples taken between PI 77-119 were negative. All these samples were RT-PCR negative for clone 16 primer. Samples taken 42 days before inoculation were negative for both primers.

  Tamarin T-1047 and T-1056 were inoculated with T-1044 serum collected on the 14th day of PI. Nine samples taken between 7 and 64 days from both these animals were positive for RT-PCR with clone 4 primers (SEQ ID NOs: 8 and 9) but negative with clone 16 primers. .

  Tamarin T-1058 was inoculated with undiluted T-1057 serum taken 22 days after induction with T-1053 serum. This inoculum was positive for the sequence detected with the clone 16 primer, but negative with the clone 4 primer. Serum samples taken from this animal were tested with primers derived from GBV-sequence [clone 16, clone 2, clone 10, clone 18] and GB-B sequence [clone 4 and clone 50]. All samples collected 9 days before inoculation were negative for all primers. The sample collected on the 14th day of PI was positive only with the primers of clones 10 and 18. Samples taken on day 21 PI were positive only with primers for clones 16, 10, and 18. The sample collected on the 28th day of PI was positive only with the clone 18 primer. Samples taken on day 35 of PI were positive with clones 2, 16, and 18 primers only. The sample collected on the 41st day of PI was positive only with the primers of clones 16 and 18. With the primers from clone 4 and clone 50, all samples tested were negative.

5). Summary of Serological Studies with Tamarin When 5 tamarins were inoculated with various preparations of HGBV, liver enzymes began to rise by 2 weeks after inoculation. This increase value lasted for the next 6-8 weeks. Specific antibody responses to one or more HGBV recombinant antigens, 1.7, 1.4, and 4.1 were observed in all five tamarins. In each case, the antibody was first detected at PI 6 to 10 weeks and lasted for an additional 2 to 7 weeks. In general, antibody levels peaked and then dropped rapidly within the next few weeks. It can be seen that the antibody can be detected some time after the liver enzyme levels return to normal. This suggests that antibody production has some effect on resolution of viral infection.

6). Summary of PCR studies on tamarins The results of the genomic walking experiment suggest that clone 4 (SEQ ID NO: 21) and clone 16 (SEQ ID NO: 26) are on separate RNA molecules. We have previously had two different types of viral genomes, one part containing clone 4 (SEQ ID NO: 21) and the other part containing clone 16 (SEQ ID NO: 26). The hypothesis was raised. The observation that an animal is positive with the primer from clone 4 and negative with the primer from clone 16 supports the existence of two different types in the viral genome. However, the fact that the sequence of clone 16 (SEQ ID NO: 26) could not be detected in some of the infected tamarins reflected the difference in the lower sensitivity limit between the primer set of clone 16 and the primer set of clone 4. It may be possible to argue that If this latter possibility is correct, a tamarin that is positive for both primers should show a difference in sensitivity for these two primer sets. To reinforce the position that the above results are explained by the presence of the two virus species and not by the difference in sensitivity of the two primer sets, a series of tamarin T-1057 and T-1053 cDNA sequences PCR was performed using a dilution series. T-1057 serum was positive at 5 × 10 ⁻³ μl serum equivalent of clone 4 primer and negative at 5 × 10 ⁻⁴ μl. RT-PCR was performed with clone 16 primer using T-1057 serum in a large amount of 20 μl, but the result was negative. If this difference is attributed to the relative sensitivity of the two primer sets (clone 4 vs. clone 16), it is expected that other specimens will also show a 4000-fold higher endpoint dilution when the PCR test is performed. However, the cDNA derived from the T-1053 serum was positive at a serum equivalent of 2.5 × 10 ⁻⁴ μl for both clone 4 (SEQ ID NO: 21) and clone 16 (SEQ ID NO: 26). 2.5 × 10 ⁻⁵ μl was found to be negative. Thus, this result cannot be explained by the difference in the sensitivity of the primer sets, and the contigB-clone 4 (SEQ ID NO: 21) and contigA-clone 16 (SEQ ID NO: 26) sequences have titers that differ by at least 4000 times in T-1057. Is consistent with the assumption that is present on a separate viral genome that is approximately the same titer at T-1053. That is, this data is consistent with the presence of two separate viruses with different relative endpoint titers in different specimens.

  From the observation that HGBV-B alone is sufficient to cause an increase in hepatic enzyme levels, and no increase in hepatic enzyme levels is observed in the viremia stage of GBV-A alone, HGBV-B Was probably the causative agent of hepatitis in these tamarins. The immune response to HGBV-B antigen appeared for a short period of 150 days after inoculation. An explanation for this may be that the epitope selected in these ELISA assays was not from the main epitope that the immune response generated. Another explanation is that in tamarins, hepatitis induction is not severe and requires a long-term response. This is consistent with histological evidence that liver inflammation stopped mildly or non-severely (results not shown) in animals killed in the acute phase or died spontaneously shortly after the acute phase.

  In 5 of the 6 animals described in this study, HGBV-B-induced viremia was dissipated by PI112 days. On the other hand, tamarin T-1048 did not recover from viremia for 136 days, and viremia was observed at the time of death (PI 137 days). Of the 4 animals that were positive for the GBV-A sequence, 3 had resolution of viremia by 77 days after the first appearance of the GBV-A sequence. On the other hand, tamarin T-1061 showed viremia for 245 days before being sacrificed. In addition, Tamarin T-1051 was viremia until sacrifice (PI 113 days), but this persistent viremia was due to the initial inoculation with T-1053 plasma, or It is unclear whether it was due to the additional inoculation with T-1053 plasma after 69 days.

  The average value of peak ALT of 6 animals that were positive for both HGBV-A and HGBV-B was higher than the average value of 4 animals that were positive only for HGBV-B. In addition, on average, peak values were reached earlier in animals positive for both GBV-A and GBV-B than animals positive for GBV-B alone. These results suggest that the strength of hepatitis may be related to the presence of significant amounts of both agents. It was observed that there was almost no increase in liver enzyme levels in GBV-B viremia in the additional passage inoculation of GBV-B to Tamarin T-1047 and T-1056. Supports the hypothesis that is essential to bring about a major rise in ALT levels. In addition to passage inoculation with HGBV-B alone, initial results when inoculating T-1058 with inoculum HGBV-A were due to the absence of detectable GB-B sequences with clone 4 and clone 50 primers. As shown, HGBV-A suggests that it can be transmitted independently without detectable HGBV-B.

F. Experimental procedure to demonstrate exposure to HGBV in the human population Samples were obtained from various human populations and examined for the presence of antibodies to HGBV by three separate ELISAs using recombinant proteins derived from HGBV-B. . As in Example 15B, the solid phase was coated with the CKS-1.7 recombinant protein (SEQ ID NO: 610) in the 1.7 ELISA, and the CKS-1.4 recombinant protein (SEQ ID NO: 611) in the 1.4 ELISA. In the 4.1 ELISA, the solid phase was coated with 4.1 recombinant protein (SEQ ID NO: 612). As noted in Example 15E, tamarins inoculated with HGBV showed specific but short antibody responses to these proteins. Given the short-term nature of this detectable immune response, it cannot be said that there has been no prior exposure to HGBV in a negative human population.

  Serological studies performed on human specimens have a dual purpose. The primary objective was to determine the serological presence of antibodies against the HGBV recombinant antigens of the invention in various human populations. These studies include the following tests (1)-(3): (1) Groups considered to be “low risk” for exposure to HGBV (eg, healthy volunteer blood donors in the United States); (2 ) Populations considered “at risk” for exposure to HGBV (eg, samples of intravenous drug users and hemophilia patients often have a serum response to parenterally transmitted hepatitis viruses (HBV and HCV)) Specimens; specimens from individuals living in developing countries often have a positive serologic response to enterally transmitted viruses (HAV and HEV); (3) known hepatitis viruses (HAV, HBV) , HCV, HDV, or HEV) and other hepatitis related viruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV) A group of specimens from individuals with “non-AE-hepatitis” that are not related to exposure to A. Some members of the group belonging to the general designation of non-AE-hepatitis have been tested for antibodies to HEV Therefore, HEV-ELISA assay (available from the Applicant) for all specimens in non-AE groups that were reactive in 1.7, 1.4, or 4.1 ELISA Anti-HEV gave positive results as described below in samples from three regions (Pakistan, USA, and New Zealand).

  Higher serological prevalence may be seen in “at risk” populations of exposure to HGBV and individuals with non-AE hepatitis than those considered “low risk” for exposure to HGBV is expected.

  The second objective of this serological study was to determine if the virus was present in the serum by examining the specimen RT-PCR that proved positive for antibodies to one or more HGBV epitopes. . It is well known that HBV and HCV cause viremia disease states that last for months or years, and that HAV and HEV generally cause short-term viremias that generally last for weeks. In the case of HBV or HCV infection, which is an acute and disappearing hepatitis, the viremia stage may be as short as several weeks. Thus RT-PCR can be used to obtain evidence that a virus is present in an infected individual. However, since the viremia state can be short-lived, an individual infected with an agent may become RT-PCR negative for that agent.

G. Determination of truncation values From previous experience in other ELISA assays using indirect methods, preliminary truncation values should be calculated based on absorption obtained from a population that is likely negative for antibodies to the protein under study. It has turned out to be good. Preliminary truncation values were calculated as the sum of the population mean absorption value and 10 standard deviations from the population mean value. Since the truncation value is used each time a test group is tested, a simpler way to express truncation is to run a negative control (normal human plasma pool-NFP) that runs 5 times for each assay. It was a method to decide from. For 1.7, 1.4, and 4.1 ELISAs, the negative control typically had an absorption between 0.030 and 0.060. As described below, the truncation value was calculated to be about 0.300 to 0.600 in absorbance, which was comparable to an absorbance signal 10 times that of the negative control. Therefore, it is necessary that the absorption value (= S / N ratio) of the sample (S) with respect to the negative control (N) absorption value is 10.0 or more for the specimen to be considered reactive.

H. Supplementary testing In principle, specimens that were considered reactive in the first test were retested in duplicate. If one or both absorptions at the time of the retest were higher than the truncation value, the specimen was considered to be repeatedly reactive. For specimens that were considered repetitive, a supplemental assay was performed to further supplement the ELISA data. If there is a sufficient amount, the repeat-reactive specimen is one in which the antibody response is directed to the CKS1.7 (SEQ ID NO: 610) CKS1.4 (SEQ ID NO: 611) or CKS4.1 (SEQ ID NO: 612) antigen by Western blot. It was confirmed that it was not intended for the E. coli protein that might have been coated on the solid phase together with the main protein in question. For a Western blot to be considered positive, a visible band is 80 kD for 1.7 protein (SEQ ID NO: 610), 60 to 70 kD for 1.4 protein (SEQ ID NO: 611), or 4.1. It needs to be detected at 42 kD for the protein (SEQ ID NO: 612). Western blots were not optimized to match or exceed the sensitivity of ELISA, so ELISA data were not immediately discarded when negative results were obtained. However, if a positive result is obtained, it reinforces the reactivity detected by ELISA.

  RT-PCR using the clone 4 primer (performed as described in Example 15D) is performed on a sample that is repeatedly reactive and has a sufficient amount, and an HGBV-specific nucleotide sequence in serum is identified. Good. If the result is positive, the sample is known to be viremia and will ultimately help establish the role of HGBV in human hepatitis. However, a negative result should not be interpreted as an incorrect ELISA result. As described in Example 15E in the Tamarin study, RT-PCR results were positive for the first few weeks after infection, and were negative by the time the antibody just started to be detected in the ELISA of the present invention. It was. Samples at this later stage may be positive in one or both ELISAs even though they are negative for RT-PCR reactions.

I. Serum response data in low-risk specimens A population of 100 sera and 100 plasma was obtained from healthy volunteer blood donors in southeastern Wisconsin, and 1.7 (SEQ ID NO: 610), 1.4 (SEQ ID NO: 610) by ELISA as described above. 611) and 4.1 (SEQ ID NO: 612) were tested for antibodies against each recombinant protein. For serum and plasma, absorption values obtained with 1.7, 1.4, and 4.1 ELISA were plotted separately (FIGS. 9-14).

  In the 1.7 ELISA, the mean absorption values for the serum sample and the plasma sample were 0.072 [standard deviation (SD) is 0.061] and 0.083 (SD = 0.055), respectively. Thus, in the 1.7 ELISA, the provisional cutoff values for serum and plasma were 0.499 and 0.468, respectively. As described above, the cut-off value can also be expressed based on the absorption value of the negative control. A specimen having an S / N ratio exceeding 10.0 was regarded as reactive. Using this truncation value, 0 out of 200 samples tested against an antibody against 1.7 (SEQ ID NO: 610) were reactive.

  In 1.4 ELISA, some specimens (3 from the serum population and 6 from the plasma population) showed absorption values exceeding 0.300 (S / N ratio 6 to 12, close to the expected truncation value) More than that). When retested, all nine of these specimens showed S / N ratios lower than 10.0. The average absorption values for serum and plasma samples were 0.072 (SD = 0.052) and 0.108 (SD = 0.062), respectively. The cutoff value in 1.4 ELISA was calculated by the above formula. That is, the cut-off values for the serum and plasma populations were 0.436 and 0.542, respectively. One specimen in the serum population was considered reactive in the first test but was negative when duplicated and retested. Two specimens in the plasma population were also initially reactive in the test but negative in the retest. A population of second normal samples 200 consisting of 100 plasma samples and 100 serum samples was tested. When tested based on the previously proposed truncation, 2 plasma and 2 serum samples were repeatedly reactive.

  In the 4.1 ELISA, the mean absorption values for the serum specimen and the plasma specimen were 0.070 [standard deviation (SD) is 0.037] and 0.063 (SD = 0.040), respectively. Thus, in the 4.1 ELISA, provisional truncation values for serum and plasma were 0.329 and 0.511, respectively. As described above, the cut-off value can also be determined based on the absorption value of the negative control: a specimen having an S / N ratio of more than 10.0 was regarded as reactive. Using this truncation value, 0 out of 100 plasma samples tested for antibodies to 4.1 (SEQ ID NO: 612) and 0 out of 100 serum samples are reactive in the first test. It was said.

  Additional 760 plasma donors were provided by the Interstate Blood Bank (Ohio) and the 1.7 and 1.4 ELISA studies were conducted. A total of 9 specimens were repeatedly reactive. None of the specimens were reactive simultaneously in both ELISAs, and all 9 specimens were repeatedly reactive in the 1.4 ELISA.

  A total of 960 specimens from plasma or blood donors residing in the United States were examined for the presence of antibodies to 1.7 and 1.4 proteins. A total of 13 specimens were repeatedly reactive in the 1.4 ELISA. None of the specimens were repeatedly reactive in the 1.7 ELISA.

  In summary, these data indicate the following: That is, in one or more ELISA assays using recombinant antigens from HGBV-B, according to previous ELISA results, 13 of 960 samples collected from blood donors in the United States were antibody reactive. From this result, it seems that HGBV may be a so-called endemic disease unique to the United States.

  These data are shown in Table 16.

J. et al. Samples considered “at risk” for hepatitis Data from this study are shown in Table 16.

(I) Specimens from West Africa Of the 1300 specimens obtained from West Africa, a total of 181 specimens were repeatedly reactive in one or more ELISAs. There was one sample that was repeatedly reactive in all three ELISAs. A total of 43 specimens were repeatedly reactive with 1.7 ELISA, 91 specimens were repeatedly reactive with 1.4 ELISA, and 51 specimens were repeatedly reactive with 4.1 ELISA.

  One of the 6 specimens that was repeatedly reactive in the 1.7 ELISA was reactive in the Western blot for the 1.7 protein (SEQ ID NO: 610). Of the 9 specimens that were repeatedly reactive in the 1.4 ELISA, 9 specimens (100%) were positive by Western blot for antibodies against the 1.4 protein (SEQ ID NO: 611). One specimen was positive by Western blot for both proteins. Of the 12 specimens that were repeatedly reactive in the 4.1 ELISA, 12 specimens (100%) were positive in the Western blot for the 4.1 protein (SEQ ID NO: 612).

  RT-PCR using primers from clone 4 for 3 specimens that were repeatedly reactive (including one specimen that was positive in 1.4 ELISA and one specimen that was positive in both ELISA and Western blot) The presence or absence of HGBV-RNA was examined as described above.

  The data obtained suggested that HGBV may be endemic in West Africa.

(Ii) Specimens from transvenous drug users (IVDU) Three out of 112 specimens were positive in 1.4 ELISA. Five specimens were reactive with 4.1 ELISA and three specimens were reactive with 1.7 ELISA. Two samples were positive in two or more ELISAs.

  Set 2: A total of 99 samples were collected from a group of intravenous drug users. This trial is part of what was done at Hines Veteran ’s Administration Hospital in Chicago, Illinois. None of the samples were reactive in the 1.7 or 4.1 ELISA. One sample was repeatedly reactive in 1.4 ELISA. The repeat-reactive specimen was examined for the presence of HGBV-RNA as described above by RT-PCR using primers from clone 4. This specimen was RT-PCR negative.

K. Table 16 summarizes data obtained from specimens collected from individuals with non-AE hepatitis .

  Various specimen populations were obtained from populations diagnosed with non-AE hepatitis and 1.7, 1.4, and 4.1 ELISAs were performed according to Example 15C. Samples used included: 180 samples from the Japanese clinic; 56 samples from the New Zealand clinic; 73 samples from the Greek clinic; 132 samples from the Egyptian clinic; 64 from the clinic in Texas, USA Samples (Set T); 72 samples from the Minnesota Research Center (Set M); 62 samples from the United States (Set # 1); 82 samples from the Pakistan clinic; 10 samples from the Italian clinic (some samples) Did not perform all feasible ELISA assays due to lack of serum).

(I) Japanese specimens These 180 specimens were from 85 different patients. Two of all 180 specimens were repeatedly reactive in 1.7 ELISA. The two reactive specimens were from two different patients. The two specimens were examined as described above by RT-PCR using primers from clone 4. There were no positive specimens.

  There were no specimens positive in the 1.4 ELISA.

  For the 4.1 ELISA, 7 out of 89 samples were repeatedly reactive in the 4.1 assay (Note: these 89 samples were obtained from 29 different patients). Of the reactive samples, 5 were from one patient. The remaining two specimens were from another single patient.

(Ii) New Zealand specimens A total of 4 specimens out of 56 specimens were repeatedly reactive in one or more of 1.7, 1.4, and 4.1 ELISAs. None of these specimens showed reactivity in two or more ELISAs. One specimen was repeatedly reactive with 1.7 ELISA and two specimens were repeatedly reactive with 1.4 ELISA. One specimen was repeatedly reactive in the 4.1 ELISA. PCR was performed on two repeatedly reactive specimens. As a result, both samples were negative. One specimen that showed repeated reactivity in 1.4 ELISA was also reactive with an antibody against HEV.

(Iii) Greek specimens A total of 5 specimens out of 73 specimens were antibody-reactive in 1.7 and / or 1.4 ELISA. These 73 samples were collected from a total of 11 patients. Two of the five samples with repeated reactivity showed repeated reactivity in both ELISAs, but these were collected from the same patient on separate days. Two specimens showing repetitive reactivity were examined by RT-PCR and were negative. None of these specimens had antibody reactivity in the 4.1 ELISA.

(Iv) Egyptian specimens A total of 11 specimens out of 132 specimens showed reactivity in 1.7, 1.4, or 4.1 ELISA. Eight specimens were positive in both 1.7 and 1.4 ELISA. Nine specimens were antibody reactive with 1.7 ELISA and 9 specimens were reactive with 1.4 ELISA. One specimen showed repeated reactivity in the 4.1 ELISA, but was negative in the 1.7 and 1.4 ELISA. One specimen that showed repeated reactivity in the 1.7 ELISA was negative for antibodies to the 1.7 recombinant protein (SEQ ID NO: 610) when examined by Western blot. Six out of nine specimens that showed repeated reactivity in the 1.4 ELISA were positive for antibodies against the 1.4 recombinant protein (SEQ ID NO: 611) by Western blot. Seven specimens that were repeatedly reactive were tested by RT-PCR. None of the samples showed reactivity. All 132 specimens were collected on 25 different individuals on different days. Eleven specimens that showed repeated reactivity were from five different individuals. In one of these individuals (patient # 101), the immune response was clearly similar to that observed with tamarin (Figure 31). Note that in FIG. 31, the ALT level was elevated when the doctor observed symptoms. Subsequent specimens had decreased ALT levels and antibodies were detected by 1.4 and 1.7 ELISA. The antibody response declined over the next few weeks, similar to the serum response situation observed with tamarin. An additional 3 patients (257, 260, and 340) showed a serum pattern similar to patient # 101 (see FIGS. 32-34). These data support the hypothesis that HGBV may be the causative agent in these hepatitis cases.

  Of the 7 samples from the above 4 patients, none of the samples was HGBV-RNA positive by RT-PCR. There are several possible reasons for this result. First, the stage of viremia may be very short, in which case the virus may have been removed from the serum by the day of the first blood draw. Secondly, since these specimens were transported from Egypt by ship, they were probably frozen, thawed or otherwise changed during the storage and transport process, resulting in the ability to detect HGBV-RNA. May have declined.

(V) Specimens from the United States (Set T)
None of the 64 specimens (Set T) from the United States were repeatedly reactive in 1.7, 1.4, 4.1 ELISA.

(Vi) Specimens from the United States (Set M)
Out of 72 samples (Set M) in the United States, a total of 4 samples showed repeated reactivity in one or more ELISAs. Two specimens showed reactivity in 1.7 and 4.1 ELISA. One sample was reactive only with 1.7 ELISA, and one sample was reactive only with 4.1 ELISA.

(vii) United States specimens (set 1)
Of 51 specimens of non-AE hepatitis United States specimens (Set 1), 3 specimens showed repeated reactivity in one or both ELISAs. One specimen was repeatedly reactive in both ELISAs. One specimen showed reactivity by 1.7 ELISA, and three specimens showed repeated reactivity by 1.4 ELISA. Samples that were positive in both ELISAs were positive by Western blot against 1.4 recombinant protein (SEQ ID NO: 22) but negative by 1.7 recombinant protein (SEQ ID NO: 23). One additional sample was positive by 1.4 ELISA and positive by Western blot against 1.4 recombinant protein (SEQ ID NO: 611). One specimen that showed repeated reactivity in the 1.4 ELISA was reactive to antibodies against HEV.

(viii) Pakistan specimens A total of 4 specimens out of 82 specimens were repeatedly antibody-reactive in 1.4 and / or 1.7 ELISA. None of the specimens showed reactivity in both ELISAs. Two specimens showed repeated reactivity with 1.7 ELISA, and two specimens showed repeated reactivity with 1.4 ELISA. Two specimens that were repeatedly reactive in the 1.4 ELISA were also reactive with antibodies to HEV. None of the 82 samples were positive in the 4.1 ELISA.

(Ix) Italy Samples None of the 10 samples showed repetitive reactivity in 1.7, 1.4, and 4.1 ELISA.

L. Statistical significance of the results of the serum response These data were obtained from three specific groups of the populations investigated for specific antibodies against the HGBV protein (ie, specimens that were repeatedly reactive with antibodies in 1.7, 1.4, or 4.1 ELISA). It shows that it can be detected in all categories. Serological results from various specimen categories (“low risk”, “at risk”, and non-AE hepatitis patients) were combined and analyzed for statistical significance by chi-square test. The data are significant when comparing serological prevalence of anti-HGBV in volunteer blood donors, including individuals considered “at risk” for exposure to HGBV, or individuals diagnosed with hepatitis of unknown classification It showed that there was a difference.

  In West African specimens, the serological prevalence was 13.9%, which was significantly higher under the p-value of 0.000 compared to the baseline group (Table 17). Similarly, there was a statistically significant difference in IVDU when the results were compared to volunteer donor data (p-value = 0.000). For countries including Japan, New Zealand, United States, Egypt, and Pakistan, there was a significant difference in the presence of antibodies in non-AE hepatitis patients compared to volunteer blood donors in the United States.

H. Summary These data suggest that the various ELISAs described herein may be useful for the diagnosis of human hepatitis in various regions, including Japan, New Zealand, the United States, Egypt, and Pakistan. These data appear to underestimate the serological presence of antibodies to HGBV in all categories of specimens tested. As additional HGBV epitopes are discovered and evaluated, the utility of tests based on the HGBV genome (s) is expected to become more important in diagnosing hepatitis in patients who are currently unable to make a diagnosis. The Note: In these initial studies, RT-PCR results were negative, but subsequent data found flavi-like virus sequences in the sera of seropositive individuals.

  As described above, there are two or more HGBV strains. These are considered to be within the scope of the present invention and are referred to as “hepatitis GB virus (HGBV)”.

(Example 16)
Serological studies of HGBV-A Recombinant protein purification procedure Bacterial cells expressing CKS fusion protein were frozen and stored at -70 ° C. Bacterial cells from each GBV-A construct were thawed and ground as described in Example 15 for the GBV-B construct. In addition, the recombinant protein was purified as described for GBV-B recombinant protein in Example 15.

  Fractions collected by this purification procedure were separated by electrophoresis and stained with Coomassie Brilliant Blue R250, with molecular weights of about 60 kD (CKS-1.5 / SEQ ID NO: 614), 65 kD (CKS-2.17 / sequence). No. 613), 55 kD (CKS-1.18 / SEQ ID NO: 390), and 66 kD (CKS-1.22 / SEQ ID NO: 390) were examined for the presence or absence of proteins. Fractions containing the relevant protein were pooled and examined again by SDS-PAGE.

  The immunogenicity and structural integrity of the pool fraction containing the purified antigen was determined by electron transfer to nitrocellulose and immunoblotting according to the method described in Example 13. Because there was no appropriate positive control, recombinant proteins were identified by their reactivity with monoclonal antibodies against the CKS portion of each fusion protein. When CKS-1.5 protein (SEQ ID NO: 614) was examined by Western blotting using an anti-CKS monoclonal antibody to detect a recombinant antigen, a single band was observed at about 60 kD. This result corresponds to the expected size of the CKS-1.5 protein (SEQ ID NO: 614). Similarly, CKS-2.17 protein (SEQ ID NO: 613), CKS-1.18 protein (SEQ ID NO: 390), and CKS-1.22 protein (SEQ ID NO: 390) were also estimated by immunoblotting. Had.

B. Procedure for coating polystyrene beads The proteins were dialyzed and the antigenicity of these proteins on polystyrene beads was evaluated as described in Example 15.

C. ELISA procedures for detecting antibodies against HGBV Various ELISAs were performed as described in Example 15.

D. Detection of HGBV-RNA in the Serum of Infected Individuals Samples that showed repeated reactivity in each ELISA were examined for the presence or absence of HGBV-RNA as described in Section D of Example 15.

E. All of the serum images of tamarin In the ELISA assay using CKS1.5 protein, CKS2.17 protein, CKS1.18 protein, or CKS1.22 protein derived from HGBV-A genome, all sera from tamarin show specific immune reaction There wasn't. However, as described in the previous examples, HGBV-A RNA was detected in some of the infected tamarins (see Summary for Tamarin Serum Image in Example 15).

F. Experimental Procedure for Serological Studies on Human Populations Example 15 evaluated the presence of antibodies against HGBV-B in various human populations by ELISA using recombinant antigens derived from HGBV-B. Thereafter, for many of the same specimens, 1.5 ELISA using CKS1.5 recombinant protein (SEQ ID NO: 614), 2.17 ELISA using CKS2.17 recombinant protein (SEQ ID NO: 613), CKS1.18 recombinant protein The presence or absence of an antibody against HGBV-A was examined by 1.18 ELISA using (SEQ ID NO: 390) and ELISA using CKS1.22 recombinant protein (SEQ ID NO: 390). Each protein was used as coated on a solid phase as in Example 15. As described in Example 15, all 5 convalescent tamarins inoculated with HGBV showed a specific antibody response to the HGBV-B recombinant protein but persisted only for a short period of time (1 .7, 1.4, and 4.1 ELISA). None of the tamarins showed detectable antibody responses in the 1.5, 2.17, 1.18, or 1.22 ELISA, but some of the human specimens from West Africa had these recombinants on Western blots. One of the samples taken on day 22 of hepatitis onset that showed a specific antibody response for one or more of the proteins and was provided by a surgeon (supplier of GB drug) was 2.17 when tested by Western blot. A specific antibody reaction against the recombinant protein was shown (see Example 3). In this example, the usefulness of the 1.5, 2.17, 1.18, and 1.22 ELISAs in detecting antibodies in various human populations was evaluated.

G. Determination of truncation values The truncation values in the 1.5, 2.17, 1.18, and 1.22 ELISAs were determined as described in Example 15.

H. Supplementary testing As described in Example 15, specimens that initially showed reactivity were in principle retested. If a sample showed reactivity for the second time, an additional test (eg Western blot) was performed to further supplement the ELISA data. In order for the Western blot results to be considered positive, observable bands were 60 kD for 1.5 protein (SEQ ID NO: 614), 65 kD for 2.17 protein (SEQ ID NO: 613), 1.18. It needs to be detected at 55 kD for the protein (SEQ ID NO: 390) and 66 kD for the 1.22 protein (SEQ ID NO: 390). Western blots were not optimized to match or exceed the sensitivity of ELISA, so ELISA data were not immediately discarded when negative results were obtained. However, if a positive result is obtained, it reinforces the reactivity detected by ELISA.

  As described in Example 15, RT-PCR using primers (performed as described in Example 15) was performed on a sample that was repeatedly reactive and had a sufficient amount, and the HGBV specificity in serum was determined. Nucleotide sequences may be identified.

I. Serum data for 252 low-risk specimens A total of 252 plasma specimens were obtained from the Interstate Blood Bank, Ohio and tested for antibodies by 1.5 ELISA using 1.5 recombinant protein (SEQ ID NO: 614). . The average absorption value in this population was 0.036 (SD = 0.022). The truncation value was calculated to be 0.168. This corresponds to S / N ratio = 10.0. 760 plasma samples (including 252 samples used to determine the cut-off value) were examined for the presence of antibodies by 1.5 ELISA. None of the specimens showed repeated reactivity. In addition, 100 plasma samples were obtained from southeastern Wisconsin and examined for the presence of antibodies by 1.5 ELISA. None of the specimens showed repeated reactivity.

  Thus, no evidence of the presence of antibodies to the 1.5 protein was found in blood donors in the United States.

  200 specimens were obtained from Wisconsin blood donors and tested for the presence of antibodies by 2.17 ELISA using a 2.17 recombinant protein (SEQ ID NO: 60). The average absorption value in this population was 0.058 (SD = 0.025). The truncation value was calculated to be 0.208. This substantially corresponds to the S / N ratio = 10.0. One of the specimens was repeatedly reactive. Thus, the serological seroprevalence in US blood donors (N = 200) was relatively low.

  The same 200 specimens as described in the above paragraph were examined for the presence of antibodies by 1.18 and 1.22 ELISA. None of the specimens repeatedly showed reactivity. Thus, for samples from volunteer blood donors, there was no evidence of antibody positivity for HGBV-A protein as determined from 1.5, 2.17, 1.18, and 1.22 ELISAs. .

J. et al. Samples considered “at risk” for hepatitis Table 18 summarizes the data from this study.

(I) Specimens from West Africa Of the 1300 specimens, 58 specimens showed reactivity in 1.5 ELISA. Of the 18 samples that were repeatedly reactive, 12 samples were positive by Western blot for an antibody against 1.5 protein (SEQ ID NO: 614). Forty-three of the 817 samples were reactive in the 2.17 ELISA. For these repeatedly reactive specimens, Western blots for antibodies against the 2.17 protein (SEQ ID NO: 613) were not performed.

  Six of the 817 samples were reactive in the 1.22 ELISA. Nine of the 353 samples were reactive in the 1.18 ELISA. When 21 specimens reactive by 2.17 ELISA were tested by Western blot, 13 specimens were reactive. Eight specimens that were repeatedly reactive by 1.18 ELISA were positive by Western blot.

  These data suggest that HGBV may be endemic in West Africa.

(Ii) Specimens from intravenous drug users A total of 112 specimens were collected from a group of intravenous drug users. This trial is part of a study conducted at the Hynes Veterans Hospital in Chicago, Illinois. One sample was repeatedly reactive in 2.17 ELISA. One additional sample showed reactivity in the 1.18 ELISA. None of these specimens were positive in the 1.5 or 1.22 ELISA.

K. Data obtained from specimens collected from individuals with non-AE hepatitis are summarized in Table 18.

  Various specimen populations (described in Example 15K) were obtained from non-AE hepatitis populations and 1.5, 2.17, 1.18, and 1.22 ELISA (described in Example 15C) were performed. . Some ELISAs were not performed in full ELISA due to lack of serum.

(I) Japanese specimens A total of 4 specimens out of 89 specimens showed repeated reactivity in 1.5 ELISA. Three of them were from one individual and one was from another individual. One specimen that was negative in the 1.5, 1.18, and 1.22 ELISAs was reactive in the 2.17 ELISA. None of the samples showed reactivity in the 1.18 ELISA. These specimens were not examined by 1.22 ELISA.

(Ii) New Zealand specimens None of the 56 specimens showed reactivity in 1.5 ELISA. These specimens were not examined by 2.17 ELISA, 1.18 ELISA, or 1.22 ELISA.

(Iii) Greek samples None of the 67 samples (from 10 patients) showed reactivity in the 1.5, 2.17, or 1.22 ELISA.

(Iv) Egyptian specimens None of the 132 specimens were reactive to 1.5 ELISA. Of the 132 specimens that could be examined, a total of 7 specimens were reactive in the 2.17 ELISA. These specimens were from 25 patients with acute non-AE hepatitis. Of the 25 patients, 3 were 2.17 ELISA and were seropositive when more than 1 day after the onset of hepatitis. There were no analytes reactive in the 1.18 or 1.22 ELISA.

(V) Specimens from the United States (Set M)
None of the 72 specimens showed reactivity in 1.5 ELISA. Three of the 72 samples showed reactivity in the 1.18 ELISA. Two samples showed reactivity in 2.17 ELISA and 4 samples showed reactivity in 1.22 ELISA. Two samples showed reactivity in one or more ELISAs.

(Vi) Specimens from the United States (Set T)
None of the 64 specimens showed reactivity in the 1.5, 1.22, or 2.17 ELISA. One specimen was reactive at 1.18.

(Vii) United States specimens (Set 1)
A total of 3 out of 62 samples showed reactivity in one or more GBV-A ELISAs. There was one specimen that showed repeated reactivity in both 2.17 and 1.22 ELISA. There was one reactive sample in the 2.17 ELISA only, and another one was reactive only in the 1.22 ELISA. There were no analytes reactive to the 1.5 or 1.18 ELISA.

  As mentioned above, there are two or more HGBV strains, or two or more different viruses can be represented by the sequences described herein. They are considered within the scope of the present invention and are referred to as “hepatitis GB virus (HGBV)”.

L. Statistical significance of the results of the serum reaction These data indicate that specific antibodies against the HGBV-A protein (ie, specimens that repeatedly react with antibodies in the 1.5, 2.17, 1.18 and 1.22 ELISAs) Detected in individuals who are considered “at risk” for exposure to and those diagnosed with non-AE hepatitis, but not so frequently from volunteers and blood sellers in the United States It was shown that In Table 19, the results of serum responses from various categories of specimens (“Low Risk”, “At Risk”, and non-AE hepatitis patients in Table 18) were combined and analyzed for statistical significance by chi-square test. . Table 18 summarizes the serological presence of antibodies against HGBV protein in the total number of specimens examined, but unlike this, the data in Table 19 show results from different individuals (persons). For the GBV-A ELISA, the serological presence of anti-HGBV is compared between volunteer blood donors and individuals considered “at risk” for exposure to HGBV (West Africa, but not IVDU) , There is a significant difference (p value = 0.000). In addition, there was a statistically significant difference in the serological presence of antibodies to HGBV-A in non-AE hepatitis patients in Egypt and the United States when compared to volunteer blood donors. This data suggests an association between exposure to HGBV-A and non-AE hepatitis. Note: In these initial studies, RT-PCR results were negative, but subsequent data found flavi-like virus sequences in the sera of seropositive individuals (see Example 19).

M.M. Summary These data suggest that the ELISA described in the present invention is useful for detecting antibodies in individuals living in West Africa and in individuals with non-AE hepatitis. The risk of suffering from hepatitis is relatively high in West African populations. Nearly 85% of people are seropositive for antibodies against hepatitis B virus and about 5% are positive for antibodies against hepatitis C virus. These data appear to underestimate the serological presence of antibodies to HGBV in all categories of specimens tested. As additional HGBV epitopes are discovered and evaluated, the utility of tests based on the HGBV genome (s) is expected to become more important in diagnosing hepatitis in patients who are currently unable to make a diagnosis. The

(Example 18)
Identification of GB-related viruses in humans Epitopes of both theoretical HGBV-A and HGBV-B were identified (Example 3). These were used as serological markers to screen human serum and plasma samples (Examples 5 and 6). There was a significant correlation between serological activity for some of these markers and the incidence of non-AE hepatitis, which is the pathogenesis of non-AE hepatitis in humans in HGBV-B Suggesting the substance (Example 5G). However, when GB human serum was analyzed by Western blotting, no reactivity to the HGBV-B epitope was shown (Example 3). Instead, at least one HGBV-A epitope was identified in GB human serum. This suggests that HGBV-A was a pathogen of hepatitis in GB. Neither HGBV-A nor HGBV-B sequences were identified by RT-PCR in patients with non-AE hepatitis (Example 5E). Thus, there remains a need to establish evidence of HGBV-A and / or HGBV-B infection in humans suffering from non-AE hepatitis.

  Several factors can contribute to the failure to identify HGBV-A and / or HGBV-B sequences in human serum or plasma sources. First, we tested a limited number of HGBV-A seropositive and / or HGBV-B seropositive samples by RT-PCR, and many of these samples have an unknown storage history. Thus, the viral RNA present in these samples may have been affected by improper storage. Second, GB infection appears to heal naturally. Therefore, the duration of the GB sequence present in the serum of an infected individual may be very narrow. That is, the chance of obtaining a serum sample containing the GB sequence will be extremely low. Finally, a limited number of PCR primer sets were used in searching for HGBV-A and / or HGBV-B sequences. Since HGBV-A and / or HGBV-B are RNA viruses, they tend to have a high mutation rate (Holland et al. (1982) Science 215: 1577-1585). That is, the HGBV-A and / or HGBV-B sequence in the tested human serum is different from the sequence of our PCR primer, so that HGBV-A and / or HGBV-B may not be detected.

  Highly conserved of HGBV-A and / or HGBV-B to indicate that these viruses may have been disrupted in our PCR studies due to the genomic variability of HGBV-A and / or HGBV-B Denatured PCR primers were designed and arranged in accordance with the NS3-like region (see FIG. 17). This is because these highly conserved regions perform the necessary functions in the viral replication cycle. Thus, these sequences should be conserved in HGBV-A and / or HGBV-B variants. PCR primers designed within this region should be able to detect HGBV-A and / or HGBV-B genomic RNA by RT-PCR. In addition, designing degenerate PCR primers capable of specifically amplifying HGBV-A, HGBV-B and HCV sequences will amplify sequences obtained from viruses associated with HGBV-A, HGBV-B and HCV We decided that it might be possible. Thus, less serum response is detected in human serum and plasma samples (Examples 5 and 6) due to the presence of antibodies that cross-react with antigens of separate HGBV-A related viruses or HGBV-B related viruses. If so, we will be able to obtain sequences from these GB-related viruses. [This is similar to the experimental procedure that Nicole and his colleagues recently used to identify special hantaviruses associated with an acute respiratory disease pandemic in the southwestern United States. Nicole et al., Science 262: 914-917 (1993)]

B. Cloning of the NS3-like region of hepatitis GB virus C (HGBV-C) In viral infection models, viremia occurs early in the infection, which often correlates with the detection of IgM antibodies to viral proteins. As described in Examples 5 and 6, specimens in which IgG antibodies against recombinant proteins derived from HGBV-A and HGBV-B were detected were immunoreactive in ELISA. Additional ELISAs were performed to see if IgM antibodies were detected for these proteins. Some of the seropositive specimens of West African individuals (Example 5E-i) were reactive with IgM antibodies to the recombinant protein (data not shown). These specimens were likely to contain viruses. In addition, for specimens in which IgM antibody against HGBV-A or HGBV-B recombinant protein is not detected in HGBV-A positive and HGBV-B positive Egyptian individuals (Example 5F-vii) suffering from acute hepatitis Also, tests were performed because acute liver disease may be associated with the presence of virus. The nucleic acids of these samples were subjected to “hemi-nested” RT-PCR using degenerate oligonucleotide primers that amplify HGBV-A, HGBV-B, and HCV-1 sequences. The instrument used was a GeneAmp (registered trademark) RNAPCR kit (manufactured by PerkinElmer), which was used according to the manufacturer's instructions. Briefly, the first set of amplifications was random-primed reverse transcription of nucleic acids extracted with ₂ mM MgCl ₂ and 1 μM primers ns3.1-s and ns3.1-a (SEQ ID NOs: 671 and 672, respectively). The reaction was performed on the cDNA product. The reaction was subjected to 3 cycles of denaturation-annealing-extension [(94 ° C, 30 seconds; 37 ° C, 30 seconds; 72 ° C over 2 minutes; 72 ° C, 30 seconds), then (94 ° C, 30 seconds; 37 cycles of 55 ° C., 30 seconds; 72 ° C., 30 seconds)] was performed 40 times, and finally stretched at 72 ° C. for 10 minutes. When the reaction was completed, it was stored at 4 ° C. The second set of amplifications used 4% of the first PCR product as a template, except that ns3.1-s and ns3-a (SEQ ID NOs: 671 and 673, respectively) were used as “heminested” primer sets. Same as above. The first and second sets of PCR products were analyzed by gel filtration.

  One sample in West Africa had a PCR product derived from a blurred half-nested reaction at approximately 386 bp (expected size of HGBV-A, HGBV-B, or HCV product). This product was cloned into the pT7 blue T-vector plasmid (Novagen) described in books in this technical field. Sequences obtained from this clone (GBcontigC [GB-C], SEQ ID NO: 673, residues 2274-2640) were converted to GBcontigA (GB-A, SEQ ID NO: 163, residues 4438-4804), GBcontigB (GB-B, SEQ ID NO: 393, residues 4218-4587), and HCV-1 (SEQ ID NO: 398). FIG. 36 shows the nucleotide sequences of these sequences, while Table 20 shows the percent homology between these sequences.

As shown in FIG. 36 and Table 20, as a result of comparison of nucleotides of GB-A, GB-B, and HCV-1, these sequences are 47.9 to 61.66% similar to each other. This is not particularly surprising considering the conserved amino acid residues present in the NTP binding helicases of these viruses (Example 2B3, FIG. 17A). When comparing the nucleotides of the NS3 PCR product obtained from the West African sample (GB-C, SEQ ID NO: 673, residues 2274-2640) and nucleotides of other viruses, GB-A (SEQ ID NO: 163, residues 4438-4804), Although related to the NS3 sequence from GB-B (SEQ ID NO: 393, residues 4218-4587), and HCV-1 (SEQ ID NO: 398), it has been suggested that they are distinctly different. BLASTIN and BLASTX analysis of the nucleic acid and protein database in the Wisconsin Sequence Analysis Package (version 8) and GB-C (SEQ ID NO: 673, residues 2274-2640) showed that some of the strains of HCV have sequence identity. It turns out that there is a limit. The highest P values for nucleotides and amino acids (ie, the probability of accidental sequence alignment) were 1.9 × 10 ⁻²⁰ and 5.3 × 10 ⁻³¹ respectively (data not shown). Taken together, these data indicate that GB-C (SEQ ID NO: 673, residues 2274-2640) is a unique GB associated with HGBV-A, HGBV-B, and HCV, which we will label here HGBV-C. Suggesting that it may be derived from other viruses.

C. GB-C uses PCR primers for the exogenous GB-C sequence, and this sequence is human, rhesus monkey, S. cerevisiae as described above, ie, eg, by the procedure described in Example 6B. cerevisiae and E. coli. It was determined whether it was detected in the genome of E. coli. PCR was performed using Perkin-Elmer-Cetus GeneAmp® and essentially following the manufacturer's instructions. That is, 300 ng genomic DNA was used for each 100 μl reaction. PCR primers (SEQ ID NO: 675 and 676) were used at a final concentration of 1.0 μM. PCR was performed for 40 cycles (94 ° C., 30 seconds; 55 ° C., 30 seconds; 72 ° C., 30 seconds), and then extended at 72 ° C. for 10 minutes. PCR products were separated by agarose gel electrophoresis, nucleic acid was directly stained with ethidium bromide, visualized by UV irradiation, transferred to Hybond-N + nylon filter by Southern transfer, and then hybridized with a radiolabeled probe. FIG. 37 shows a PhosphoImage (Molecular Dynamics, Sunnyvale, Calif.) Obtained by Southern blotting of a PCR product after hybridization with a radiolabeled probe prepared from GB-C (SEQ ID NO: 673, residues 2274-2640). ). GB-C (SEQ ID NO: 673) detects ~ 350fg (1 genome copy equivalent, lane 5) and ~ 35fg (0.1 genome copy equivalent, lane 6) of the GB-C plasmid template in 300ng human genomic DNA. Except for humans (FIG. 19, lane 1), rhesus monkeys (lane 2), cerevisiae (lane 3), E. coli. It was not detected in each genomic DNA of E. coli (lane 4). (Lane 7 contains ~ 3.5 fg [0.01 genomic copy equivalent] of PCR product from GB-C plasmid template in 300 ng of human genomic DNA.) Thus, 0.1 genomic copy equivalent can be detected. Even using genomic PCR, GB-C (SEQ ID NO: 673) is human, rhesus monkey, S. cerevisiae. cerevisiae and E. coli. It cannot be detected in the genome of E. coli. This result is consistent with the expectation that GB-C (SEQ ID NO: 673) is of exogenous (ie viral) origin.

D. GB-C was subjected to RT-PCR on additional HGBV-A and HGBV-B immunoreactive human serum samples that could be detected in additional human serum samples and examined for the presence of GB-C sequences. As described in Example 7, nucleic acids extracted from serum samples were reverse transcribed with random hexamers and amplified for 35-40 cycles on cDNA (94 ° C., 30 seconds; 55 ° C., 30 seconds; 72 ° C. 30-90 seconds) and then stretched at 72 ° C. for 10 minutes. PCR primers specific for GB-C (g131-sl and g131-al, SEQ ID NOs: 675 and 676) were used at a concentration of 1.0 μM. PCR products were separated by agarose gel electrophoresis, nucleic acid was directly stained with ethidium bromide, visualized by UV irradiation, transferred to a Hybond-N + nylon filter by Southern blot, and then hybridized with a radiolabeled probe. A total of 48 HGBV-immunopositive samples from West Africa were tested. Eight samples from West Africa, including the first sample in which GB-C was identified, were found by RT-PCR to be positive for GB-C sequences. A total of 10 West African serum samples that were negative for GB serotest were tested, but none had detectable GB-C sequences. PCR products from 4 positive samples were cloned and sequenced as described above. After examining 156 nucleotides, two of the four clones tested were identical to the GB-C sequence (SEQ ID NO: 673, residues 2274-2640) and two clones (SEQ ID NOs: 677 and 678) were GB-C. (SEQ ID NO: 673, residues 2274-2640) and 88.4% and 83.6% homologous sequences (FIG. 38). However, despite the divergence at the nucleotide level, it was expected that each clone was very similar and only one amino acid difference was seen in the expected translation of SEQ ID NO: 678. Showed in translation.

  Additional serum samples were obtained from non-AE hepatitis individuals from Greece, Egypt and the United States and tested for GB-C sequences as described above. None of these samples contained the GB-C sequence. There are several possible reasons why no GB-C sequence was detected in the sample (see theory section above). However, the sequence changes described above between GB-C (SEQ ID NO: 673, residues 2274-2640) and two GB-C variants (SEQ ID NOs: 678 and 677) are from closely related West Africa. If the HGBV-Cs in the GGBV-C may differ by 15.1% at the nucleotide level, the GB-C specific PCR primers (g131-sl, g131-al, SEQ ID NOs: 675 and 676) will be separated geographically This suggests that it may not hybridize sufficiently with the body GB-C virus to produce a detectable PCR product. In this case, GB-C sequences may be detectable in West African serum samples by PCR primers designed in a more highly conserved region of the genome (5'UTR).

E. Extension of HGBV-C sequences Additional GB-C sequences were obtained by the PCR walking method described in Example 2A. That is, total nucleic acids were extracted from West African human serum used to initially identify GB-C (SEQ ID NO: 673, residues 2274-2640). This nucleic acid was reverse transcribed as described above. The resulting cDNA was amplified in a 50 μl PCR reaction (PCR1) according to the method described by Sorensen et al. Except that ₂ mM MgCl ₂ was used. The reaction was subjected to 35 cycles of denaturation-annealing-extension steps (94 ° C., 30 seconds; 55 ° C., 30 seconds; 72 ° C., 90 seconds) followed by extension at 72 ° C. for 10 minutes. The biotinylated product was isolated by streptavidin coated paramagnetic beads (Promega) according to the method described by Sorensen et al. The streptavidin purified product was then subjected to nested PCR (PCR2) by denaturation-annealing-extension for a total of 35 cycles as described above according to the method described by Sorensen et al. Table 21 shows the obtained product and the PCR primers used to purify it.

  In addition, a 1.3 kb product (C.16) was generated using oligonucleotide primers (SEQ ID NOs: 669 and 670) under the conditions of PCR1 described above. This product was isolated from an agarose gel with those listed in Table 21 and cloned into pT7 blue T-vector plasmid (Novagen) by methods well known in the art.

  The cloned product was sequenced as described in Example 5. The sequence was assembled by the program in the GCG package (version 7). An outline of the constructed contig is shown in FIG. GB-C is 9034 bp in length and its full length was sequenced. This is shown in SEQ ID NOs: 400-606. These SEQ ID NOs correspond to three forward translation frames.

(Example 19)
CKS-based expression and detection of immunogenic HGBV-C polypeptides The HGBV-C sequences obtained in the walking test described in Example 17 (Table 13) are shown in Table 22 (10 units, NEB) and cloned into CKS expression vectors pJO200, pJO201 and pJO202. Two additional PCR clones, C3 / 2 and C8 / 12, were also expressed (FIG. 39). PCR product C3 / 2 was generated with the primer of SEQ ID NO: 681, and PCR product C8 / 12 and the complement of SEQ ID NO: 685 were generated using the primer (SEQ ID NO: 693 and its complement) as described in Example 9. . This PCR product was cloned into pT7 blue as described above, then released with the restriction enzymes listed in Table 22, and cloned into the above pJO200, pJO201 and pJO202.

  Two human sera whose presence of antibodies to one or more CKS / HGBV-A or CKS / HGBV-B fusion proteins was suggested by a 1.7, 4.1, or 2.17 ELISA (see Examples 15, 16) Selected and analyzed by Western blot. One of these sera (240D) is from a non-AE hepatitis (Egypt) individual, while the other (G8-81) is a West African individual “at risk” for exposure to HGBV. (See Example 15). CKS / HGBV-C fusion protein was expressed and transferred to a nitrocellulose sheet as described above. Pre-block the blot as described, and 10% E. coli. One human serum sample diluted 1: 100 in blocking buffer containing E. coli lysate and 6 mg / ml XL1-blue / CKS lysate was incubated overnight. The blot was washed twice with TBS, reacted with HRPO-conjugated goat anti-human IgG and developed as described above. The results are shown in Table 22.

  Several HGBV-C proteins showed reactivity with one or the other of these two sera, and three of them (C.1, C.6, C.7) were selected for ELISA assay (Examples). 20). Thus, it was found that samples already known to be reactive with HGBV-A and / or HGBV-B protein also react with HGBV-C protein. The reactivity with multiple proteins from the three HGBV viruses may be due to cross-reactions resulting from the sharing of epitopes between the viruses. Alternatively, this may be due to multiple virus infections, and possibly other unidentified factors.

ａ：ＰＣＲ産物は、前出の各表および実施例に記載のもの。
ｂ：制限分解はｐＴ７ブルーＴ−ベクターからＰＣＲフラグメントを遊離させるために行った。ＮＤ＝実施せず。

a: PCR products are those described in the above tables and examples.
b: Restriction digestion was performed to release the PCR fragment from the pT7 blue T-vector. ND = not implemented.

(Example 20)
Serological studies of GBV-C Recombinant protein purification procedure Bacterial cells expressing CKS fusion protein were frozen and stored at -70 ° C. Bacterial cells from each GBV-C construct were thawed and disrupted as described in Example 15 for the GBV-B construct. In addition, the recombinant protein was purified as described for GBV-B recombinant protein in Example 15.

  Fractions collected by this purification procedure were separated by electrophoresis and stained with Coomassie Brilliant Blue R250, with molecular weights of about 75 kD (CKS-C.1 / SEQ ID NO: 404), 71 kD (CKS-C.6 / sequence). No. 404) and 49 kD (CKS-C.7 / SEQ ID No. 404) were examined. A band indicating the protein of the expected molecular weight is CKS-C. 6 and CKSC. Observed for 7 recombinant proteins. CKS-C. For one protein, a band was observed at a position corresponding to a molecular weight of 62 kD, not the expected molecular weight of 75 kD. It is unclear why the expected and observed bands are different. Fractions containing the protein of interest were pooled and examined again by SDS-PAGE.

  The immunogenicity and structural integrity of the pool fraction containing the purified antigen was determined by electron transfer to nitrocellulose and immunoblotting according to the method described in Example 13. Because there was no appropriate positive control, recombinant proteins were identified by reactivity with monoclonal antibodies against the CKS portion of each fusion protein. CKS-C. When Western blotting was performed using an anti-CKS monoclonal antibody to detect a recombinant antigen of one protein (SEQ ID NO: 404), a single band was observed at about 65 kD. This result is shown in CKS-C. It is different from the expected size of 1 protein (SEQ ID NO: 404) of 75 kD. In addition, CKS-C. 6 protein (SEQ ID NO: 404) and CKS-C. 7 protein (SEQ ID NO: 404) had an expected size as a result of examination by immunoblotting.

B. Procedure for coating polystyrene beads The above proteins were dialyzed and the immunogenicity of these proteins on polystyrene beads was evaluated as described in Example 15.

C. ELISA procedures for detecting antibodies against HGBV Various ELISAs were performed as described in Example 15.

D. Detection of HGBV-RNA in the Serum of Infected Individuals HGBV-RNA was examined according to the description in Section D of Example 15 for specimens that repeatedly showed reactivity in each ELISA.

E. Serum image of tamarin CKSC derived from HGBV-C genome. 1 protein, CKSC. 6 protein, or CKSC. In an ELISA assay using 7 protein, none of the sera from tamarin showed a specific immune response. See the description of the serum image of tamarin in Example 15.

F. Supplemental Testing As described in Example 15, specimens that initially showed reactivity were typically retested. If a sample is repeatedly reactive, additional tests (eg, Western blots) can be performed to further supplement the ELISA data. In order for the Western blot result to be considered positive, an observable band was obtained from C.I. 65 kD for one protein (SEQ ID NO: 404), C.I. 71 kD for 6 proteins (SEQ ID NO: 404), or C.I. It needs to be detected at 49 kD for 7 proteins (SEQ ID NO: 404). The Western blot was not optimized to match or exceed the sensitivity of the ELISA, so the ELISA data was not immediately discarded even if a negative result was obtained. However, a positive result reinforces the reactivity detected by ELISA.

  As described in Example 15, for a sample having a sufficient amount of repetitive reactivity, RT-PCR (performed using the primers corresponding to SEQ ID NOs: 8 and 9 according to the description of Example 10). HGBV-C specific nucleotide sequences in serum can be identified.

G. Experimental Procedure Example 15 utilized ELISA using recombinant antigens from HGBV-B to evaluate the presence of antibodies against HGBV-B and HGBV-A in various human populations. As described in Example 14, CKS-C. C. using a recombinant protein (SEQ ID NO: 404). 1 ELISA, CKS-C. C.6 using recombinant protein (SEQ ID NO: 404). 6 ELISA, and CKS-C. C.7 using recombinant protein (SEQ ID NO: 404). Using 7 ELISA, testing for antibodies against HGBV-C was performed on many of the same specimens. As described in Example 15, all five of the convalescent tamarins inoculated with HGBV had HGBV-B recombination (as detected using the 1.7, 1.4 and 4.1 ELISAs). Produced specific but short-lived antibodies that respond to proteins. C. 1, C.I. 6 and C.I. None of the tamarins produced a detectable antibody that responded to the 7 ELISA, but some of the human specimens were C. cerevisiae when tested by Western blotting (see Example 13). 1, C.I. 6 and C.I. 7 Produced specific antibodies that react with the recombinant protein. In this example, C.I. for detecting antibodies in various human populations. 1, C.I. 6 and C.I. The usefulness of 7 ELISA was evaluated.

H. Determination of truncation value 1, C.I. 6 and C.I. The 7 ELISA truncation value was determined as described in Example 15.

I. Serological data obtained using low-risk specimens A population containing 100 sera and 100 plasma was obtained from donors of healthy volunteers living in southwestern Wisconsin. CKS-C. 1 (SEQ ID NO: 404) protein, C.I. CKS-C.6 in 6 ELISA. 6 (SEQ ID NO: 404) protein, and C.I. CKS-C. Antibodies against three recombinant proteins from HGBV-C containing the 7 (SEQ ID NO: 404) protein were tested.

  C. For 1 ELISA, the mean absorption values of serum and plasma samples were 0.049 (standard deviation (SD) = 0.040) and 0.038 (SD = 0.029), respectively. The cutoff values for serum and plasma were calculated as 0.214 and 0.286, respectively. As noted above, the truncation value can also be expressed as a function of the absorption value of the negative control, and specimens with S / N values greater than 10.0 were considered reactive. Using this cut-off value, 100 plasma specimens had C.I. None of the antibodies against one protein (SEQ ID NO: 404) have been found to have an initial or repeated reactivity. Initial reactivity and repetitive reactivity were recognized in the antibody against 1 protein (SEQ ID NO: 404).

  C. For 6 ELISA, the mean absorption values for serum and plasma samples were 0.102 (standard deviation (SD) = 0.046) and 0.105 (SD = 0.047), respectively. A cut-off value was set so that a specimen having an S / N value of 10 or more was judged to be reactive. Using this truncation value, 3 specimens (2 from the serum population and 1 from the plasma population) had C.I. Repeated reactivity was found against the antibody against 6 proteins (SEQ ID NO: 404).

  C. For 7 ELISA, the mean absorption values of serum and plasma samples were 0.061 (standard deviation (SD) = 0.040) and 0.050 (SD = 0.555), respectively. A cut-off value was set so that a specimen having an S / N value of 10 or more was judged to be reactive. Using this truncated value, C.I. None of the specimens were considered to be repeatedly reactive with the antibody against 7 protein (SEQ ID NO: 404).

  Therefore, about 1% of American blood donors (N = 200) have C1, C6 or C.I. It has been demonstrated that antibodies against 7 proteins exist.

J. et al. Samples considered “at risk” for hepatitis Data from this study are summarized in Table 23.

(I) Specimens from West Africa A total of 20 out of 137 specimens were reactive in one or more of the ELISAs using GBV-C protein. A total of 12 of the 97 are C.I. 1 ELISA was found to be repeatedly reactive, 3 out of 52 were C.I. In 6 ELISAs, it was found to be repeatedly reactive and 5 out of 137 specimens were C.I. Repeated reactivity was observed in 7 ELISA. C. Three of the samples that were reactive in 1 were tested by Western blot and found to be reactive.

  These data suggest that HGBV is endemic in West Africa.

(Ii) Samples of intravenous drug users As part of a study conducted at the Hynes Veterans Hospital in Chicago, Illinois, a total of 112 samples were obtained from a group of intravenous drug users. A total of 2 out of 112 specimens were repeatedly reactive with one or more proteins. One specimen is C.I. It was found to be repeatedly reactive in 1 ELISA and one specimen was C.I. It was found to be repeatedly reactive in 7 ELISA. C. None of the specimens were positive in 6 ELISA.

K. Samples obtained from individuals with non-AE hepatitis Data from this study are summarized in Table 23.

  Various specimen populations (described in Example 15.K) were obtained from individuals with non-AE hepatitis and 1.5, 2.17, 1.18, and 1.22 ELISA (described in Example 15.C). ). Due to the insufficient amount of specimen, not all specimens could be tested in all ELISAs.

(I) Japanese specimens Among the 89 specimens in total, C.I. None were found to be repeatedly reactive in 1 ELISA. Due to the small amount of specimen, C6 or C.I. No testing for antibodies in the 7 ELISA was performed.

(Ii) Greek samples A total of 67 samples were obtained from C.I. 1 and C.I. Tested using 7 ELISA. None of the specimens were only reactive.

(Iii) Egyptian specimens A total of 18 of 132 specimens were found to be reactive in one or more ELISAs. C. None were found to be reactive in 1 ELISA. A total of 15 specimens were C.I. 6 specimens were found to be reactive in 6 ELISAs and 3 specimens were C.I. It was found to be reactive in 7 ELISA.

(Iv) US specimens (M set)
A total of 6 specimens were found to be reactive in one or more ELISAs. Two specimens were C.I. Repeated reactivity was observed in 1 ELISA. Four specimens were C.I. Repeated reactivity was observed in 6 ELISA. C. There were no repetitive specimens in 7 ELISA.

(V) Specimens from the United States (T set)
Among 64 specimens, C.I. 1 ELISA or C.I. None were found to be reactive in the 6 ELISA. One specimen is C.I. It was repeatedly reactive in 7 ELISA.

(Vi) Samples from various clinical sites in the United States (Set 1)
In total, 3 out of 62 specimens were found to be reactive in one or more ELISAs. One specimen is C.I. 1 ELISA and C.I. Repeated reactivity was observed in both 6 ELISAs. Two specimens were C.I. Repeated reactivity was observed in 7 ELISA.

  As mentioned above, there can be more than one strain in HGBV, ie more than one distinct virus can be represented by the sequences disclosed herein. These are considered to be within the scope of the present invention and are referred to as “hepatitis GB virus (“ HGBV ”)”.

L. Statistical Significance of Serum Response Results These data indicate that HGBV-C protein (i.e., C. cerevisiae) from individuals considered to be at risk of HGBV exposure and individuals diagnosed with non-AE hepatitis. 1 and C.6 and C.7 antibodies that are repeatedly reactive with antibodies) are detected, and a low percentage of these antibodies are detected from volunteers or blood donors from the United States. It was done. Table 24 shows the results of the serum responses for the various categories of specimens (“low risk”, “at risk” and non-AE hepatitis patients shown in Table 23), grouped together, chi-square test Statistical significance was analyzed by. Unlike the data in Table 23, which summarizes the serological presence of antibodies to HGBV protein in the total number of specimens tested, the data in Table 24 reflects the results obtained for different individuals (persons). For the GBV-C ELISA, the data compare the serological presence of anti-HGBV in volunteer blood donors with individuals considered “at risk” who are exposed to HGBV but not IVDU (West Africa). Indicates that there is a significant difference (p value = 0.000). Also, the presence of serological antibodies to HGBV-C in individuals suffering from non-AE hepatitis in Egypt and the United States was statistically significant when compared to volunteer blood donors. These data suggest that exposure to HGBV-C is associated with non-AE hepatitis. Note: RT-PCR results were negative in these early studies, but subsequent data revealed the flavi-like virus sequence in the sera of serologically positive individuals (see Example 19) did.

(Example 21)
Presence of HGBV-C in non-AE hepatitis humans The generation of an HGBV-C specific ELISA resulted in immunopositive sera from patients suffering from non-AE hepatitis (from HGBV-C serology). Example) can be identified. This serum was tested by RT-PCR for HGBV-C sequences, along with several immunologically positive HGBV-A and HGBV-B obtained from individuals proven to be non-AE hepatitis (Table 25). did. In order to detect HGBV-C variants as much as possible, RT-PCR was performed using the denatured NS3 oligonucleotide primer in the first amplification, followed by the nested GB-C specific primer. A second amplification was performed. Specifically, the first round of amplification uses 2 mM MgCl ₂ and 1 μM primers ns3.2-s1, ns3.2-a1 (SEQ ID NOs: 711 and 712, respectively) and are described in Example 6. Serum cDNA products prepared as described above were used. Repeated 40 cycles of denaturation-annealing-extension to the reaction [3 times at 94 ° C for 30 seconds, 37 ° C for 30 seconds, temperature rise to 72 ° C over 2 minutes, 72 ° C for 30 seconds) Thereafter, the cycle (94 ° C. for 30 seconds, 50 ° C. for 30 seconds, 72 ° C. for 30 seconds) was repeated 37 times], followed by extension at 72 ° C. for 10 minutes. The resulting reaction was maintained at 4 ° C. The second round of amplification was performed using 2 mM MgCl ₂ , 1 μM GB-C specific primers (SEQ ID NOs: 675 and 676) and 4% of the first PCR product as template. The second round of amplification employed a temperature cycling scheme designed to also amplify specific products with oligonucleotide primers that contain base pair mismatches with the template to be amplified [Roux, Bio / Techniques, 16: 812-814 (1994)]. That is, the reaction was repeated 43 times (94 ° C for 20 seconds, 94 ° C for 20 seconds, 55 ° C (0.3 ° C decrease for each cycle) for 30 seconds, 72 ° C for 1 minute). 10 minutes at 40 ° C. for 30 seconds and 72 ° C. for 1 minute), and final extension was performed at 72 ° C. for 10 minutes. The PCR product was isolated by agarose gel electrophoresis, nucleic acid was directly stained with ethidium bromide, visualized by ultraviolet irradiation, transferred to Hybond-N + nylon filter by Southern transfer, and then radiolabeled for GB-C. Hybridization was performed on the probe. PCR products were cloned and sequenced as described in the art.

  Using the above method, GB-C. 4, GB-C. 5, GB-C. 6, and GB-C. 7 was obtained. These sequences are homologous to GB-C (SEQ ID NO: 400, bases 4167-4365) at a rate of 82.1-86.6%. FIG. 40 shows GB-C.1 matched to the region homologous to GB-C in the expected codon triplet. 4, GB-C. 5, GB-C. 6, and GB-C. 7 sequence differences are shown. As shown in this figure, most of the nucleotide differences do not cause amino acid changes from GB-C. This overall sequence maintenance at the amino acid level is described in GB-C. 4, GB-C. 5, GB-C. 6, and GB-C. This suggests that 7 was obtained from HGBV-C, a different strain of the same virus. Furthermore, the degree of sequence differences at the nucleotide level indicates that these PCR products are not the result of contamination with any of the previously identified GB-C sequences.

  For three of these individuals (GB-C.4, GB-C.5, GB-C.6, and GB-C.7 sources), hepatitis A, hepatitis B, or hepatitis C There was no evidence of infection. The presence of GB-C sequences in individuals suffering from hepatitis of unknown etiology suggests that one of the causative agents of human hepatitis is HGBV-C. Time-lapse samples were available for two of the individuals (including GB-C.4 and GB-C.5). To track the sequence of HGBV-C in these samples, a clone-specific RT-PCR was developed. That is, nucleic acid extracted from serum was reverse transcribed with random hexamers as in Example 7. The obtained cDNA was amplified 40 times at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds, and then extended at 72 ° C. for 10 minutes. GB-C. 4 and GB-C. 5 specific PCR primers (GB-C.4-s1 and GB-C.4-a1, or GB-C.5-s1 and GB-C.5-a1) were used at a concentration of 1.0 μM . PCR product was isolated by agarose gel electrophoresis, nucleic acid was directly stained with ethidium bromide, visualized by ultraviolet irradiation, transferred to Hybond-N + nylon filter by Southern transfer, and then hybridized to radiolabeled probe did.

  In the serum of Egyptian patients suffering from acute non-AE hepatitis, GB-C. 4 was found. The patient was seropositive for the HGBV-A protein (see the HGBV-A ELISA example). RT-PCR of a series of 5 samples from this Egyptian patient showed viremia, which persisted for at least 20 days after serum ALT levels were normal (Table 26). The presence of GB-C sequences after normalization of serum ALT suggested that in some people, it can lead to chronic infection. However, since no more samples from this patient were available, no conclusions can be drawn about the chronicity of HGBV-C. In order to solve this problem, an additional sample is required.

  GB-C. 5 was obtained from a Canadian patient suffering from aplastic anemia associated with hepatitis. Each sample of this patient is C.I. Serologically positive in 7 ELISA (Example 20). From samples obtained from Canadian patients during aplastic anemia (13 days after symptom manifestation) and at death (14 days, FIG. 41), GB-C. GB-C.5 using 5 specific primers (GB-C.5-s1 and GB-C.5-a1). 5 was detected. However, GB-C. 5-specific PCR was performed at GB-C. 5 sequences could not be detected. Therefore, GB-C. It is unclear whether 5 was present at a level below the detection limit. If present, HGBV-C would have been the causative agent of the patient's aplastic anemia. However, GB-C. 5 was detected by RT-PCR only at the critical stage of poor regeneration, so GB-C. 5 may have been introduced from a blood product administered to address anemia. In this case, the association of HGBV-C to aplastic anemia would be similar to HCV [Hibbs et al., JAMA, 267, 2051-2054 (1992)].

  In view of the distant relationship between HGBV-C and HCV, it was of interest whether or not human samples containing HGBV-C could be recognized by current methods for detecting HCV infection. The usual test for individuals exposed to or infected with HCV is by antibody testing utilizing antigens obtained from three or more regions of HCV-1. This test can detect antibodies against all known genotypes of HCV in most individuals [Sakamoto et al. Gen. Virol. 75, 1761-1768 (1994), Stuyver et al. Gen. Virol. 74, 1093-1102 (1993)]. A second generation ELISA for HCV was performed on the sample containing HGBV-C described in Example 10 (Table 25). One of the four samples containing HGBV-C was seropositive for HCV antigen. Only a limited number of human sera that are seronegative for HCV have been shown to be positive for HCV genomic RNA by highly sensitive RT-PCR assays [Sugitani, 1992, # 65]. A similar RT-PCR assay (as described in Example 9) confirmed the presence of HCV viremia in seropositive samples. However, there was no HCV viremia among seroresponsive HCV negative samples. Thus, although one of four individuals containing the HGBV-C sequence has evidence of HCV infection, this assay for the presence of HCV could not accurately predict the presence of HGBV-C. This one HCV positive patient also seems to have been infected with HGBV-C. It is unclear whether the patient's hepatitis was due to the presence of HCV, HGBV-C, or both viruses. The discovery of HCV and HGBV-C in the same patient may suggest that there is a common risk of receiving these infections.

  Using the PCR procedure described above, in a “normal” unit of blood obtained from two volunteer American donors in 1994, the GB-C sequence (the previous GB-C shown in FIG. ˜85% homology with the isolate, data not shown). These units were found to be negative for HBV and HCV as a result of the test, and had normal serum ALT values. However, these units were tested positive in the 1.4 ELISA. The discovery of HGBV-C in at least two “normal” blood units of ~ 1000 units immunologically screened suggests that the virus is now present in the US blood supply Yes. However, we show that these blood units can be identified using ELISA probes developed from HGBV proteins and nucleotide probes from HGBV sequences.

  It should be noted that there are numerous sequence variations of the various HGBV sequences (FIG. 41). PCR-based assays for viral nucleic acids, albeit very sensitive, rely on sequence matching between oligonucleotide primers and viral templates. Therefore, since the PCR primers utilized in this study were located in regions of the HGBV-C genome that were not well conserved in various isolates, all of the tested HGBV-C viremia samples were employed here. Could not be detected by RT-PCR. As discovered in the untranslated region of HCV 5 '[Cha et al. Clin. Microbiol. 29, 2528-2534 (1991)], PCR primers from well-conserved regions of the HGBV-C genome will allow more accurate detection of HGBV-C viremia samples. .

１ ウイルスＡ、ＢまたはＣからの組換えＨＧＢＶタンパクに対する免疫学的応答性検出。
２ 切捨て値と報告された値との対比。値が１以上のもの（下線で示す）は陽性と考えられる。
３ 肝炎に関連した再生不良貧血。

1 Detection of immunological responsiveness to recombinant HGBV protein from virus A, B or C.
2 Contrast between rounded down value and reported value. A value of 1 or more (indicated by an underline) is considered positive.
3 Aplastic anemia associated with hepatitis.

１ 正常検体の上限：４５Ｕ／ｌ。
２ 正常と報告された値との対比。値が１０以上のものは陽性と考えられる。

1 Upper limit of normal specimen: 45 U / l.
2 Contrast with value reported as normal. A value of 10 or more is considered positive.

(Example 22)
Sequence comparison and systematic analysis Information on the degree of intimacy of the virus can be obtained by comparing the nucleotide to the predicted amino acid sequence, ie, alignment. By performing alignment between the HGBV sequence and the sequence of another virus, the degree of similarity and homology between sequences can be quantitatively evaluated. This information can be used to develop principles for the classification of HGBV viruses. In general, the calculation of similarity between two amino acid sequences is based on the degree of similarity between the side chains of the amino acid pair in the alignment. Similarity is based on the physicochemical characteristics of the side chains of amino acids, ie, size, shape, charge, hydrogen bonding ability, and chemical reactivity. That is, similar amino acids have side chains with similar physicochemical characteristics. For example, phenylalanine and tyrosine both contain aromatic side chains and can therefore be considered chemically similar. A discussion of amino acid chemistry can be found in basic biochemistry textbooks, for example, Biochemistry, 3rd edition, edited by Lubert Stier, W.C. H. Freeman and Company, New York, 1988. The calculation of homology between two aligned amino acid sequences is generally an arithmetic calculation in which the number of identical amino acid pairs in the alignment is counted and this number is divided by the length of the sequence in the alignment. Similar to the method used to align amino acid sequences, the determination of the degree of homology between two aligned nucleotide sequences counts the number of identical nucleotide base pairs in the alignment and this number is the sequence in the alignment. It is an arithmetic calculation that divides by the length of. Calculation of similarity between two aligned nucleotide sequences uses sometimes different values for transitions and transitions between paired (ie matched) pairs at various positions in the alignment. However, the similarity and homology scores between a pair of nucleotide sequences are usually very close, i.e., within 1-2%.

  As stated previously, the homology between the HGBV agonist and the hepatitis C genotype amino acid sequence is limited. In order to more accurately determine the degree of intimacy between HGBV agonist and HCV, the HGBV-A, B and C overall large open reading frame (ORF) sequences, and several representatives Amino acid sequence alignment was performed using the amino acid sequence of the large ORF of the HCV isolate. Furthermore, the degree of intimacy between the HGBV agonist and HCV at the nucleotide level is determined by the overall genomic nucleotide sequence of HGBV-A, B and C, and the overall genomic nucleotides of some representative HCV isolates. Determined using sequence. Alignment between amino acid and nucleotide sequences was performed using the program GAP of the Wisconsin Sequence Analysis Package (version 8) available from Genetic Computer Group, Inc. at 53711, Madison, Wis., Science Drive 575. The gap creation and gap extension penalties were 5.0 and 0.3, respectively, for nucleic acid sequence alignments and 3.0 and 0.1, respectively, for amino acid sequence comparisons. The GAP program calculates the degree of similarity and homology between two aligned sequences in terms of Needleman and Wunsch's algorithm (J. Mol. Biol. 48, 443-453, 1970).

  Nucleotide and amino acid sequences of selected numbers of major hepatitis C virus (HCV) genotypes were obtained from GenBank. This is shown below along with their registration numbers.

  Table 28 shows the pairwise comparison results between the predicted amino acid sequence of a large open reading frame (ie putative precursor polyprotein) and the nucleotide sequence between each of the HCV genotypes and the HGBV isolate. 29 respectively. The genotype designation for each HCV isolate is shown in the top row. This genotyping is based on the nomenclature for HCV isolates described in Simmonds, P et al. (1994) Hepatology, 19, 1321-1324.

  The data shown in Table 28 shows that the lower limit of amino acid sequence homology between HCV genotypes is 69%. This value is very close to the value shown by Simmons et al. [Simmonds, P et al., Hepatology, 19, 1321-1324 (1994)]. Simmons et al. Compared amino acid sequence similarity of 67.1% to 68.6%, comparing the coding regions of eight complete HCV genomes from two major groups. However, this author did not describe how the similarity was calculated. This value (69%) is very close to the value reported by Okomoto et al. [Virology, 188, 331-341, 1992] for comparison of HCV-J8 with other major HCV isolates. However, these researchers also did not describe how to calculate homology. A comparison of the HGBV polyprotein sequence with each HCV genotype shows that the sequence of the polyprotein encoded by HGBV shows no more than 33% homology to any of the HCV polyproteins (Table 28). It is revealed. A comparison of nucleotide sequences (Table 29) shows that there is a maximum of 44.2% homology between any HGBV and any HCV isolate, as opposed to a minimum nucleotide sequence between HCV isolates. Is 64.9%. Therefore, between HGBV-A, B, and C and HCV, the homology between the nucleotide and the predicted amino acid sequence is far from the range of homology established for known HCV genotypes. The HGBV virus cannot be considered a genotype of hepatitis C virus.

  The relationship between hepatitis C virus and hepatitis GB virus is based on a systematic analysis of their aligned nucleotides, deduced amino acid sequences (ie large open reading frames), or parts of these sequences. You can find out by doing. Application of this method to hepatitis C virus has been shown to depict six equally divergent main groups of sequences due to the variability of HCV isolates [Simmonds, P et al. Gen. Virol. (1993) 74, 2391-2399 and Simmonds, P. et al. Gen. Virol. (1994) 75, 1053-1061]. As a result of this analysis, a nomenclature for hepatitis C virus was established [Simmonds, P et al., Hepatology, 19, 1321-1324 (1994)]. They are classified into genotypes based on evolutionary gaps between sequences.

  To determine the systematic relationship between hepatitis GB virus and hepatitis C virus, alignment of amino acid sequences within the putative helicase gene of NS3 and the putative RNA-dependent RNA-polymerase (RdRp) of NS5B was performed. Also included in the alignment were related sequences from other viruses in Flaviviridae and viruses that were shown to have evolutionary intimacy for members of Flavivirae in the helicase and polymerase genes [Kunin E. (Koonin, EV) and Dolja, V. V (1993), Crit. Rev. Biochem. Mol. Biol. 28, 375-430, and Koonin, E. V. (1991), J. MoI. Gen. Virol. 72, 2179-2206].

  Amino acid sequence alignment was performed using the program PILEUP of the Wisconsin Sequence Analysis Package (version 8). Systematic distance between aligned pairs of sequences is provided by PHYLIP, courtesy of J. Felsenstein (Felsenstein Jay (1989), Cadistics 5, 164-166). Determined using PRODIST program in package (version 3.5c, 1993). This calculated distance was used to construct phylogenetic trees using the program NEIGHBOR (adjacent connection setting). A system diagram was plotted using the program DRAWTREE. Table 30 shows the virus sequences used and the corresponding Genbank registration numbers. The evolutionary distances between each HCV genotype and each HGBV virus for alignments performed in helicase, RdRp, or complete large open reading frame are shown in Tables 31, 32 and 33, respectively. The calculated distance between the HCV genotype and the HGBV virus and other viruses listed in Table 30 is not shown. The phylogenies made for helicase, RdRp, or complete large open reading frame sequence amino acid alignments are shown in FIGS. 42, 43 and 44, respectively.

  The putative RdRp encoded in the NS5B region of HGBV-A, B and C, several HCV genotypes RdRp, two infectious viruses, several representative flaviviruses, and several Alignment of amino acid sequences with positive strand RNA plant viruses indicated that they have conserved sequence features related to RdRp of positive strand RNA viruses (data not shown). Based on similar analysis, helicases encoded by HGBV-A and HGBV-B show significant homology to RNA positive helicases of these positive strands (data not shown). Exceptions are CARMV, TCV, and MNSV, which are presumed to carry no helicase gene [Guilley, H, et al. (1985) Nucleic Acids Res. 13, 6663-6677]. These results were not unexpected in view of the association of these viral helicases and RdRp genes to supergroups as shown by previous systematic analysis [Koonin, EV and Dolja, V. V (1993), Crit. Rev. Biochem. Mol. Biol. 28, 375-430]. However, a survey of systematic distances between HGBV and HCV isolates based on the sequence of helicase or RdRp sequences (Tables 31 and 33) shows that there is a considerable gap between these two groups. . The calculated gap shows a close relationship between HCV genotypes, with a maximum distance between the two genotypes of 0.3696 (RdRp distance). However, the distance calculated from RdRp between HGBV-A, -B, or -C and any member of the HCV group is 0.96042-1.42611. Similarly, values calculated from helicase alignments for any two HCV genotypes range from 0.044555 to 0.19706, whereas any member of the HCV group and HGBV-A, -B, or The distance between -C ranges from 0.69130 to 0.87120. In addition, the alignment of the predicted amino acid sequence of the complete large open reading frame of the HCV genotype and GB virus has a narrow range of evolutionary separation of HCV isolates (0.17918-0.39646), while arbitrary It indicates that the distance between GB virus and any HCV isolate is at least 1.686650. Thus, hepatitis GB virus exhibits an evolutionary gap that is outside the range indicated by the hepatitis C virus genotype.

  A systematic analysis of HGBV and HCV sequences has shown that “how can the divergence of HGBV sequences from HCV sequences be compared with divergence between HCV sequences? "Is there less divergence in HGBV sequences?" If the divergence from the HCV sequence of the HGBV sequence is less than the divergence between the HCV sequences, the condition that must be satisfied is that the sequence of HGBV-A, HGBV-B, and / or HGBV-C is To be as close to at least one of the HCV sequences as the most distantly related pair (ie, the minimum distance from any HGBV sequence to any HCV sequence is observed between HCV sequences). Less than or equal to the maximum value). The current sequence data does not satisfy this condition. In Table 31 (RbRp alignment), the minimum HCV-HGBV distance is 2.83 times the maximum HCV-HCV distance, and in Table 32, the minimum HCV-HGBV distance is 3 of the maximum HCV-HCV distance. .51 times. Thus, the data do not support the idea that the HGBV sequence is a member of a group whose divergence is limited by the previously characterized members of the HCV group.

  These relative distances can be determined using tests based on bootstrap [Efron, B. (1982), “Jackknife, bootstrap, and other resampling schemes”, Society Industrial. and Applied Mathematics, Philadelphia, Elton, B. and Gong, G. (1983) “Fun studies of jackknife, bootstrap, and cross-validation” Am. Stat. 37, 36-48]. The results obtained from bootstrap sampling are shown in Table 32. This table shows a comparison of HCV-HGBV divergence (minimum of all HCV-HGBV distances) to HCV divergence (maximum of all HCV-HCV distances) using the PROTDIST program. Based on the calculated PAM distance. At 1000 bootstrap resampling of the column in sequence alignment, the maximum divergence between HCVs did not exceed the minimum divergence of HGBV sequences from HCV sequences (Table 34). Therefore, in each measurement based on the alignment of coding regions from two separate genes, there is no example that the HGBV sequence falls within the divergence range of the gene sequence of the HCV genotype. Even with careful guessing, there is less than one in 100,000 possibilities for data for HGBV to be drawn from the same sequence pool as the HCV sequences.

  Assuming that the HCV sequences utilized in this study are representative of the most divergent HCV genotypes, these results indicate that HGBV-A, B, and C are not HCV genotypes. Yes. Also, the relationship between HGBV-A and HGBV-C appears to be closer than their relationship to HGBV-B, which is representative of a series of viruses in which HGBV-A and HGBV-C are different. It is suggested that. Similarly, HGBV-B is the only one that represents its own virus line. The relative swivel distance between the analyzed viral sequences is readily apparent by examining the rootless phylogeny shown in FIGS. In the system diagram, the length of the branch is proportional to the evolutionary distance. It is clear that the HCV virus has a close evolutionary relationship, consistent with choosing to use a portion of the encoded genomic sequence or the entire genome for the analysis ( FIG. 44). Although the degree of divergence between HGBV-A, HGBV-B, and HGBV-C and other members of Flaviviridae is most closely related to hepatitis C virus, the GB agonist is HCV It cannot be considered as a genotype of, indicating that it would actually represent a new group within the Flaviviridae family.

  The present invention provides reagents and methods for determining the presence of HGBV-A, HGBV-B, and HGBV-C in a test sample. Polynucleotides or polypeptides (or fragments thereof) specific for HGBV-A, HGBV-B, and HGBV-C described herein, or antibodies produced from these polypeptides and polynucleotides are generally used. Can be combined with the assay reagents described above, and can be incorporated into the assay procedures for detecting antibodies against the viruses described above, and are considered within the scope of the present invention. Alternatively, produced from polynucleotides or polypeptides (or fragments thereof) specific for HGBV-A, HGBV-B, and HGBV-C described herein, or from these polypeptides and polynucleotides (or fragments thereof) The antibodies that can be used can be used separately to detect HGBV-A, HGBV-B, and HGBV-C viruses, respectively.

  Other uses or variations of the present invention will be apparent to those of skill in the art upon reviewing the disclosure herein. Accordingly, it is intended that the invention be limited only by the scope of the appended claims.

It is the graph which plotted the quantity (mU / ml) of the liver enzyme (ALT or ICD) of tamarin T-1053 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of liver enzyme (ALT or ICD) of tamarin T-1048 against time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of liver enzyme (ALT or ICD) of tamarin T-1057 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of the liver enzyme (ALT or ICD) of tamarin T-1061 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of the liver enzyme (ALT or ICD) of tamarin T-1047 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of the liver enzyme (ALT or ICD) of tamarin T-1042 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of liver enzyme (ALT or ICD) of tamarin T-1044 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of the liver enzyme (ALT or ICD) of tamarin T-1034 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of liver enzyme (ALT or ICD) of tamarin T-1055 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of liver enzyme (ALT or ICD) of tamarin T-1051 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of liver enzyme (ALT or ICD) of tamarin T-1038 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. It is the graph which plotted the quantity (mU / ml) of liver enzyme (ALT or ICD) of tamarin T-1049 with respect to time (week after inoculation). In the graph, ALT CO indicates an ALT cutoff value, and ICD OD indicates an ICD cutoff value. Fig. 4 shows a flowchart of the steps involved in representative difference analysis (RDA) used for clone identification. Figure 2 shows an ethidium bromide stained 2.0% agarose gel of the product obtained by representative difference analysis (RDA) performed on plasma of inoculated and acute HGBV infected tamarins. Shown are autoradiograms of Southern blots of genomic DNA, amplicon DNA, and product obtained from the first three subtraction / hybridizations. Same autoradiogram as shown in FIG. 15, except that another radiolabeled probe was used. Figure 5 shows an ethidium bromide stained 1.5% agarose gel of polymerase chain reaction (PCR) amplification product from genomic DNA. The autoradiogram obtained by the Southern blot of the 1.5% agarose gel of FIG. 17 is shown. Shown is an ethidium bromide stained 1.5% agarose gel of RT-PCR products obtained from normal human serum and pre-inoculation and acute tamarin plasma. FIG. 20 shows an autoradiogram obtained by Southern blotting of the same gel as shown in FIG. Figure 6 shows an Northern radiogram of a Northern blot of total cellular RNA extracted from livers of uninfected tamarin and HGBV infected tamarin. Figure 6 shows an Northern radiogram of a Northern blot of total cellular RNA extracted from livers of uninfected tamarin and HGBV infected tamarin. FIG. 5 shows that each recombinant polynucleotide isolate is present in a continuous RNA species. Figure 2 shows a dot plot analysis of the nucleic acid sequence of HGBV-A. Figure 2 shows a dot plot analysis of the nucleic acid sequence of HGBV-B. Figure 2 shows a dot plot analysis of the nucleic acid sequence of HGBV-A versus HGBV-B. Conserved residues in the putative NTP binding helicase domain of putative translation products of HGBV-A, HGBV-B and HCV-1 NS3 are shown. The conserved residues of the RNA-dependent RNA polymerase domain of putative translation products of HGBV-A, HGBV-B and HCV-1 NS5b are shown. Figure 2 shows a Coomassie stained 10% SDS-polyacrylamide gel of CKS fusion protein whole cell lysate. Three CKS fusion proteins are immunoreactive against HGBV-infected tamarin sera. Figure 2 shows a Coomassie stained 10% SDS-polyacrylamide gel of CKS fusion protein whole cell lysate. Three CKS fusion proteins are immunoreactive against HGBV-infected tamarin sera. Tamarin T-1048 1) Amount of liver enzyme (ALT) (mU / ml) with respect to time (weeks after inoculation) (indicated by solid line); 2) ELISA absorption value of CKS-1.7 recombinant protein ( 3) ELISA absorption value of CKS-1.4 recombinant protein (indicated by white circle connected by dotted line); 4) ELISA absorption value of CKS-4.1 recombinant protein (shown by black circle connected by dotted line) 5) Negative PCR results using the primer of SEQ ID NO: 21 (shown by the white squares); 6) Positive PCR results using the primer of SEQ ID NO: 21 (the black squares) 7) Negative PCR result using the primer of SEQ ID NO: 26 (indicated by a white diamond); 8) Positive PCR result using the primer of SEQ ID NO: 26 (indicated by a black diamond); 9) Inoculation data (indicated by arrowhead ) Is a graph that plots. Tamarin T-1057 1) Amount of liver enzyme (ALT) (mU / ml) with respect to time (weeks after inoculation) (indicated by solid line); 2) ELISA absorption value of CKS-1.7 recombinant protein ( 3) ELISA absorption value of CKS-1.4 recombinant protein (indicated by white circle connected by dotted line); 4) ELISA absorption value of CKS-4.1 recombinant protein (shown by black circle connected by dotted line) 5) Negative PCR results using the primer of SEQ ID NO: 21 (shown by the white squares); 6) Positive PCR results using the primer of SEQ ID NO: 21 (the black squares) 7) Negative PCR result using the primer of SEQ ID NO: 26 (indicated by a white diamond); 8) Positive PCR result using the primer of SEQ ID NO: 26 (indicated by a black diamond); 9) Inoculation data (indicated by arrowhead ) Is a graph that plots. Tamarin T-1061 1) liver enzyme (ALT) amount (mU / ml) with respect to time (weeks after inoculation) (indicated by solid line); 2) ELISA absorption value of CKS-1.7 recombinant protein ( 3) ELISA absorption value of CKS-1.4 recombinant protein (indicated by white circle connected by dotted line); 4) ELISA absorption value of CKS-4.1 recombinant protein (shown by black circle connected by dotted line) 5) Negative PCR results using the primer of SEQ ID NO: 21 (shown by the white squares); 6) Positive PCR results using the primer of SEQ ID NO: 21 (the black squares) 7) Negative PCR result using the primer of SEQ ID NO: 26 (indicated by a white diamond); 8) Positive PCR result using the primer of SEQ ID NO: 26 (indicated by a black diamond); 9) Inoculation data (indicated by arrowhead ) Is a graph that plots. Tamarin T-1051 1) liver enzyme (ALT) amount (mU / ml) with respect to time (weeks after inoculation) (indicated by solid line); 2) ELISA absorption value of CKS-1.7 recombinant protein ( 3) ELISA absorption value of CKS-1.4 recombinant protein (indicated by white circle connected by dotted line); 4) ELISA absorption value of CKS-4.1 recombinant protein (shown by black circle connected by dotted line) 5) Negative PCR results using the primer of SEQ ID NO: 21 (shown by the white squares); 6) Positive PCR results using the primer of SEQ ID NO: 21 (the black squares) 7) Negative PCR result using the primer of SEQ ID NO: 26 (indicated by a white diamond); 8) Positive PCR result using the primer of SEQ ID NO: 26 (indicated by a black diamond); 9) Inoculation data (indicated by arrowhead ) Is a graph that plots. Tamarin T-1034 1) Liver enzyme (ALT) amount (mU / ml) with respect to time (weeks after inoculation) (indicated by solid line); 2) ELISA absorption value of CKS-1.7 recombinant protein ( 3) ELISA absorption value of CKS-1.4 recombinant protein (indicated by white circle connected by dotted line); 4) ELISA absorption value of CKS-4.1 recombinant protein (shown by black circle connected by dotted line) 5) Negative PCR results using the primer of SEQ ID NO: 21 (shown by the white squares); 6) Positive PCR results using the primer of SEQ ID NO: 21 (the black squares) 7) Negative PCR result using the primer of SEQ ID NO: 26 (indicated by a white diamond); 8) Positive PCR result using the primer of SEQ ID NO: 26 (indicated by a black diamond); 9) Inoculation data (indicated by arrowhead ) Is a graph that plots. 1) Amount of liver enzyme (ALT) (mU / ml) (shown in solid line) over time (weeks after inoculation) of human test sample of patient 101; 2) ELISA absorption of CKS-1.7 recombinant protein Values (indicated by black circles connected by a dotted line); 3) A graph plotting ELISA absorption values (indicated by white circles connected by a dotted line) of CKS-1.4 recombinant protein. 1) Liver enzyme (ALT) amount (mU / ml) (indicated by solid line) over time (weeks after inoculation) of human test sample of patient 257; 2) ELISA absorption of CKS-1.7 recombinant protein Values (indicated by black circles connected by a dotted line); 3) A graph plotting ELISA absorption values (indicated by white circles connected by a dotted line) of CKS-1.4 recombinant protein. 1) Liver enzyme (ALT) amount (mU / ml) (shown in solid line) over time (weeks after inoculation) of human test sample of patient 260; 2) ELISA absorption of CKS-1.7 recombinant protein Values (indicated by black circles connected by a dotted line); 3) A graph plotting ELISA absorption values (indicated by white circles connected by a dotted line) of CKS-1.4 recombinant protein. 1) Amount of liver enzyme (ALT) (mU / ml) (shown in solid line) over time (weeks after inoculation) of human test sample of patient 340; 2) ELISA absorption of CKS-1.7 recombinant protein Values (indicated by black circles connected by a dotted line); 3) A graph plotting ELISA absorption values (indicated by white circles connected by a dotted line) of CKS-1.4 recombinant protein. Contig. A, Contig. B and conserved residues of putative NTP binding helicase domains of putative translation products of HCV-1 NS3 are shown. Contig. A, Contig. B and conserved residues of the RNA-dependent RNA polymerase domain of the predicted translation product of HCV-1 NS5b are shown. The nucleotide alignment of HGBV-A, HGBV-B, HGBV-C and HCV-1 is shown. FIG. 2 shows PhosphoImage (Molecular Dynamics, Sunnyvale, Calif.) Obtained from a Southern blot of PCT products after hybridization with a radiolabeled probe derived from GB-C. The nucleotide alignment of HGBV-C and two variant clones is shown. The outline of Contig after the assembly of HGBV-C is represented. The nucleotide alignment of HGBV-C and 4 variant clones is shown. Figure 2 shows a PhosphoImage (Molecular Dynamics, Sunnyvale, CA) of a Southern blot of PCT products generated from a Canadian hepatitis patient after hybridizing with a radiolabeled probe from Canadian patient GB-C.5. Figure 2 shows a phylogenetic tree generated from alignment of the helicase domains of the described viruses. SCOTT, phylogenetic tree generated from alignment of the RNA-dependent RNA polymerase domains of the described viruses. Figure 2 shows a phylogenetic tree generated from alignment of the large open reading frames (putative precursor polyprotein) of the described virus.

Claims (14)

A hepatitis GB virus (HGBV) polypeptide comprising an amino acid sequence encoded by a positive-strand RNA genome comprising an open reading frame (ORF) encoding a polyprotein comprising the amino acid sequence of SEQ ID NO: 397. 2. The HGBV polypeptide according to claim 1, wherein the polypeptide is a purified polypeptide, a recombinant polypeptide or a synthetic polypeptide. A diagnostic reagent comprising a hepatitis GB virus polypeptide encoded by a positive strand RNA genome comprising an open reading frame (ORF) encoding a polyprotein comprising the amino acid sequence of SEQ ID NO: 397. A purified antibody that specifically binds to at least one epitope of a hepatitis GB virus (HGBV) antigen, wherein said epitope consists of at least 5 amino acids, said HGBV antigen comprising (a) a polyprotein comprising SEQ ID NO: 397 Said purified antibody encoded by the encoding positive-strand RNA viral genome and (b) not immunoreactive with an antibody that specifically binds to HGBV-A or HGBV-C. The antibody according to claim 4, wherein the antibody is a polyclonal antibody or a monoclonal antibody. 6. The antibody of claim 4 or 5, which is bound to a signal generating compound. An assay kit for detecting the presence of an antibody of hepatitis GB virus (HGBV) in a test sample, comprising a container of a polypeptide having at least one HGBV antigen, wherein the HGBV antigen comprises (a) SEQ ID NO: 397 Said assay kit, characterized in that it is not immunoreactive with an antibody that is encoded by a positive-strand RNA viral genome that encodes a polyprotein containing and that specifically binds to HGBV-A or HGBV-C. A method for detecting an antibody in a test sample suspected of containing an antibody of hepatitis GB virus (HGBV), comprising:
(a) under a time and under conditions sufficient to form an antigen / antibody complex, the test sample comprises (i) at least one HGBV epitope consisting of at least 5 amino acids, and (ii) SEQ ID NO: 397 Contacting a polypeptide that is not immunoreactive with an antibody that is encoded by a positive-strand RNA viral genome that encodes the polyprotein comprising and (iii) specifically binds to HGBV-A or HGBV-C;
(b) A method for detecting the antibody, comprising detecting the complex containing an antibody of HGBV. 9. The method of claim 8, wherein the polypeptide is produced by recombinant techniques or synthetic means. 9. The method of claim 8, wherein in step (b), the complex is contacted with an indicator prior to detecting the complex. 11. The method of claim 10, wherein the indicator is a signal generating compound. A method for detecting HGBV antigen in a test sample suspected of containing hepatitis GB virus (HGBV) comprising:
(a) an antibody or a fragment thereof that specifically binds the test sample to at least one epitope consisting of at least five amino acids of HGBV antigen for a time and under conditions sufficient to form an antigen / antibody complex In contact with
(b) (i) encoded by a positive-strand RNA viral genome encoding a polyprotein comprising SEQ ID NO: 397, and (ii) not immunoreactive with an antibody that specifically binds to HGBV-A or HGBV-C A method for detecting the antigen, comprising detecting the complex containing the HGBV antigen in a test sample. 13. The method according to claim 12, wherein in step (b), the complex is contacted with an indicator and incubated before detecting the complex. 14. The method of claim 13, wherein the indicator is a signal generating compound.

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