EP1137779A2 - Feststellung von senv genotypen - Google Patents

Feststellung von senv genotypen

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Publication number
EP1137779A2
EP1137779A2 EP99971855A EP99971855A EP1137779A2 EP 1137779 A2 EP1137779 A2 EP 1137779A2 EP 99971855 A EP99971855 A EP 99971855A EP 99971855 A EP99971855 A EP 99971855A EP 1137779 A2 EP1137779 A2 EP 1137779A2
Authority
EP
European Patent Office
Prior art keywords
seq
senv
nucleic acid
virus
acid molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99971855A
Other languages
English (en)
French (fr)
Inventor
Daniele Primi
Gianfranco Fiordalisi
Giovanni Lorenzo Mantero
Sonia Mattioli
Alessandra Sottini
Fabrizio Bonelli
Laura Vaglini
Paolo Olivero
Andrea Dal Corso
Marco Bonelli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Diasorin International Inc
Original Assignee
Diasorin International Inc
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Filing date
Publication date
Priority claimed from ITMI982437 external-priority patent/IT1303732B1/it
Priority claimed from ITMI990923 external-priority patent/IT1312552B1/it
Application filed by Diasorin International Inc filed Critical Diasorin International Inc
Priority to EP99971855A priority Critical patent/EP1137779A2/de
Publication of EP1137779A2 publication Critical patent/EP1137779A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a nucleic acid molecule representing the genome of a virus/viral agent, said nucleic acid molecule having at least one of the following features: (a) it contains at least one open reading frame (ORF) which encodes a polypeptide having an amino acid sequence disclosed herein, (b) it comprises a DNA sequence as described herein; (c) it hybridizes preferably under stringent conditions to the complementary strand of the nucleic acid molecule described herein, but not to the complementary strand of the genome of TT virus; (d) it is degenerate with respect to the hybridizing nucleic acid molecule; (e) it is at least 60% identical with the nucleic acid molecule representing the genome of the virus/viral agent of the invention; and (f) parts of it can be amplified under suitable conditions by PCR employing primers as defined herein.
  • ORF open reading frame
  • the invention further relates to vectors comprising said nucleic acid molecule, methods of producing (poly)peptide(s) encoded by said nucleic acid molecule, to viruses/viral agents carrying a genome represented by said nucleic acid molecule and antibodies to said (poly)peptide(s) and/or said viruses/viral agents. Additionally, the invention relates to compositions, preferably pharmaceutical compositions, vaccines, diagnostics and diagnostic kits comprising or employing the compounds of the invention.
  • hepatitis This hepatopathy is one of the most important diseases transmitted from donor to recipient by transfusion or other administration of blood products, by organ transplantation or haemodialysis. Hepatitis is also transmitted via ingestion of contaminated water or nutrition and can be community- acquired (person-to-person contact). Viral agents suspected of causing such hepatopathies include hepatitis A-(HAV), hepatitis B- (HBV), hepatitis C- (HCV), hepatitis D- (HDV), hepatitis E- (HEV) or, hepatitis G-virus (HGV/HGBV-C) as well as cytomegalovirus (CMV) and Epstein-Barr virus (EBV).
  • HAV hepatitis A-(HAV), hepatitis B-
  • HCV hepatitis C-
  • HDV hepatitis D-
  • HEV hepatitis E-
  • HGV/HGBV-C hepatit
  • HCV hepatitis C virus
  • TTV is an unenveloped, single-stranded DNA virus with 3739 nucleotides (Okamoto, et al., Hepatology Res. 10 (1998), 1-16). Two genetic groups of this virus have been identified, differing by 30% in nucleotide sequences. TTV DNA was detected in 47% of patients with fulminant non-A-G hepatitis and 46% of patients with chronic liver diseases of unknown etiology, suggesting that TTV may be the cause of some cryptogenic liver diseases. More recent findings however have casted doubt on the potential pathogenicity of TTV.
  • TTV hepatitis G-virus
  • the rate of TTV infection in the normal, healthy population is at least three times higher than the prevalence of HGV agent in the general population which appear to be 1.7% in the USA (Linnen et al., Science 271 (1996), 505-508) and 3.2% in Scotland (Jarvis et al., Lancet 348 (1996), 1352-1355).
  • the majority of patients with TTV infection had a history of blood transfusion or intravenous drug use, confirming the importance of the parenteral route of transmission, the agent is also detected in a large proportion of individuals with "community-acquired" infection
  • TTV may not be a primary hepatitis virus.
  • the present invention relates to a nucleic acid molecule representing the genome of a virus/viral agent, said nucleic acid molecule having at least one of the following features:
  • (b) it comprises the DNA sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:
  • (c) it comprises portions of at least 80 nucleotides, preferably at least 100 nucleotides, more preferably at least 500 nucleotides and most preferably at least 2000 nucleotides which hybridize (under stringent conditions) to the complementary strand of the nucleic acid molecule of SEQ ID NO: 26, SEQ ID NO: 26
  • (f) parts of it can be amplified under suitable conditions by PCR employing as primers oligonucleotides as defined in SEQ ID NO: 41 and SEQ ID NO: 42 and/or as defined in SEQ ID NO: 115 and SEQ ID NO: 71 or as defined in SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 32, SEQ ID NO: 73, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128; SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 , SEQ ID NO: 132, SEQ ID NO: 135,
  • the term “genome” defines not only sequences which are open reading frames (ORFs) encoding proteins, polypeptides or peptides, but also refers to non-coding sequences. Accordingly, the term “nucleic acid molecule” comprises coding and, wherever applicable, non-coding sequences. The nucleic acid molecule of the invention furthermore comprises nucleic acid sequences which are degenerative to the above nucleic acid sequences. In accordance with the present invention, the term “nucleic acid molecule” comprises also any feasible derivative of a nucleic acid to which a nucleic acid probe may hybridize.
  • nucleic acid probe itself may be a derivative of a nucleic acid molecule capable of hybridizing to said nucleic acid molecule or said derivative thereof.
  • nucleic acid molecule further comprises peptide nucleic acids (PNAs) containing DNA analogs with amide backbone linkages (Nielsen, P., Science 254 (1991), 1497-1500).
  • PNAs peptide nucleic acids
  • ORF open reading frame
  • nucleic acid sequences which encodes a (preferably expressed) polypeptide, in connection with the present invention, is defined either by (a) the specific nucleotide sequences encoding the polypeptides specified above in (aa) or in (ab) or (b) by nucleic acid sequences hybridizing under stringent conditions to the complementary strand of the nucleotide sequences of (a) and encoding a polypeptide deviating from the polypeptide of (a) by one or more amino acid substitutions, deletions, duplications, insertions, recombinations, additions or inversions and wherein the amino acid sequence shows at least 35% identity with the amino acid sequence of said polypeptide of (aa) or encoding a polypeptide deviating from the polypeptide of (a) by one or more amino acid substitutions, deletions or inversions and wherein the amino acid sequence shows at least 40% identity with the amino acid sequence of said polypeptide of (ab).
  • the nucleic acid molecule representing the genome of a virus as defined herein is the nucleic acid molecule representing the genome of a pathogenic virus.
  • pathogenic virus means a virus which is capable of a viral pathogenesis, wherein said pathogenesis is defined as the method by which said virus/viral agent produces or contributes to disease in a host.
  • pathogenesis refers herein to acute as well as/or to chronic infections as they occur in humans or other hosts. Pathogenesis therefore means the mechanism by which said virus injures discrete populations of cells in different organs or an organ to produce the signs and symptoms of disease of a host.
  • Said diseases comprise, inter alia, hepatopathologies, like hepatitis or hepatocarcinomas.
  • pathogentic virus means in accordance with the invention a virus/viral agent which is capable of negatively influencing the physiological state of a host even without primariiy causing distinct signs and/or symptoms of disease. Therefore, the term
  • pathogenic virus also refers to a virus/viral agent which aggravates a clinical condition in a host, e.g., after or during infection with another pathogenic organism
  • pathogenic virus not only comprises viruses/viral agents which are capable of causing a primary infection but also comp ⁇ ses viruses/viral agents which cause secondary infections. Additionally, the term “pathogenic virus” means a virus/viral agent which causes or contributes directly to a disease or disease symptoms in a host but also comprises viruses/viral agents which are indirectly involved in pathological processes in a patient.
  • pathogenic virus in context of this invention, therefore, comp ⁇ ses viruses/viral agents which have a direct disease association, aggravate a (even pre-existing) clinical condition in a host/patient and/or are indirectly involved in a pathological condition of a host/patient.
  • hybridizes as used in accordance with the present invention may relate to stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, "Molecular Cloning, A
  • Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6xSSC, 1%
  • fragments representing the genome or parts of the genome of a virus, and having a length of at least 12 nucleotides, preferably at least
  • nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include fragments, derivatives and allelic variants of these molecules.
  • derivative means in this context that the nucleotide sequence of these nucleic acid molecules differs from the sequences of the above-described nucleic acid molecules in one or more nucleotide positions and they are highly homologous to said nucleic acid molecules. Additionally or alternatively, the term “derivative” means a compound that does not or not solely consist of DNA, PNA or RNA as well as any chemically or biologically modified nucleic acid.
  • nucleotide or amino acid sequence identities/homologies can be determined conventionally by using known computer programs such as BLASTIN,
  • Gap Cost 10. It was found that these parameters are the best suited to calculate the percentage of identity over the full length of the reference nucleotide or amino acid sequences, especially considering the different levels of homology among the different sequences analyzed.
  • the deviations from the sequences of the nucleic acid molecules described above can, for example, be the result of nucleotide substitution(s), deletion(s), addition(s), insertion(s) duplicatron(s), inversion(s) and/or recombination(s) either alone or in combination, that may naturally occur or be produced via recombinant DNA techniques well known in the art; see, for example, the techniques described in Sambrook, loc. cit. and Ausubel, loc. cit.
  • the allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants.
  • proteins, peptides or protein fragments encoded by the various derivatives, allelic variants, homologs or analogs of the above-described nucleic acid molecules may share specific common characteristics, such as molecular weight, immunological reactivity, conformation, etc., as well as physical properties, such as electrophoretic mobility, chromatographic behavior, sedimentation coefficients, pH optimum, temperature optimum, stability, solubility, spectroscopic properties, etc.
  • the nucleic acid molecule of the invention contains two ORFs and most preferably, it contains three ORFs. Additionally, nucleic acid molecules representing a genome of a virus and containing four or more ORFs are also preferred embodiments and within the scope of the present invention.
  • the nucleic acid molecules of the present invention do not hybridize to the complementary strand of the genome of TT virus, at least in the region comprised in SEQ ID NO: 26 (comprising the regions encoding for a viral ORF1 , ORF3 and ORF2 with the exception of the about 15 5'terminal nucleotides of ORF2) in the region comprised in SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 125, SEQ ID NO: 186, SEQ ID NO: 191 and SEQ ID NO: 199 (comprising the regions encoding further viral ORFsl , ORFs2 and ORFs3) or in the region comprised in SEQ ID NO: 95 (comprising yet another ORF1 , ORF2 and ORF3 as well as an ORF4).
  • SEQ ID NO: 26 comprising the regions encoding for a viral ORF1 , ORF3 and ORF2 with the exception of the about 15 5'terminal nucleotides
  • the genome of TT virus comprises, e.g., genes as deposited in GENEBANK under accession numbers AB011 482, AB01 1486, AB011 487, AB011 488, AB011 489, AB011 490, AB011 491 , AB008 394, AB011 493, AB011 494, AF 055897, AF 06045, AF060546, AF 060 547, AF060 548, AF060 549, AF060 550.
  • Nucleic acid molecules which do not hybridize to the complementary strand of the genome of TT virus can be deduced by the person skilled in the art without further ado, for example by employing hybridization strategies as described in Britten and Davidson in "Nucleic acid hybridisation", edited by Hames and Higgins (1985), IRL Press Oxford.
  • nucleic acid molecules representing the genome of a virus and having a nucleotide sequence at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably 80% and particularly preferred 95% identical to the DNA sequences as defined in SEQ ID NO: 26, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 125, SEQ ID NO: 186, SEQ ID NO: 191 and SEQ ID NO: 199 above, to nucleic acid molecules having a nucleotide sequence which is at least 60%, preferably 70%, more preferably 80% and particularly preferred 95% identical with the DNA sequences as defined in SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 75, SEQ ID NO: 87, SEQ ID NO: 118, SEQ ID
  • nucleic acid molecules which contain an ORF or a fragment thereof which is at least 50%, preferably at least
  • SEQ ID NO: 183, SEQ ID NO: 192 or SEQ ID NO: 200 which is at least 50%, preferably at least 60% and more preferably at least 75% identical with the nucleic acid molecule of SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 89, SEQ ID NO: 184 or SEQ ID NO: 193, at least 60%, preferably at least 70% and more preferably at least 75% identical with SEQ ID NO: 77, SEQ ID NO: 120, SEQ ID NO: 177 or SEQ
  • ID NO: 201 which is at least 50%, preferably at least 60% and more preferably at least 75% identical with the nucleic acid molecule of SEQ ID NO: 30, SEQ ID NO: 40,
  • SEQ ID NO: 78 SEQ ID NO: 90, SEQ ID NO: 121 , SEQ ID NO: 185, SEQ ID NO:
  • SEQ ID NO: 202 which is at least 50%, preferably at least 60% and more preferably at least 75% identical with the nucleic acid molecule of SEQ ID NO: 79; see feature (e), supra.
  • the present invention also relates to nucleic acid molecules representing the genome of a virus and comprising a nucleic acid molecule encoding an amino acid sequence which is at least 35%, preferably at least 40%, more preferably at least 50% and most preferably at least 75% identical with SEQ ID NO: 21 , SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 91 ,
  • this invention relates to nucleic acid molecules representing the genome of a virus and encoding an amino acid sequence which is at least 40%, preferably at least 45%, more preferably at least 50% and most preferably at least 75% identical with SEQ ID NO: 81 , SEQ ID NO: 121 , SEQ ID NO: 181 or SEQ ID NO: 188; see feature (e), above. Additionally, the present invention relates to nucleic acid molecules representing the genome of a virus wherein parts of said genome can be amplified under suitable conditions by PCR employing as primers oligonucleotides as defined in SEQ ID NO:
  • SEQ ID NO: 205 or the complementary strand of such an oligonucleotide.
  • Suitable conditions for the hybridization of said primers to said genome are known to the person skilled in the art and comprise, preferably, stringent conditions. As described herein below, said conditions are described in standard textbooks, such as
  • primers/probes that specifically recognize and/or detect nucleic acid sequences of the invention.
  • probes/primers are also comprised within the scope of the present invention. Examples of such primers/probes are primers/probes that overlap with the above primers/probes specified by their SEQ ID.
  • the present invention relates furthermore to nucleic acid molecules representing the complementary strand of the above referenced genome and/or ORF(s) or to nucleic acid molecules representing an anti-sense molecule to the above referenced genome and/or ORF(s).
  • nucleic acid molecules of a new viral agent distantly related to TT virus have been found. These nucleic acid molecules are detectable only in a minimal fraction of healthy blood donors but have a high dissemination in the blood of intravenous drug users, a population group with a rather high prevalence of parenterally transmittable diseases like chronic liver diseases of unknown etiology
  • SEN-virus As shown herein, SEN-virus or its subtype(s) appear to be selectively segregated in the sera of patients suffering from Non A-Non E hepatitis (NANE hepatitis), in the sera of about 70% of intravenous drug users but could not be detected in sera obtained from healthy blood donors or patients suffering from an autoimmune disease (rheumatoid anthritis and primary biliary cirrhosis) or from hepatitis B (see appended Examples 6, 14 and furthermore, Examples 17 and 19).
  • NANE hepatitis Non A-Non E hepatitis
  • hepatitis delta patients are positive for SENV is not surprising, since most of the patients screened who are positive for both viral agents are intravenous drug users. However, some HBV patients have been screened as also SENV-positive (see appended example 31). These were only a few hepatitis B patients and they were selected not to have a high risk behavior. Further testing of larger cohorts of hepatitis B patients revealed that some of these patients can be co-infected with certain SENV subtypes. While SEN-virus or its subtypes have a low prevalence among healthy blood donors, the majority of patients suffering from advanced liver disease of unknown origin appear to be positive for this (these) viral agent(s).
  • SEN-virus comprises different types and subtypes and that some of these subtypes are more pathogenic than others.
  • other pathogenic manifestations than hepatopathies may be caused by this viral agent and/or its subtypes.
  • pathogenic manifestations comprise, but are by no means limited to disorders of the gastrointestinal tract and/or proliferative disorders. These disorders comprise Crohn's disease (or other inflammatory bowel diseases), lupus erythematosus, as well as cancers, like hepatocarcinomas and coloncarcinomas (see appended examples 17, 19 and 26).
  • other pathogenic agents or genetic or other dispositions may be involved in the onset of said hepatopathies.
  • the present invention furthermore relates to a nucleic acid representing the genome of said virus which deviates by insertion, substitution, deletion, inversion or duplication from the genome specified above and comprises at least one ORF, preferably two ORFs and most preferably the three different ORFs specified above. Additionally, this invention comprises a nucleic acid representing the genome of said virus which deviates by insertion, substitution, deletion, inversion or duplication from the genome specified above that comprises at least four ORFs as specified herein- above.
  • the present invention relates to a nucleic acid molecule encoding a viral (poly)peptide or a fragment thereof, wherein said nucleic acid molecule
  • (a) contains at least one ORF which encodes a polypeptide having the amino acid sequence of SEQ ID NO: 21 , SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO.
  • SEQ ID NO: 122 SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 180, SEQ ID NO: 181 , SEQ ID NO: 182, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198 or a fragment thereof of at least 6 amino acids;
  • (b) has the sequence identified in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 28 , SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO:
  • (e) is degenerate with respect to the nucleic acid molecule of (d) but does not hybridize to the complementary strand of the genome of TT virus encoding TTV ORF1 and/or TTV ORF2;
  • (f) encodes a polypeptide which is at least 35%, preferably at least 40%, more preferably at least 50% and most preferably at least 75% identical with SEQ ID NO: 21 , SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 124 or SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 or encodes a polypeptide which is at least 40%, preferably at least 50%, more preferably at least 60% and most preferably at least 75% identical with SEQ ID NO: 81 , SEQ ID NO:
  • (g) is at least 50%, preferably at least 60%, more preferably at least 75% and most preferably at least 90% identical with the sequence specified in (b) or at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 90% identical with the sequence specified in (c); or (h) it deviates from any of the above molecules by insertion, substitution, deletion, inversion, duplication, recombination, addition or a combination thereof; or
  • parts of it can be amplified under suitable conditions by PCR employing as primers oligonucleotides as defined in SEQ ID NO: 41 and SEQ ID NO: 42 and/or as defined in SEQ ID NO: 1 15 and SEQ ID NO: 71 or as defined in SEQ
  • said nucleic acid molecule contains two ORFs and most preferably, it contains three ORFs. Additionally and also preferably, said nucleid acid molecules may comprise four or more ORFs.
  • said fragment in feature (a) comprises at least 8 amino acids, more preferably at least 10 amino acids, more preferably at least 12 amino acids, more preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 30 amino acid, and most preferably at least 35 amino acids.
  • said fragment in feature (b) comprises at least 15, more preferably at least 18 nucleotides, even more preferably at least 21 nucleotides, and most preferably at least 28 nucleotides.
  • the invention also relates to a nucleic acid molecule encoding the above viral polypeptide or a fragment thereof wherein said polypeptide deviates by one or more amino acid substitutions, amino acid deletions, amino acid insertions, amino acid inversions, amino acid additions, amino acid recombinations or amino acid duplications or a combination thereof from the above-identified sequence.
  • the present invention provides for a nucleic acid molecule specifically hybridizing to the complementary strand of the nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 26, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 125, SEQ ID NO: 186, SEQ ID NO: 191 or SEQ ID NO: 199 specified above or to said nucleic acid molecule or being identical to said nucleic acid molecule or to said complementary strand thereof wherein said nucleic acid molecule comprises at least 12 nucleotides, preferably at least 15, more preferably at least 18 nucleotides, more preferably at least 21 nucleotides and most preferably at least 25 nucleotides.
  • Said nucleic acid molecule comprises coding, non-coding sequences, sequences which are complementary to the coding strand and, whenever applicable, antisense sequences.
  • viral-non coding sequences are 5' and 3' terminal sequences, like 3'UTRs or 5' IRES (internal ribosome entry site), or 5' and 3' repetitive sequences. They may comprise regulatory sequences for the initiation of transcription. Furthermore, such non-coding regions can be intron sequences.
  • the nucleic acid molecule may also extend 5' or 3' to the above specified SEQ ID NO: 26, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 125, SEQ ID NO: 186, SEQ ID NO: 191 or SEQ ID NO: 199 either when having the identical nucleotide sequence in the overlapping parts of said sequence or the complementary sequence, when hybridizing thereto or when hybridizing to its complementary strand.
  • said fragment of the nucleic acid molecule of the present invention encodes an epitope that reacts with antibodies specific for SEN virus. This means that these epitopes do not react with antibodies specific for hepatitis, A, B, C, D, E, G, Non B-E or TT virus.
  • the nucleic acid molecule of the present invention is a probe or primer.
  • Said probe or primer can comprise the sequence(s) as defined in SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 32, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128; SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 , SEQ ID NO: 132, SEQ ID NO:
  • SEQ ID NO: 13 can also be used for detecting the PCR product obtained with SEQ ID NO: 11 and SEQ ID NO: 12 and/or with SEQ ID NO: 126 and SEQ ID NO: 127.
  • SEQ ID NO: 98 can be used for detecting the PCR product obtained with SEQ ID NOs: 115 and 71
  • SEQ ID NO: 130 can be employed to detect the PCR product obtained with SEQ ID NOs: 128 and 71
  • SEQ ID NO: 173 might be employed to detect the PCR product obtained with SEQ ID NOs: 172 and 71
  • SEQ ID NO: 176 can be used to verify the PCR product obtained with SEQ ID NOs: 174 and 175
  • SEQ ID NO: 205 can be used to detect the PCR product obtained with SEQ ID NOs: 203 and 204.
  • primers or their complements can be used in PCR reactions or similar methods in combination with primers derived from known viral sequences such as, inter alia, primers as shown in SEQ ID NO:1 , SEQ ID NO: 33, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 86 or SEQ ID NO: 178.
  • the nucleic acid molecule of the invention may be DNA such as cDNA or RNA such as mRNA. Additionally, the nucleic acid molecule of the invention may be PNA (peptide nucleic acid). Its origin may be natural, synthetic or semisynthetic or it may be a derivative.
  • said nucleic acid molecule may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination.
  • said nucleic acid molecule is part of a vector.
  • the present invention therefore also relates to a vector comprising the nucleic acid molecule of the present invention.
  • the vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the vector of the present invention may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, translation initiation codon, and translation sites. This vector may also include insertion sites for introducing an insert into the vector.
  • the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells.
  • Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV- HSV thymikine kinase promoter, SV40, RSV-promoter (Rous sarcome virus), human elongation factor 1 ⁇ -promoter, CMV enhancer or SV40-enhancer.
  • promoters including, for example, the tac-lac-promoter or the trp promoter.
  • elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1
  • the vector may further comprise nucleic acid sequences encoding for secretion signals.
  • sequences are well known to the person skilled in the art.
  • leader sequences capable of directing the polypeptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • the vector can also comprise regulatory regions from the viral agent of the invention.
  • the vector of the present invention may also be a gene transfer or targeting vector.
  • Gene therapy which is based on introducing therapeutic genes, for example for vaccination into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • nucleic acid molecules and vectors of the invention may be designed for direct introduction, e.g. by biolistic methods, or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention.
  • the present invention also relates to a host cell transfected or transformed with the vector of the invention or a non-human host carrying the vector of the present invention, i.e. to a host cell or host which is genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid molecule.
  • the term "genetically modified" means that the host cell or host comprises in addition to its natural genome a nucleic acid molecule or vector according to the invention which was introduced into the cell or host or into one of its predecessors/parents.
  • the nucleic acid molecule or vector may be present in the genetically modified host cell or host either as an independent molecule outside the genome, preferably as a molecule which is capable of replication, or it may be stably integrated into the genome of the host cell or host.
  • the host cell of the present invention may be any prokaryotic or eukaryotic cell.
  • Suitable prokaryotic cells are those generally used for cloning like E. coli or Bacillus subtilis.
  • eukaryotic cells comprise, for example, fungal or animal cells.
  • suitable fungal cells are yeast cells, preferably those of the genus Saccharomyces and most preferably those of the species Saccharomyces cerevisiae.
  • Suitable animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, and most preferably non-neuronal cells, such as e.g. CHO, Hela, NIH3T3, MOLT-4, Jurkat, K562, HepG2.
  • suitable cell lines known in the art are obtainable from cell line depositories, like the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • the invention relates to a host cell which is in vitro infected or transfected with the virus/viral agent of the present invention.
  • the host cell which is in vitro infected or transfected with the virus of the invention is a hepatic cell, a macrophage, a lymphocyte, an epithelial cell or an osteocyte or a cell (line) derived therefrom.
  • Host may be mammals, most preferably monkey or apes. Said mammals may be indispensable for developing a cure, preferably a vaccine against the viral agent.
  • the present invention relates to a method of producing a (poly)peptide encoded by the nucleic acid molecule of the invention comprising culturing the host cell of the present invention under suitable conditions that allow the synthesis of said (poly)peptide and recovering and/or isolating the (poly)peptide produced from the culture.
  • the transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the (poly)peptide of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.
  • the protein of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoressis and the like; see, Scopes, "Protein Purification", Springer- Verlag, N.Y. (1982). Substantially pure proteins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the proteins may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures.
  • the present invention relates to a (poly)peptide encoded by the nucleic acid molecule of the invention or produced by or obtainable by the above-described method.
  • the term "(poly)peptide” denotes either a peptide or a polypeptide.
  • the (poly)peptides of the present invention may be recombinant (poly)peptides expressed in host cells like bacteria, yeasts, or other eukaryotic cells, like mammalian or insect cells. Alternatively, they may be isolated from viral preparations. In another embodiment of the present invention, synthetic (poly)peptides may be used. Therefore, such a (poly)peptide may be a (poly)peptide as encoded by the nucleic acid molecule of the invention which only comprises naturally occurring amino acid residues, but it may also be a (poly)peptide containing modifications.
  • covalent derivatives such as aliphatic esters or amides of a carboxyl group, O-acetyl derivatives of hydroxyl containing residues and N-acyl derivatives of amino group containing residues.
  • covalent derivatives such as aliphatic esters or amides of a carboxyl group, O-acetyl derivatives of hydroxyl containing residues and N-acyl derivatives of amino group containing residues.
  • Such derivatives can be prepared by linkage to reactable groups which are present in the side chains of amino acid residues and at the N- and C-terminus of the protein.
  • the (poly)peptide can be radiolabeled or labeled with a detectable group, such as a covalently bound rare earth chelate, or conjugated to a fluorescent moiety.
  • the (poly)peptide of the present invention can be, for example, the product of expression of a nucleotide sequence encoding such a (poly)peptide, a product of chemical modification or can be purified from natural sources, for example, viral preparations. Furthermore, it can be the product of covalent linkage of (poly)peptide domains.
  • amino acid sequences representing the (poly)peptide(s) of the present invention can be easily chemically synthesized using synthesizers which are well known in the art and are commercially available.
  • the present invention relates to a virus or viral agent carrying the genome represented by the nucleic acid molecule of the present invention.
  • the present invention relates to a virus or viral agent which has a genomic sequence which comprises at least 3 ORFs, wherein parts of said genomic sequence can be amplified under suitable conditions by PCR employing as primers oligonucleotides as defined in SEQ ID NO: 41 and SEQ ID NO: 42, as defined in SEQ ID NO: 115 and SEQ ID NO: 71 , as defined in SEQ ID NO: 126 and SEQ ID NO: 127, as defined in SEQ ID NO: 128 and SEQ ID NO: 71 , as defined in SEQ ID NO: 172 and SEQ ID NO: 71 , as defined in SEQ ID NO: 174 and SEQ ID NO: 175 or as defined in SEQ ID NO: 203 and SEQ ID NO: 204 or as defined in any one of SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 32
  • this virus or viral agent is a SEN virus.
  • SEN virus means a virus, virus type, virus class, virus genotype or virus subtype, e.g., SENV-A, SENV-B, SENV-C, SENV-D, SENV-E, SENV-F, SENV-G or SENV-H which comprises a genome having at least one ORF, preferably two, most preferably the three and/or particularly preferred the four different ORFs identified above.
  • the SEN virus/viral agent of the present invention can be a derivative, such as non-pathogenic derivative, of the virus/viral agent specified above, preferably an attenuated virus.
  • An attenuated virus/viral agent is a selected strain of a virulent virus which does not cause the clinical signs associated with the parent virus but still replicates well enough to induce immunity.
  • the person skilled in the art knows of the methods commonly used to obtain such attenuated viral strains. Methods may follow conventional protocols as described, for example in Mahy, BWJ and Kangro, H. O., "Virology Method Manual", Academic Press London (1996). Attenuation may be achieved by heat or chemical treatment or by genetic engineering in which the virus is deprived of its pathogenic genes or parts of them.
  • Attenuated strains are usually obtained by passage in cell culture or in a host different from the natural host.
  • an attenuated strain may be constructed utilizing the genomic information of SEN virus provided herein, and employing recombinant techniques. These attenuated strains are useful for vaccines or for the isolation of antigens.
  • the SEN virus of the present invention can be a chimeric virus comprising at least one ORF, preferably two ORFs, more preferably three ORFs and most preferably four different ORFs of this invention in combination with one or more ORFs from other viral agents, such as, inter alia, TT virus and/or ORF(s) from viruses specific for hepatitis A, B, D, E, G, Non-A-Non-E or any other virus suitable for genetic manipulation.
  • viral agents such as, inter alia, TT virus and/or ORF(s) from viruses specific for hepatitis A, B, D, E, G, Non-A-Non-E or any other virus suitable for genetic manipulation.
  • TT virus and/or ORF(s) from viruses specific for hepatitis A, B, D, E, G, Non-A-Non-E or any other virus suitable for genetic manipulation.
  • the person skilled in the art is enabled by modern, molecular biological methods, such as in vivo and in vitro
  • VLPs immunogenic virus-like particles
  • the present invention relates to a method for producing the virus or viral agent of the invention comprising culturing the above-mentioned host cell under suitable conditions and isolating the virus from the culture.
  • supernatants from said cultures can be harvested or infected cells can be lysed in order to obtain the produced virus.
  • Common methods are, for example, described in Harden MR, "Approaches to anti-viral agents", VCH Pub. (Eds.), (1986).
  • the virus of the present invention is transmissible by blood.
  • the invention relates to a (poly)peptide isolated from viral preparations, wherein said viral preparation comprises a virus of the invention.
  • viral (poly)peptides can be isolated and/or prepared using chromatographic methods, like affinity chromatography employing antibodies directed against these (poly)peptides.
  • the present invention additionally relates to an antibody or a fragment or derivative thereof or an antiserum of an aptamer or another receptor specifically recognizing an epitope on the nucleic acid, the (poly)peptide or on the virus or viral agent of the invention.
  • the general methodology for producing antibodies is well-known and has, for monoclonal antibodies, been described in, for example, K ⁇ hler and Milstein, Nature 256 (1975), 494 and reviewed in J.G.R. Hurrel, ed., "Monoclonal Hybridoma Antibodies: Techniques and Applications", CRC Press Inc., Boco Raron, FL (1982), as well as that taught by L. T. Mimms et al., Virology 176 (1990), 604-619.
  • the term "antibody” relates to monoclonal or polyclonal antibodies.
  • Polyclonal antibodies (antiserum) can be obtained according to conventional protocols.
  • Antibody fragments or derivatives comprise F(ab') 2 , Fab, Fv or scFv fragments; see, for example, Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, NY.
  • the antibody of the invention is a monoclonal antibody.
  • the derivatives of the invention can be produced by peptidomimetics.
  • Said other receptors may, for example, be derived from said antibody etc. by peptidomimetics.
  • the specificity of the recognition implies that other known viruses such as TTV are not bound.
  • a suitable test for assessing the specificity would imply contacting the above recited compound comprising the epitope of the invention as well as corresponding compounds e.g. from TTV, for example in an ELISA format and identifying those antibodies etc. that only bind to the compound of the invention but do not or to no significant extent cross-react with said corresponding compounds.
  • the invention further relates to a derivative of the (poly)peptide of the invention which is specifically recognized by the antibody or fragment or derivative thereof or an aptamer of the invention.
  • Such derivatives can be (semi)synthetically, e.g. chemically produced. Production methods may also employ peptidomimetics. Such production methods are well known in the art and can be applied by the person skilled in the art without further ado.
  • the present invention relates to a hybridoma producing the antibody of the present invention.
  • the preparation of a hybridoma is well known to the artisan, see, for example, Harlow and Lane, loc. cit.
  • the present invention also relates to a fusion protein comprising the (poly)peptide of the present invention.
  • said fusion protein can comprise at least one further domain, said domain being linked by covalent or non-covalent bonds.
  • the linkage can be based on a genetic fusion according to the methods known in the art (Sambrook et al., loc. cit., Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)) or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686.
  • the additional domain present in the fusion protein comprising the (poly)peptide of the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker comprises plural, hydrophiiic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N- terminal end of the peptide, polypeptide or antibody or vice versa.
  • the above described fusion protein may further comprise a cleavable linker or cleavage site, which, for example, is specifically recognized and cleaved by proteinases or chemical agents.
  • said at least one further domain may be of a predefined specificity or function.
  • the (poly)peptides of the invention may be further modified by conventional methods known in the art. This allows for the construction of fusion proteins comprising the (poly)peptide of the invention and other functional amino acid sequences, e.g., nuclear localization signals, transactivating domains, DNA-binding domains, hormone-binding domains, protein tags (e.g. GST, GFP, h-myc peptide, FLAG, HA peptide) which may be derived from heterologous proteins.
  • protein tags e.g. GST, GFP, h-myc peptide, FLAG, HA peptide
  • the present invention relates to a mosaic polypeptide comprising at least two epitopes of the (poly)peptide of the invention or the virus of the invention wherein said mosaic polypeptide lacks amino acids normally intervening between the epitopes in the native SEN virus genome.
  • mosaic polypeptides are useful in the applications and methods described herein, since they may comprise within a single peptide or (poly)peptide a number of immunologically relevant epitopes possibly presented linearly or as multi- antigen peptide system in a case of lysines. Relevant epitopes can be separated by spacer regions.
  • the present invention relates to the nucleic acid molecule, the (poly)peptide, the derivative of the (poly)peptide, the virus, the antibody or fragment or derivative thereof, the aptamer or other receptor, the fusion protein, the mosaic polypeptide, or the primer of the invention which is detectably labeled.
  • a variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention. Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden, RH and von Knippenburg (Eds), Volume 15 (1985), “Basic methods in molecular biology”; Davis LG, Dibmer MD; Battey Elsevier
  • labels There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.
  • Commonly used labels comprise, inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, ⁇ - galactosidase, alkaline phosphatase), radioactive isotopes (like 32 P or 125 l), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).
  • fluorochromes like fluorescein, rhodamine, Texas Red, etc.
  • enzymes like horse radish peroxidase, ⁇ - galactosidase, alkaline phosphatase
  • radioactive isotopes like 32 P or 125 l
  • biotin digoxygenin
  • colloidal metals chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums
  • Labeling procedures like covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, random priming, nick- translations, tailing (using terminal transferases) are well known in the art.
  • Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
  • the present invention relates to a solid phase which is attached to
  • Solid phases are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, animal red blood cells, or red blood cell ghosts, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes.
  • Suitable methods of immobilizing nucleic acids, (poly)peptides, proteins, antibodies, viruses, etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like.
  • the solid phase can retain one or more additional receptor(s) which has/have the ability to attract and immobilize the region as defined above.
  • This receptor can comprise a charged substance that is oppositely charged with respect to the reagent itself or to a charged substance conjugated to the capture reagent or the receptor can be any specific binding partner which is immobilized upon (attached to) the solid phase and which is able to immobilize the reagent as defined above.
  • detection assays comprise radioisotopic or non-radioisotopic methods. These comprise, inter alia, R1A (Radioimmuno Assay) and IRMA (Immune
  • the present invention relates to a diagnostic composition
  • a diagnostic composition comprising
  • nucleic acid molecules of the invention may also comprise PNAs, modified DNA analogs containing amide backbone linkages. Such PNAs are useful, inter alia, as probes for DNA/RNA hybridization.
  • the (poly)peptide of the invention may be, inter alia, useful for the detection of anti-viral antibodies in biological test samples of infected individuals. It is also contemplated that antibodies of the invention may be useful in discriminating acute from non-acute infections.
  • the diagnostic composition optionally comprises suitable means for detection.
  • the (poly)peptides and antibodies or fragments or derivatives thereof or aptamers etc. described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • immunoassays which can utilize said (poly)peptide are competitive and non- competitive immunoassays in either a direct or indirect format.
  • examples of such immunoassays as already described above are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay.
  • RIA radioimmunoassay
  • the (poly)peptides, antibodies, mosaic polypeptides and/or fusion proteins etc. can be bound to many different carriers.
  • Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble or insoluble for the purposes of the invention. Appropriate labels and methods for labeling have been identified above.
  • Said diagnostic compositions may be used in methods for detecting expression of a nucleic acid molecule of the invention by detecting the presence of mRNA coding for a (poly)peptide or viral protein of the invention which comprises, for example, obtaining mRNA from viral preparations and contacting the mRNA so obtained with a probe/primer comprising a nucleic acid molecule capable of specifically hybridizing with a polynucleotide of the invention under suitable conditions (see also supra), and detecting the presence of mRNA hybridized to the probe/primer.
  • Further diagnostic methods leading to the detection of nucleic acid molecules in a sample comprise, e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR), Southern blotting in combination with nucleic acid hybridization, comparative genome hybridization
  • CGH CGH
  • RDA representative difference analysis
  • the present invention further relates to a kit comprising
  • the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of scientific or diagnostic assays or the like.
  • parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
  • the kit of the present invention may be advantageously used, inter alia, for carrying out the method of producing a (poly)peptide of the invention and could be employed in a variety of applications referred herein, e.g., as diagnostic kits, as research tools or vaccination tools. Additionally, the kit of the invention may contain means for detection suitable for scientific medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
  • the present invention relates to a composition
  • a composition comprising (a) the nucleic acid molecule
  • composition is a pharmaceutical composition.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors.
  • dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively.
  • compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously.
  • the compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as interleukins, interferons and/or CpG- containing DNA stretches depending on the intended use of the pharmaceutical composition.
  • compositions as described hereinabove which comprise further agents which are used in treatment of viral infections, hepatopathies, inflammatory diseases or proliferative disorders, like cancer.
  • the pharmaceutical composition of the present invention is a vaccine.
  • Vaccines may be prepared, inter alia, from one or more (poly)peptides, derivatives of the (poly)peptides, nucleic acid molecules, mosaic polypeptides, fusion proteins, viruses, non-pathogenic/attenuated derivatives of the viruses, antibodies, fragments of said antibodies, derivatives of the antibodies or aptamers or other receptors of the invention.
  • nucleic acid molecules of the invention may be used for gene vaccination or as DNA vaccines.
  • Routes for administration of gene vaccines are well known in the art and DNA vaccination has been successfully used to elicit alloimmune, anti-tumor and antiidiotype immune responses (Tighe M. et al., Immunology Today 19 (1998), 89-97).
  • inoculation with nucleic acid molecules/DNA has been found to be protective in different modes of viral disease (Fynan, E.F. et al, Proc. Natl. Acad. Sci. U.S.A. 90 (1993), 1 1478-1 1482; Boyer, I.D.;
  • the (poly)peptides, derivatives of the (poly)peptides, fusion proteins comprising the
  • polypeptide, or the mosaic polypeptide of the invention used in a pharmaceutical composition as a vaccine may be formulated e.g. as neutral or salt forms.
  • Vaccines can be, inter alia, used for the treatment and/or the prevention of an infection with the virus of the present invention and are administered in dosages compatible with the method of formulation, and in such amounts that will be pharmacologically effective for prophylactic or therapeutic treatments.
  • the vaccine comprises an attenuated viral agent as described herein above.
  • a vaccination protocol can comprise active or passive immunization, whereby active immunization entails the administration of an antigen or antigens (like, for example, the virus/viral agent, the (poly)peptides, the derivatives of the (poly)peptides, the fusion proteins, the mosaic polypeptides and/or the nucleic acid molecules of the present invention) to the host/patient in an attempt to elicit a protective immune response.
  • Passive immunization entails the transfer of preformed immunoglobulins or fragments thereof (for example, the above antibodies, the derivatives or fragments thereof) or the aptamers of the present invention to a host/patient.
  • vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection also may be prepared.
  • the preparation may be emulsified or the protein may be encapsulated in liposomes.
  • the active immunogenic ingredients often are mixed with pharmacologically acceptable excipients which are compatible with the active ingredient.
  • Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used.
  • the vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • such adjuvants can include aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-DMP), N-acetyl-nomuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-
  • thr-DMP N-acetyl-muramyul-L-alanyl-D-isoglutaminyl-L-alanine-
  • CGP 19835A also referred to as MTP-PE
  • RIBI MPL + TDM + CWS
  • the vaccines usually are administered by intravenous or intramuscular injection. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral or nasal formulations.
  • traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides.
  • Oral formulation include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tables, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
  • DNA vaccines can be administered by biolistic methods, such as particle bombardment.
  • Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective.
  • the quantity to be adminstered generally is in the range of about 5 micrograms to about 250 micrograms of antigen per dose, and depends upon the subject to be dosed, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient required to be administered also may depend upon the judgment of the practitioner and may be unique to each subject.
  • the vaccine may be given in a single or multiple dose schedule.
  • a multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, (a) subsequent dose(s) after several months.
  • the dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner's judgment. It is contemplated that the vaccine containing the immunogenic compounds of the invention may be administered in conjunction with other immunoregulatory agents, for example, with immunoglobulins or with cytokines.
  • the present invention relates to a method of detecting the presence of the virus or viral agent in a sample comprising
  • Said primers may be of any length which allows stable hybridization to said DNA under suitable conditions (as defined herein above).
  • said pair of primers comprise primers selected from the group consisting of the sequence(s) as defined in SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 32, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID
  • these pairs of primers comprise the sequences as defined in SEQ ID NO: 41 and SEQ ID NO: 42, in SEQ ID NO: 115 and SEQ ID NO: 71 , in SEQ ID NO: 126 and SEQ ID NO: 127, in SEQ ID NO: 128 and SEQ ID NO: 71 , in SEQ ID NO: 172 and SEQ ID NO: 71 and/or in SEQ ID NO: 174 and SEQ ID NO: 175.
  • Suitable further DNA amplification techniques are known in the art and comprise, inter alia, Ligase Chain reaction, Strand Displacement Amplification, Nucleic Acid
  • NASBA Sequence based Amplification
  • Q-beta replicase based techniques Q-beta replicase based techniques.
  • hybridization is effected under stringent conditions; see, e.g., Sambrook et al., loc. cit.
  • the invention relates to a method of detecting the presence of the virus of the invention in a sample comprising
  • the invention relates to a method for detecting the antibody or fragment or derivative thereof or an aptamer or other receptor of the invention in a sample, comprising
  • Detection per se in the above recited method can be done according to conventional protocols.
  • hybridized or amplified DNA can be detected in a gel, for example, by reference to the molecular weight of the nucleic acid molecule, by using anti-double strands antibodies or by detecting proteinaceous complexes formed with secondary antibodies.
  • Other options for the detection step are immediately apparent to the person skilled in the art. Suitable formats for the detection step comprise ELISAs, RIAs and the like.
  • Methods of detection of viruses/viral infections are well known in the art and comprise, besides immunoassays, microscopy techniques, like immunodetections in light- and electron-microscopy (see, for example, Fields "Virology" 3 rd edition,
  • the invention relates to a method as described above, wherein said nucleic acid molecules, at least one of said primers or said antibody or fragment or derivative thereof or aptamer or other receptor is detectably labeled.
  • Such labels are well known to the person skilled in the art, have been referred to herein above, and may comprise a tag, a fluorescent marker or a radioactive marker.
  • Any detection method for detecting the presence of the virus of the invention or the presence of antibodies generated against the virus of the invention may be assisted by computer technology. Detection methods can therefore be automated by various means, including image analysis or flow cytometry. For a further understanding of markers and preferred embodiments thereof, it is referred to corresponding passages herein above.
  • the present invention relates to a method as described above wherein said sample is or is derived from blood, serum, sputum, feces or another body fluid.
  • the sample to be analyzed may be treated such as to extract, inter alia, nucleic acid molecules, (poiy)peptides, or antibodies.
  • the present invention relates to a preparation of substantially isolated polyclonal antibodies specifically immunoreactive with the nucleic acid molecule, the (poly)peptide of the invention or the virus of the invention. How to obtain such a preparation of polyclonal antibodies is well known to the artisan and can be carried out without any undue experimentation (see Harlow and Lane, loc. cit.). Said polyclonal antibodies may be monospecific or polyspecific.
  • the present invention relates to a method of producing antibodies to the nucleic acid molecule, the (poly)peptide, the derivative or the virus of the invention comprising immunizing an experimental animal with said nucleic acid, (poly)peptide, derivative, virus/viral agent or the non-pathogenic derivative of the invention and isolating serum or specific antibodies produced.
  • the antibody obtained from the animal may be further manipulated, e.g., cleaved to obtain suitable fragments or may be the starting compound for genetically engineered antibodies or derivatives thereof, such as scFv fragments having the same binding specificity. Alternatively, it may be the starting compound for the production of corresponding synthetic compounds manufactured, for instance, by peptidomimetics.
  • the production of antibodies is well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, antibodies or fragments such as Fab fragments, and fragments produced by a Fab or scFv expression library.
  • various hosts including goats, rabbits, rats, mice, and others, may be immunized by injection with nucleic acids, (mosaic) polypeptides of the present invention or any fragment or oligopeptide or derivative thereof which has immunogenic properties or forms a suitable epitope.
  • nucleic acids nucleic acids
  • (mosaic) polypeptides of the present invention or any fragment or oligopeptide or derivative thereof which has immunogenic properties or forms a suitable epitope.
  • virus/viral agent or derivatives thereof may be used for immunization.
  • Techniques for producing and processing polyclonal antibodies are known in the art and are described in, among others, Mayer and Walker, eds., "Immunochemical Methods in Cell and Molecular Biology", Academic Press, London (1987).
  • Polyclonal antibodies also may be obtained from an animal, preferably a mammal, previously infected with the virus of the invention.
  • Methods for purifying antibodies are known in the art and comprise, for example, immunoaffinity chromatography.
  • various adjuvants or immunological carriers may be used to increase immunological responses.
  • adjuvants include, but are not limited to, Freund's, complete or incomplete adjuvants, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions and dinitrophenol.
  • An example of a carrier, to which, for instance, a peptide of the invention may be coupled is keyhole limpet hemocyanin (KLH).
  • the (mosaic) (poly)peptides, fragments, or oligopeptides, derivatives used for the production of antibodies have an amino acid sequence consisting of at least five amino acids, such as six, seven, eight or nine and more preferably at least 10 amino acids and most preferably at least 15 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of a natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of (poly)peptides or fragments thereof of the invention may be fused with those of another protein such as keyhole limpet hemocyanin and antibody may be produced against the chimeric molecule. This includes specific antibodies to the (poly)peptide of the invention. A different strategy would be to couple the short peptides as haptens to a carrier such as KLH or BSA using conventional methods and immunizing the animals with such immunoconjugates.
  • a carrier such as KLH or BSA
  • the present invention relates to a method of immunizing an individual against the virus or viral agent of the invention, preferably SEN viruses comprising administering to said individual at least one dose of the vaccine of the invention.
  • Said individual is preferably a human.
  • boost injection such as one, two or three boost injections.
  • routes, doses and time intervals it is referred to previous passages in this specification.
  • the invention relates to a method of propagating the virus/viral agent of the invention comprising culturing the host cell of the invention under conditions suitable to promote the propagation of said virus.
  • Viral propagation methods are generally known in the art (see, e.g., Fields, "Virology", 3 rd ed., 1996, Lippincott Raven Publishers, Philadelphia) and can be adapted by the person skilled in the art using or adapting routine experimentation.
  • the invention relates to a method of propagating the virus of the invention in an animal comprising inoculating said animal with said virus, part(s) of said virus and/or material infected by said virus.
  • the replication of said virus can be monitored in the blood or in various organs.
  • Infected cells can be isolated and these cells can be utilized for viral preparations.
  • Animal models used for viral preparations and for virus propagations are well known in the art and comprise, but are not limited to, mice, rats, rabbits, guinea pigs, ducks, cats, dogs, chimpanzees, marmosets.
  • the present invention relates to the use of an anti-viral agent for the preparation of a pharmaceutical composition for the treatment of a disease which is related to and/or caused by the virus or viral agent of the invention.
  • Said disease is preferably selected from the group consisting of hepatopathies, inflammatory diseases and proliferative disorders.
  • Antiviral agents are well-known in the art and comprise, inter alia, interferons (like interferon alpha) or drugs like tribavirin, lamivudine, aciclovir and/or ursodeoxycholic acid.
  • Other compounds used in the treatment of viral infections comprise amantadine hydrochloride, cytarabine and levamisole hydrochloride (see, e.g., Martindale; "The
  • the invention relates to the use of the antibody or fragment or derivative thereof or an aptamer or other receptor of the invention for the preparation of a pharmaceutical composition for the treatment or a disease which is related to and/or caused by the virus or viral agent of the invention, preferably for the treatment of hepatopathies, inflammatory diseases and/or proliferative disorders.
  • Said hepatopathy can be, inter alia, acute or chronic hepatitis of unknown etiology (NANE-hepatitis), said inflammatory disease may be Crohn's disease or Lupus erythematosus and said proliferative disorder comprises cancer, like, inter alia, liver or colon cancer (liver or colon carcinomas).
  • NANE-hepatitis acute or chronic hepatitis of unknown etiology
  • said inflammatory disease may be Crohn's disease or Lupus erythematosus
  • said proliferative disorder comprises cancer, like, inter alia, liver or colon cancer (liver or colon carcinomas).
  • the invention relates to methods of treating or preventing a disease in a mammal, preferably a human that is afflicted with the virus/viral agent of the invention comprising administering to said mammal one or more doses of an anti-viral agent, antibody, fragment or derivative thereof, aptamer or other receptor, all as described before.
  • diseases to be treated with the method of the invention have also been identified before.
  • the regimen of administration are preferably conventional uses as have been described hereinbefore.
  • the above compound(s) may be administered together with further medicaments optionally directed to further etiologic agents.
  • Figure 1 PCR products obtained with primers TTV-1 and NG063 using DNA extracted from the sera of the indicated patients as template.
  • the arrows indicate the size of the bands obtained.
  • Figure 2 Alignment of nucleic acid sequences obtained with primers TTV-1 and NG063 from patients SEN, 37 and 28. Identical bases are indicated by asterisks.
  • Figure 3 Alignment of nucleic acid sequences obtained with primers TTV-1 and NG063 from patients SEN and patient 26. Identical bases are indicated by vertical lines.
  • Figure 5 Gene walking poiymerase chain reaction strategy for sequence extension. PCR products obtained with DNA extracted from the sera of patient SEN and from an healthy blood donors with primers NG063 and CWP. The 1200 bp bands obtained with DNA extracted from patient SEN is indicated by the arrow.
  • FIG. 7 DNA Enzyme Immuno Assay (DEIA) analysis of PCR products obtained with primers 3S and 2AS using DNA obtained from patient SEN (sample 1) and from 37 healthy blood donors (samples 2-38).
  • EIA DNA Enzyme Immuno Assay
  • PCR product were hybridized with biotinilated probe SEN 37 in a microwell plate and the bound material was revealed as previously described with a colorimetric method.
  • Figure 8 DNA Enzyme Immuno Assay (DEIA) analysis of PCR products obtained with primers 3S and 2AS using DNA obtained from 41 intravenous drug addicts subjects. Ten ⁇ l of PCR product were hybridized with biotinilated probe SEN 37 in a microwell plate and the bound material was revealed as previously described with a calorimetric method.
  • DEIA DNA Enzyme Immuno Assay
  • Figure 9 Alignments of sequences NAE8-1 , NAE35-5, NAE9-3 and NAE10-4 obtained by PCR with primers 2AS and 3S from four patients suffering from hepatitis of unknown etiology.
  • FIG. 14 Alignment of the CWPSEN 001 and SENV-B nucleotide sequences. Identical nucleotides are indicated with vertical bars.
  • Figure 15 Alignment of the CWPSEN 001 and SENV-B deduced amino acid sequence. Identical residues are indicated with vertical bars.
  • Figure 16 Alignment of the deduced amino acid sequence of ORF 1 of TTV and of
  • Figure 17 Nucleotide sequence of SENV-A and its translation ain the three reading frames.
  • Figure 19 Alignment of the nucleotide sequences according to major ORFs of SENV-A and of TTV. Identical nucleotides are indicated with vertical bars.
  • Figure 20 Alignment of the predicted amino acid sequences encoded by the main coding regions of TTV and SENV-A sequences. Identical residues are indicated with vertical bars.
  • FIG. 22 Alignment of the nucleotide sequences encoding SENV-A and SENV-B. Bold characters indicate the initiator and terminator codons of the three different open reading frames (ORFs).
  • Figure 25 Alignment of the deduced ORF 2 protein sequences of SENV-A and SENV-B.
  • Figure 26 Alignment of the deduced ORF 3 protein sequences of SENV-A and
  • Figure 31 Alignment of the deduced protein sequences encoded by the DNA fragments amplified with primers L1 S and L3AS. conserveed amino acids are indicated with asterisks.
  • Figure 32 Unrooted phylogenic tree derived from alignments of deduced protein sequences encoded by the DNA fragments amplified with primers L1A and L3AS. The most homologous region of TTV and SENV-A, as well as a random sequence derived from HGV have been included in the a analysis.
  • Figure 35 Alignment of the deduced ORF 1 protein sequences of SENV-C and TTV.
  • Figure 36 Alignment of the deduced ORF 2 protein sequences of SENV-C and
  • Figure 40 Alignment of the deduced ORF 3 protein sequences of SENV-C and SENV-B.
  • Figure 41 Alignment of the nucleotide sequences encoding SENV-C and SENV-A (ORF1 region).
  • Figure 44 Alignment of the ORF 2 nucleotide sequences encoding SENV-C and SENV-A.
  • Figure 45 Alignment of the ORF 2 nucleotide sequences encoding SENV-C and SENV-B.
  • Figure 46 Alignment of the ORF 2 nucleotide sequences encoding SENV-C and TTV.
  • Figure 47 Alignment of the ORF 3 nucleotide sequences encoding SENV-C and
  • Figure 48 Alignment of the ORF 3 nucleotide sequences encoding SENV-C and SENV-B.
  • Figure 51 Phylogenic distances calculated on the basis of the alignments of the indicated amino acid sequences.
  • Figure 54 Optical density values obtained with DNA extracted from indicated clinical samples and amplified with SENV specific primers.
  • Figure 56 Percentage of SENV-A positive samples among different groups of patients. The numbers in brackets indicate the number of patients analyzed for each group.
  • Figure 57 Percentage of SENV-B positive samples among different groups of patients. The numbers in brackets indicate the number of patients analyzed for each group.
  • Figure 58 Percentage of SENV-C positive samples among different groups of patients. The numbers in brackets indicate the number of patients analyzed for each group.
  • Figure 59 Percentage of SENV-D positive samples among different groups of patients. The numbers in brackets indicate the number of patients analyzed for each group.
  • Figure 60 DNA extracted from serum samples which are positive for either SENV- A, SENV-B, SENV-C or SENV-D were tested with reaction 1 , reaction 2 or reaction 3 (For detailed information on the three different reactions see example 20). The data are expressed as optical values of a DEIA assay. Positive values are indicated in bold.
  • FIG 61 Western blot of recombinant SENV-A ORF2 protein (30k mw) .Western blot with serum ID 2174 (dil. 1 :100), tracer anti-human IgG-HRP (dil. 1 :500).
  • Lane 1 Low Molecular Weight Marker
  • Lane 2 Loaded ORF2
  • Lane 3 Flow Through
  • Lane 4 Pool 4-1 1
  • Lane 5 Pool 19-25
  • Lane 6 Pool 33- 39
  • Lane 7 Pool 48-54
  • Lane 8 Pool 61-64.
  • Figure 62 Prevalence of different SENV subtypes among different, indicated chorts of patients.
  • Figure 64 Comparative analysis of the percentage of identities at the amino acid level of the different ORFs. Alignment were obtained using the PALIGN program of the PCGENE package using the following parameters: Open Gap Cost: 10 and Unit Gap Cost: 2.
  • Figure 65 Comparative analysis of the percentage of identities at the nucleotide level of the different ORFs. Alignment were obtained using the NALIGN program of the PCGENE package using the following parameters:
  • HIV viremia squares
  • SENV viremia dots
  • FIG. 68 Detection of SENV in the supernatant of PBC culture.
  • PBC from a SENV positive patient were cultured for 5 days in the presence of PHA.
  • Supernatant fractions were collected at the indicated days and tested for SEN presence. The data are expressed as optical density values.
  • Figure 69 Detection of SENV RNA in liver tissue. Reactivities of PCR products obtained with SENV primers using RNA and cDNA derived from neoplastic (TT) and peritumoral (TS) surgical liver tissues obtained from 2 hepatocarcinoma patients.
  • TT neoplastic
  • TS peritumoral
  • Figure 70 Map of the clones deposited with DSMZ, Mascheroder Weg 1 b, D- 38124 Braunschweig, GERMANY. Bold lines represent the non-coding nucleotide sequences conserved among TTV and all the SEN viruses.
  • Example 1 Estimation of prevalence and diversity of TTV in the Italian population using specific TTV primers Following the discovery of TTV Nishizawa, Hepatology Res 10 (1998), 1 -16, the prevalence and the diversity of this viral agent in the Italian population was studied.
  • the nested PCR method originally described by Okamoto et al., Hepatology Res 241 (1998), 1 -16 was used under the employment of the sense primers NG059 and NG061 and anti-sense primer NG063 as defined in Okamoto et al., loc. cit.
  • PCR was performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ -mercaptoethanol, 1.5 mM MgCI 2 ), 300 ng of each PCR primer, 200 ⁇ M deoxyribonucleoside triphosphates (dATP, dCTP, dTTP and dGTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer). The reaction was performed in a DNA thermal cycler with mineral oil.
  • PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • the PCR products were loaded on a 2% agarose gel with 2 ⁇ g of ethidium bromide per ml to determine the sizes of the amplified products.
  • TTV was detected in 10% of intravenous drug users and in only 1% of blood donors healthy controls Samples were obtained from the Transfusion Unit of the Ospedale Civile of Brescia, Italy. DNA sequencing analysis was carried out in 10 TTV-positive samples which verified that these amplicons represented the TTV genome. Furthermore, this analysis revealed a great degree of variability between the different pantry TTV isolates.
  • Example 2 Single step PCR for the estimation of prevalence and diversity of TTV in the
  • PCR primers used in this assay were NG063 anti-sense Okamoto et al., loc. cit. and primer sense TTV-1 S, deduced on the basis of sequences found in the Italian population.
  • the sequence of primer TTV-1 S was:
  • PCR was performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ -mercaptoethanol, 1.5 mM MgCI 2 ), 300 ng of each PCR primer, 200 ⁇ M deoxyribonucleoside triphosphates (dATP, dCTP, dTTP and dGTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer). The reaction was performed in a DNA thermal cycler with mineral oil.
  • PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • the PCR products were loaded on a 2% agarose gel with 2 ⁇ g of ethidium bromide per ml to determine the sizes of the amplified products.
  • Negative controls containing ail of the reagents but lacking template DNA were routinely processed exactly as described above to monitor for contamination and were negative in all experiments.
  • TTV infection was detected in 55% of intravenous drug users and in 13% of blood donors, confirming therefore the high incidence of this viral agent in the normal population.
  • PCR was carried out on samples derived from intravenous drug users as described in Example 2, supra.
  • amplicons derived from two intravenous drug users had a length of 580 bp.
  • the PCR product of two intravenous drug users contained both fragments of 513 bp and 580 bp (Fig. 1).
  • amplicons containing poly A tails were excised from the agarose gel and the DNA content was purified and ligated to a pGEM vector (Promega, Madison, WI, USA). Using DNA extracted from with the different obtained PCR products transformed E.
  • ORF capable of encoding 187 amino acids was recognized in sequence SEN 8001 in a reading frame starting at the second nucleotide. No further ORF in the other two reading frames starting at the first or third nucleotide, or in any of the three reading frames in the complementary sequence which could code for > 100 amino acids could be deduced.
  • the amino acid sequence of this ORF had no significant homology with any of the protein sequences contained in the database (GENEBANK) and had only 32 % of homology with the ORF encoded by the TTV sequence.
  • the 581 bp amplicon derived from two additional intravenous drug users had a sequence that was 95 % homologous to the one derived from patient SEN, demonstrating that this viral agent is present in different individuals and that is rather conserved.
  • the alignment of clone SEN8001 and the clones N37 (SEQ ID NO: 4) and N28 (SEQ ID NO: 5) is documented in Fig. 2.
  • Clone 26 A (SEQ ID NO: 6) was derived from an intravenous drug user whose amplicon seemed to migrate slightly slower than the 580 band detected in patient SEN.
  • Fig. 3 shows the alignment of sequence SEN8001 with the one obtained from clone 26A. The homology between the two clones was 81 %, furthermore clone 26A contained an insertion of 15 nucleotides. Thus clone 26A (SEQ ID NO: 6) encodes for a subtype of SENV.
  • clone 26A had 76 % of homology with clone SENV and, as expected, an insertion of a stretch of 5 amino acids (SEQ ID NO: 7).
  • Fig. 4 shows the alignment between the ORFs encoded by clones SEN8001 , N37, N28 and 26A.
  • the "targeted gene walking polymerase chain reaction” strategy was used, as originally described by Parker et al., Nuc. Ac. Research 19 (1991), 3055-60.
  • the method is based upon the observation that a primer may initiate PCR at either unknown or specific target sequences which bear only partial homology at the 3' end.
  • the technique can be used to "walk” along the DNA sequence or to search for nucleotide sequences that have been designed by the user.
  • the method allows the production of micrograms of DNA of unknown sequences that occur upstream or downstream from a single region of targeted DNA.
  • a series of PCR reaction all containing the same NG063 targeted primer as used in Example 2 were set up.
  • walking primer one in each different PCR reaction
  • oligonucleotides that had been previously synthesized for various different target amplifications were utilized.
  • the primers used in these experiments were derived from sequences of the human T-cell receptor, from hepatitis G virus, from hepatitis B virus and from hepatitis C virus. The most useful primer was the primer denoted CWP, as described below.
  • PCR For each couple of primers, duplicate PCR reactions, one containing DNA extracted from positive sample and the other one containing DNA extracted from negative blood donor were set up. PCR was performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI pH 8.8, 16.6 mM (NH ) 2 S0 4 , 10mM ⁇ - mercaptoethanol, 1.5 mM MgCI 2 ), 300 ng of each PCR primer, 200 ⁇ M deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, dGTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer).
  • QIAamp blood kit QIAamp blood kit
  • PCR buffer 67 mM Tris HCI pH 8.8, 16.6 mM (NH ) 2 S0 4 , 10mM ⁇ -
  • PCR was performed in a DNA thermal cycler with mineral oil. PCR consisted of a preheating at 94°C for 5 min, 50 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • the PCR products were loaded on a 2% agarose gel with 2 ⁇ g of ethidium bromide per ml to determine the sizes of the amplified products. Only bands that were present in the positive sample and absent in the reaction containing the negative sample were excised from the agarose gel, cloned and sequenced. Most of the clones contained genomic DNA, however, upon sequencing, one band of 1200 bp (Fig.5 (SEQ ID NO: 8)) obtained with primers
  • SEQ ID NO: 178 was found to contain the SENV sequence extended of 700 bases in 5' (clone CWP SEN 001 ; SEQ ID NO: 8).
  • the "targeted gene walking strategy” was employed. However, instead of using two primers, only the primer 3S (5'TCC ATA TTC ATA GGC CCC AA3' SEQ ID NO: 11 ) derived from the sequence SEN8001 was utilized. By using this strategy, a clone containing the SENV sequence extended of 200 bases in 3' was obtained. After assembling the overlapping sequences with the ASSEMLGEL program of the PC-GENE package an unique sequence of 1353 bases encoding for part of the SENV genome was obtained. This sequence was named CWP SEN001 (SEQ ID NO: 9).
  • the SENV ORF characterized contains two potential N-glycosylation sites at positions 54 and 233, one thyrosine sulfatation site at position 160, three potential protein kinase C phosphorylation sites at positions 89, 235 and 431 , four potential casein kinase II phosphorylation sites at positions 119, 161 , 315 and 319 and two potential amidation sites at positions 83 and 228. N-Myrisoylation sites were not detected. In comparison, the region of highest homology with SENV of the TTV ORF
  • FIG. 6 contained 4 N-glycosilation sites at positions 175, 191 , 206 and 234, one tyrosine sulfatation site at position 275, seven protein kinase C phosphorylation sites at positions 77, 99, 158, 207, 223, 311 and 325, eight casein kinase II phosphorylation sites at positions 17, 119, 144, 158, 232, 271 , 294 and 359, one tyrosine kinase phosphorylation site at position 103, seven N-myrisoylation sites at positions 128, 190, 195, 240, 267, 281 and 282 and one amidation site at position 82.
  • SENV are different viruses that may be distantly related.
  • PCR products were loaded on a 2% agarose gel with 2 ⁇ g of ethidium bromide per ml to determine the sizes of the amplified products.
  • Negative controls containing all of the reagents but lacking template DNA were routinely processed exactly as described above to monitor for contamination and were negative in all experiments.
  • PCR was performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ -mercaptoethanol, 2 mM MgCI 2 ), 300 ng of each PCR primer, 200 ⁇ M deoxyribonucleoside triphosphates (dTTP, dATP, dCTP, dGTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer). The reaction was performed in a DNA thermal cycler with mineral oil. PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • PCR products were loaded on a 2% agarose gel with 2 ⁇ l of ethidium bromide per ml to determine the sizes of the amplified products.
  • Negative controls containing all the reagents but lacking DNA template were routinely processed exactly as described above to monitor for contamination and were negative in all the experiments.
  • SENV was detected in none of the 37 blood donors (Fig. 7) and in none of the 32 patients suffering from rheumatoid arthritis tested. However, the viral agent was detected in 29 of the 41 (67%) intravenous drug users analyzed (Fig. 8). Thus, these data document the parenteral route of transmission of SENV.
  • Example 7 SENV and SENV subtypes as pathogens involved in hepatitis of unknown ethiology
  • DNA extracted from the sera of 20 patients suffering from hepatitis nonA- nonE was amplified.
  • the amplification was carried out with primers 3S (SEQ ID NO: 11 ) and 2AS (SEQ ID NO: 12).
  • PCR was performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum with QIAamp blood kit (QIAGEN), PCR buffer (67 mM
  • PCR consisted of a preheating at 94°C for 5 min, 50 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • NAE8-1 SEQ ID NO: 14
  • NAE9-3 SEQ ID NO: 15
  • NAE10-4 SEQ ID NO: 14
  • NAE 35-5 SEQ ID NO: 17
  • AutoRead sequencing Kit Pharmacia Biotech
  • fluorescent M13 Universal primer and fluorescent M13 Reverse Primer Pharmacia Biotech
  • Figure 9 shows the alignments of the sequences derived from these four patients suffering from NonA-NonE hepatitis. The four sequences have 98.28% identity, suggesting that a virus encoded by this particular genome is highly prevalent in NonA-NonE hepatitis patients.
  • Figures 10, 11 , 12 and 13, respectively, show the alignments of clones NAE8-1 and NAE9-3, NAE10-4 and NAE 35-5 with clone SEN8001. These sequences have an homology with clone SEN8001 that varies between 65,90% to 69,77%, suggesting that clones NAE8-1 NAE9-3, NAE10-4 and NAE35-5 are a new subtype of SENV that is named SENV-B.
  • SENV-B appears to be selectively segregated in the sera of NonA-NonE patients suggests that this SENV subtype may be involved in the transmission of hepatitis of unknown hetiology.
  • the numerous mismatches between the SEN 37 biotinilated probe and the SENV-B sequences can account for the low reactivity of the PCR products derived from NonA-NonE patients in the DEIA hybridization assay and implies that SENV-B is not a causative agent of NANE-hepatitis.
  • NAE 8-1 sequence SEQ ID NO: 14
  • DNA extracted from the serum of patient NAE 8 was amplified with the primer NAE 2AS (SEQ ID NO: 18) derived from SEQ ID NO: 14 and primer 6S (SEQ ID NO: 19) derived from the CWPSEN 001 sequence (SEQ ID NO: 9).
  • PCR was performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ -mercaptoethanol, 2 mM MgCI 2 ), 300ng of each PCR primer, 200 ⁇ M deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, dGTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer). The reaction was performed in a DNA thermal cycler with mineral oil.
  • PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • the PCR products were loaded on a 2% agarose gel with 2 ⁇ g of ethidium bromide per ml to determine the sizes of the amplified products.
  • a gel piece corresponding to the 1100 bp band was cut out.
  • the DNA was extracted, cloned into a pGEM vector (Promega, Madison, WI, USA), introduced into E. Coli and sequenced using standard methods.
  • SENV-B SEQ ID NO: 20
  • the DNA sequence of the insert was determined, using the AutoRead sequencing Kit (Pharmacia Biotech) with fluorescent M13 Universal primer and fluorescent M13 Reverse primer (Pharmacia Biotech).
  • Fig. 14 shows the alignment of SENV-B and the CWPSEN001 nucleotide sequences.
  • the two sequences show 52,11% homology and the ALIGN program introduced 6 gaps in the SENV-B sequence in order to obtain the best score.
  • the alignment of the CWPSEN001 amino acid sequence and the SENV-B predicted amino acid sequences (SEQ ID NO: 21) is shown in Fig. 15.
  • the two protein sequences show regions of high homology, confirming their phylogenic relationship. However, the region spanning amino acid 185 to 285 of the SENV-B sequence is highly divergent from its CWP SENV001 counterpart. In total, the two sequences share 55% of identity.
  • Fig. 16 shows the alignment of the predicted amino acid sequence of the deduced ORF of SENV-B with ORF 1 of TTV.
  • the two sequences show only 29% of identity. Furthermore, differences are uniformly scattered along the alignment of the two viral amino acid sequences.
  • SENV- or some of its subtypes may have important consequences for public health.
  • other genetic variants of this viral agent may vary in their ability to cause disease particularly, but not exclusively, of hepatic nature.
  • the reaction was performed in a DNA thermal cycler with mineral oil. PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • the primer used for this reaction was primer 8S (SEQ ID NO: 22) deduced from the 3' region of sequence CWP SEN001.
  • the second reaction was performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum from patient SEN with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ - mercaptoethanol, 2 mM MgCI ⁇ ), 300ng of a single PCR primer), 200 ⁇ M concentration of each deoxyribonucleoside triphosphate (Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer).
  • the reaction was performed in a DNA thermal cycler with mineral oil.
  • PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • the primer used for this reaction was primer 7AS (SEQ ID NO: 23) deduced from the 5' region of sequence CWP SEN001.
  • the PCR products were loaded on a 2% agarose gel with 2 ⁇ g of ethidium bromide per ml to determine the sizes of the amplified products.
  • the SENV-A sequence shows an identity of 54% to the nucleotide sequence of TTV.
  • Fig. 18 shows the alignment between these two sequences. The sequences are highly homologous at the 5' and at the 3' termini, however, they diverge substantially in the coding regions, showing that the two viruses are clearly distinct but belong to the same family.
  • SEQ ID NO: 26 relates to the nucleotide sequence of SENV-A,. excluding the 5' and 3' portions which are highly homologous to TTV.
  • the alignment of the nucleotide sequences encoding for the major ORF of SENV-A and TTV is shown in Figure 19. The identity between the two coding regions is only of 48% with the insertion of numerous gaps.
  • ORF2 spans nucleotide 231 to 730 (SEQ ID NO: 28). This 166 amino acids sequence (SEQ ID NO: 27), rich in proline, was recognized in the first reading frame and its alignment with the ORF2 of TTV demonstrated that the two proteins have only 30% identity.
  • ORF3 spanning nucleotides 2545 to 280/1 (SEQ ID NO: 30) overlaps with the 3' end of ORF1 but it was recognized on the first reading frame.
  • the protein comprises only 87 amino acids (SEQ ID NO:29) but it contains an interesting nuclear binding region and a TATA motif was recognized 35 nucleotide upstream of the ATG starting codon, suggesting therefore that this potential reading frame is indeed translated. No equivalent ORF was detected in the TTV nucleotide sequence.
  • PCR reaction were performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum from patient NAE 8 with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI, pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ -mercaptoethanol, 2 mM MgCI 2 ), 300ng of each PCR primer, a 200 ⁇ M concentration of each deoxyribonucleoside triphosphate (dATP, dGTP, dCTP, dTTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer).
  • the reaction was performed in a DNA thermal cycler with mineral oil. PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and an incubation at
  • SENV-B SEQ ID NO: 34.
  • SENV-A SENV-B contains three open reading frames.
  • ORF 2 (SEQ ID NO: 35), starts from nucleotide 259 and ends at nucleotide 727
  • ORF1 (SEQ ID NO: 36) starts at nucleotide 831 and ends at nucleotide 2870
  • ORF3 (SEQ ID NO: 37) spans nucleotide 2598 to 2847.
  • the SENV-B sequence shares 68.12% of identity to the nucleotide sequence of SENV-A.
  • Figure 22 shows the alignment of these two sequences. Interestingly, the sequences are highly homologous at the 5' and the 3' termini while they diverge in the coding regions.
  • the alignment of the amino acid sequences encoding for the major ORF (ORF1) of SENV-A and full-length ORF1 of SENV-B and of TTV ORF1 are shown in Figures 23 and 24, respectively.
  • the identity between the SENV-A and SENV-B ORF1 is 56.07%, while the ORF1 of TTV shares only 34.76 % of identity with the corresponding full-length ORF of SENV-B.
  • FIGS. 25 and 26 show the alignment of the ORF 2 and ORF3 sequences of SENV-A and SENV-B.
  • the identity between the two ORF2 is 33.33%, while the one between the two ORF3 is 27.71%, indicating that these proteins are specific subtypes and confer genotype different tropism or different biological functions to these SENV subtypes.
  • SENV-B ORF2 shares 22.84% amino acid identity with TTV ORF2.
  • the nucleotide sequences encoding the SENV-B ORF1 , ORF2 and ORF3 are shown in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively.
  • the alignment of the nucleotide sequences of SENV-A and SENV- B ORF1 and ORF2 regions are shown in Figures 27 and 28.
  • the two ORF1 of SENV-A and SENV-B share 63.40% of identity, while the ORF2 of both SENV subtypes share a nucleotide identity of 49,68%.
  • Figures 29 and 30 show the nucleotide alignment of the SENV-B ORF1 and ORF2 sequences, respectively, with the corresponding region of TTV.
  • the two ORF1 sequences share only 49.85% of identity, and the best alignment requires the insertion of numerous gaps.
  • the ORF2 segments of the two viral agents, TTV and SENV-B have a even lower identity (41.83%). Therefore, SENV-B can be considered as being a subtype of SENV-A, and both SENV subtypes are only distantly related to TTV.
  • L1S SEQ ID NO: 41
  • L3AS primers SEQ ID NO: 42
  • the primers were designed on the basis of the alignment between SENV-A and SENV-B ORF1 sequences and on the basis of their nucleotide composition which made them the best candidates for amplifying conserved regions of the two SENV subtypes.
  • PCR reactions using DNA extracted from the sera of 6 non-A non-E hepatitis patients (NAE) were set up.
  • PCR reactions were performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum from NAE patients with QIAamp blood kit
  • PCR buffer 67 mM Tris HCI, pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ - mercaptoethanol, 2 mM MgCI 2
  • dATP deoxyribonucleoside triphosphate
  • PCR was performed in a DNA thermal cycler with mineral oil. PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 55°C for 1 min, and
  • Fig. 31 The alignment of the translated product of these sequences (SEQ ID NO: 57-70) is shown in Fig. 31.
  • Fig. 31 also depicts the sequences of the corresponding region of SENV-A (encoded by nucleotides 1372-1830 of SEQ ID NO: 24) and SENV-B (encoded by nucleotides 1379-1879 of SEQ ID NO: 34).
  • the evolutionary relationship between different viruses can be established by calculating the phylogenic distances between aligned nucleotide or aligned deduced amino acid sequences of their large open reading frames or of portions of these sequences.
  • evolutionary distances were determined by alignments of nucleotide sequences of the analyzed segment of ORF1 (Fig. 31).
  • SENV-A comprising the sequence encoded by nucleotides 1372-1830 of SEQ ID NO: 24
  • SENV-B comprising the sequence encoded by nucleotides 1379-1879 of SEQ ID NO: 34 as well as SEQ ID NO: 55 and 56
  • SENV-C comprising SEQ ID NO: 43-50
  • SENV-D comprising SEQ ID NO: 51-54.
  • the unrooted tree graphically demonstrates the significant degree of divergence of the SENV genotypes from TTV.
  • primers L2AS SEQ ID NO: 71
  • TTV15S SEQ ID NO: 72
  • primer SEN-C1S SEQ ID NO: 73
  • primer 66AS SEQ ID NO: 74
  • PCR reactions were performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum from patient NAE 8 with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI, pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ - mercaptoethanol, 2 mM MgCI 2 ), 300ng of each PCR primer, a 200 ⁇ M concentration of each deoxyribonucleoside triphosphate (dATP, dGTP, dCTP, dTTP from
  • PCR was performed in a DNA thermal cycler with mineral oil. PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 60°C for 1 min, and
  • SENV-C SENV-C (SEQ ID NO: 75).
  • SENV-A and SENV-B this SENV-C sequence contains three open reading frames, but an additional ORF was also detected.
  • nucleotide sequences of ORF1 (nt 579-2840), 2 (nt 232-705) , 3 (nt 2548-2814) and 4 (nt 2588-2944) (SEQ ID NO: 76- 79) encode for proteins of 753, 157, 88, and 118 amino acids (SEQ ID NO: 80-83), respectively.
  • ORF2 of SENV-A, SENV-B and of TTV is shown in Figures 36, 37 and 38, respectively.
  • the identities between the SENV-C ORF2 with the ORF2 of SENV-A and SENV-B are 46.50% and 34.62%, respectively, while the identity between
  • SENV-C ORF2 and TTV ORF2 is 35.67%.
  • SENV-C nucleotide sequence shares 67.76% and 66.71% of identity to the nucleotide sequence of SENV-A and SENV-B, respectively.
  • PCR reactions were performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum from patient NAE 8 with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI, pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ - mercaptoethanol, 2 mM MgCI 2 ), 300ng of each PCR primer, a 200 ⁇ M concentration of each deoxyribonucleoside triphosphate (dATP, dGTP, dCTP, dTTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer).
  • the reaction was performed in a DNA thermal cycler with mineral oil.
  • PCR consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min. Using this strategy two amplicons of about 1350 bp and 1614 bp were obtained. The PCR products were loaded on a 2% agarose gel with 2 ⁇ g ethidium bromide per ml in order to determine the size of the amplified products.
  • the ORF1 SENV-D sequence shares 64.39%, 58.73% and 60.43% of identity to the ORF1 nucleotide sequence of SENV-A, SENV-B and SENV-C respectively.
  • the identity of the SENV-D ORF1 nucleotide sequence with the nucleotide sequence encoding for TTV ORF1 is 49.01 %.
  • the alignment of the nucleotide sequences encoding SENV-D ORF2 with the nucleotide sequence encoding ORF2 from SENV-A, SENV-B, SENV-C or TTV demonstrates that these sequences share 57.59%, 57.32%, 52.11 % and 47.47 %, respectively, of identity.
  • Figure 49A summarizes the percentage of nucleotide identities between the whole genomes ( Figure 49A) or the sequence encoding the different ORFs (Figure 49C, 49D and 49E) of SENV-A, SENV-B, SENV-C, SENV-D and the genome of TTV.
  • Figure 49B was also carried out considering only the complete coding regions of SENV (SEQ ID NO:s 26, 94, 95 and 96). Thus, even considering the conserved 5' and 3' untranslated regions between SENV and TTV, the nucleotide sequences of these two viral agents never share more than 60% of identity.
  • Figure 50 summarizes the percentage of identities at the amino acid levels of the ORFs encoded by SENV-A, SENV-B, SENV-C, SENV-D and TTV genomes.
  • SENV ORFs2 and ORFs3 are much less conserved than SENV ORFsl .
  • the evolutionary relationship among different viruses can be examined by calculating the phylogenic distances between aligned nucleotide or aligned deduced amino acid sequences of large open reading frames or of a portion of these sequences.
  • evolutionary distances were determined for alignments of amino acid sequences of ORF1 and ORF2.
  • Figure 51 shows the results of this analysis. The relative evolutionary distances between the viral sequences analyzed are readily apparent from the numeric values.
  • FIG. 52 shows also the unrooted phylogenic tree derived from alignments of the amino acid sequences.
  • primers BCD 1 S SEQ ID NO: 97
  • L 2AS SEQ ID NO: 71
  • PCR reactions were performed with a 50- ⁇ l mixture containing template, 1/10 th of DNA extracted from 100 ⁇ l of serum with QIAamp blood kit (QIAGEN), PCR buffer (67 mM Tris HCI, pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 10mM ⁇ -mercaptoethanol, 2 mM MgCI 2 ), 300ng of each PCR primer, a 200 ⁇ M concentration of each deoxyribonucleoside triphosphate (dATP, dGTP, dCTP, dTTP from Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Perkin Elmer).
  • the reaction was performed in a DNA thermal cycler with mineral oil.
  • PCR consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min. PCR products were analyzed on 2% agarose gels. In order to evaluate the specificity of the amplified products, aliquots of 5 ⁇ l of each amplified product were hybridized in a DEIA assay (DNA Enzyme Immunoassay;
  • each amplified product could be simultaneously tested for the presence of total SENV sequences as well as for each specific genotype/subtype.
  • SENV had a low prevalence among blood donors, while it was surprisingly found that the majority of patients with advanced liver disease of unknown origin were positive for this (these) viral agent(s). Furthermore, the high prevalence of SEN virus among intravenous drug users also suggests that the virus can be transmitted with blood.
  • SENV-A and SENV-C ORFsl were also characterized by the presence of a leucine pattern spanning positions 617-638 and 729-750, respectively.
  • SENV-C ORF1 also contained a microbodies C-terminal signal spanning position 751-753.
  • SENV-C and SENV-D contained 20 and 21 bipartite nuclear targeting sequences at the N-termini. None of these sequences were found in SENV-A and SENV-B ORFsl .
  • SENV-A, B, C and D ORFs2 contain 0, 0, 0 and 1 potential N-glycosylation sites, 0, 1 , 1 and 0 tyrosine sulfatation sites, 1 , 0, 0 and 2 proteinkinase C-phosphorylation sites, 1, 1 , 0 and 1 caseinkinase ll-phosphorylation sites, and 1 , 6, 7 and 3 N-myristoylation sites, respectively.
  • SENV-A and SENV-C ORFsl were also characterized by the presence of a leucine pattern spanning positions 617-638 and 729-750, respectively, which cannot be detected in ORF1 of TTV.
  • ORFs3 of SENV-A, B, C and D were characterized by the presence of 1 , 4, 3 and 0 cAMP and cGMP-dependent proteinkinase phosphorylation sites, 1 , 4, 6 and 4 proteinkinase C-phosphorylation sites, 2, 2 1 and 3 caseinkinase il-phosphorylation sites, 1 , 0, 1 and 1 amidation sites and by 2, 2, 4 and 4 bipartite nuclear targeting sequences, respectively.
  • the ORF1 of SENV-A cloned into the EcoRl restriction site of ⁇ GEM * -T Easy (Promega) was amplified by PCR in order to add at the 5' and the 3' termini two restriction site sequences.
  • the upper primer has a non-complementary BamHI recognition sequence at the 5' end: TTG GAT CCA TGA ACT ATG CCA TGC ACT GCG (SEQ ID NO: 109), whilst the lower primer has an EcoRl recognition sequence at the 5' end: ATG AAT TCT TAC GTG AGG TGT GCT AAA GAT AGT GGG (SEQ ID NO: 110).
  • the amplicon obtained was cloned into pET-30a E. coli expression vector (Novagen) in order to express the ORF1 as a fusion protein with an histidine tail at the N-terminus.
  • This tail allows an easier purification onto a metal chelating column (HI TRAP Chelating Pharmacia, # 17-0409-01 , used according to manufacturer's protocols).
  • the induction protocol of transformed E. coli BL21 strain was performed according to the pET system manual (Novagen): briefly, an overnight culture of recombinant E. coli was inoculated at a dilution of 1 :50 in fresh medium. The cells were incubated on a shaker at 37°C until the culture reached an optical density of OD600 0.5-0.6. The cells were then induced adding IPTG from a
  • the pellet is resuspended in a urea buffer (8M Urea, 50 mM Tris-HCl, 100 mM NaCl pH 8.0) and heated at
  • the residual pellet (if present) is resuspended into SDS-PAGE loading buffer.
  • ORF1 protein The majority of recombinant ORF1 protein is found in the urea fraction and was purified by affinity chromatoghaphy on a metal chelation resin. A partially purified fusion protein of 80354 dalton of molecular weight was obtained (see table # 1 ;
  • the ORF2 of SENV-A cloned into the EcoRl site of pGEM * -T Easy vector (Promega) was PCR amplified in order to insert two restriction site recognition sequences at the 5' and 3' end of the sequence.
  • the forward primer has a BamHI site at the 5' end: ATG GAT CCG GGC TAT GGG CAA GGC TCT TAG GG (SEQ ID NO: 1 1 1 );
  • the reverse primer has an EcoRl recognition sequence at the 5' end: TAG AAT TCT TAC TGT TGG TCG TCT TCT TCG ACG G (SEQ ID NO: 1 12).
  • the sequence obtained was cloned into pET30c E.
  • the ORF3 of SENV-A was excised from the EcoRl site of a recombinant pGEM * -T Easy vector and amplified by PCR in order to add two unique restriction site recognition sequences at the 5' and 3' termini.
  • the forward primer used has a BamHI recognition sequence at the 5' end: ATG GAT CCC CAA CAG TAG ATG AAC TCT
  • ATC TTC AAA AAC C (SEQ ID NO: 113), the reverse primer has an EcoRl binding sequence at the 5' end: TAG AAT TCT GGA ACA TGT CAT ACT TTA CGT GAG
  • the protein extraction protocol was the same as for the ORF1 and the recombinant protein was found in the soluble Tris fraction.
  • primer BCD 1 S (SEQ ID NO: 97) was modified and the resulting primer
  • NEW BCD 1S (SEQ ID NO: 115) was used in combination with primer L 2AS (SEQ ID NO: 115) was used in combination with primer L 2AS (SEQ ID NO: 115).
  • PCR consisted of preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • LD (SEQ ID NO: 102), specific for SENV-D sequences.
  • each amplified product could be simultaneously tested for the presence of total
  • Sera from the specific, highly selected patient groups were analyzed. Data are represented in Figure 54.
  • One group (patients No: 1 to 12) comprised individuals that developed acute Non A-Non E (NANE, post transfusion) hepatitis after a single blood transfusion.
  • the second group (patients No: 13 to 22) comprised individuals with chronic Non A-Non E hepatitis (NANE, chronic hepatitis), while the third group (patients No: 23 to 31) (HCV positive donors) consisted of blood donors infected with hepatitis C virus (HCV).
  • 20 healthy blood donors were included in the analysis (patient No. 32 to 52).
  • SENV viruses Considering the heterogenicity of SENV viruses, the possibility that some SENV subtypes may be more pathogenic than others is contemplated.
  • NANE Non A-Non E patients
  • amplicons obtained from healthy blood donors were sequenced.
  • SENV-B has been found in healthy blood donors and therefore its potential role in transmitting disease appears to be marginal.
  • SENV- A, SENV-D and SENV-E are very rarely detected in healthy blood donors, however, they appear to be associated with hepatitis transmission.
  • Example 18 Identification and molecular characterization of SENV-E
  • SENV-E two different PCR reactions were set up:
  • primers L 2AS SEQ ID NO: 71
  • TTV15S SEQ ID NO: 72
  • primers NEW BCD 1S SEQ ID NO: 115
  • primer 662AS SEQ ID NO: 86
  • SENV-A, SENV-B and SENV-C share a common sequence in the 3' untranslated region of their genomes, it was likely that a region of sequence similarity would also exist in the most 3' region of the genomes of SENV-C and SENV-E.
  • PCR was carried out as described hereinabove and consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min. Employing this strategy, two amplicons of about 1350 and 1614 bp were obtained.
  • gel slices corresponding to these 1350 bp and 1914 bands were cut out and purified DNA extracts were connected to a pGEM vector (Promega, Madison, WI, USA) according to standard protocols and introduced to E. coli.
  • the DNA sequences of the inserts were determined by using the AutoRead sequencing Kit (Pharmacia Biotech) with fluorescent M13 Universal primer and fluorescent M13 Reverse. Sequences were determined on three clones of the respective PCR products, and the consensus sequence was adopted.
  • SENV-E SEQ ID NO: 118
  • SENV-E contains three open reading frames (ORF1 , ORF2 and ORF3) respectively of 743, 152 and 97 amino acids (SEQ ID NOs: 122, 123 and 124).
  • ID NO: 125 relates to the nucleotide sequence of SENV-E, excluding the 5' and 3' portions which are highly homologous to TTV.
  • ORF1 major ORF
  • SENV-E is indeed a member of the SENV family, although it appears to be more distantly related to the other members of said family, which have, so far, been identified.
  • the phylogenetic distances between the ORF1 proteins, estimated by the PROTDIST program of the PHYLIP package, ( Figure 55) revealed that SENV-E is the most divergent among the SEN viruses so far identified.
  • SENV-E is still more closely related to the other four identified SENV (A to D) than TTV (see Figure 55).
  • Example 19 Different biological effects of SENV subtypes
  • DNA samples (extracted from the different patients) were amplified by PCR with primers NEW BCD 1S (SEQ ID NO: 115) and primer L 2AS (SEQ ID NO: 71). Again, this PCR reactions were carried out as described hereinabove and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • LA SEQ ID NO: 96
  • SENV-A The results in Figure 56 show the percentage of patients harboring SENV-A.
  • SENV-A was not detected in patients suffering from several diseases, like neurological disorders and rheumathoid arthritis neither in healthy blood donors. However, this virus was present in a high proportion of groups of patients that have received exogenous blood demonstrating the parenteral route of transmission of this agent. Surprisingly, SENV-A was also found to be present in a high proportion of patients affected by Crohn's disease and hepatocarcinomas. Thus, this particular viral agent may be involved in Crohn's disease, a pathology of unknown etiology for which the involvement of a transmissible agent is suspected.
  • Figure 57 shows the incidence of SENV-B in the different groups of patients. It is evident that the profile of the positive samples is very different from the one obtained from SENV-A. It is striking, for instance, that the percentage of SENV-B positive samples among HIV+ intravenous drug users (IVDU) is by far much lower as compared to SENV-A. Furthermore, SENV-B appears to be randomly present also in categories of patients not exposed to exogenous blood.
  • IVDU intravenous drug users
  • SENV-A and SENV-D appear to be strongly associated to the transmission of hepatitis of unknown etiology. Furthermore, SENV-A appears to be also associated to Crohn's disease. SENV-C and SENV-D, on the other hand, appear to play an important role in Lupus erythematosus.
  • SENV-B appears not to be related to any of the screened pathological conditions.
  • Example 6 describes a method for selectively amplifying and detecting SENV-A. By aligning additional SENV-A sequences it was found that a new set of primers is also suitable for SENV-A amplification. This new set of primers was named NEW SEN 3S (SEQ ID NO: 126) and NEW SEN 2AS (SEQ ID NO: 127). These primers were used in PCR reactions (Reaction 1) as described in example 6. The specificity of the amplified products was assessed in a DEIA assay using the SEN 37 biotinylated probe (SEQ ID NO: 13).
  • SENV-D a method for selectively amplifying and detecting SENV-D was developed. To this end all the available SENV sequences were aligned and primers and a specific probe for SENV-D were identified.
  • DNA extracted from the different patients infected with SENV-A, SENV-B, SENV-C and SENV-D was amplified by PCR with primers D10S (SEQ ID NO: 128) and primer L 2AS (SEQ ID NO: 71) (Reaction 2) or with primers D10S (SEQ ID NO: 128) and primer D2AS (SEQ ID NO: 129) (Reaction 3).
  • the PCR was carried out as described hereinabove and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • PCR reaction was carried out in a volume of 100 ⁇ l using the Perkin-Elmer GeneAmp® kit containing 10mM Tris-HCl pH 8.3, 50 mM KCI, 1.5 mM MgCI 2 , 0.001% gelatin, 0.2 mM of dNTP mixture and 40 pmoles of the forward primer: TTG GAT CCA TGA ACT ATG CCA TGC ACT GCG (SEQ ID NO:109) and 40 pmoles of the reverse primer: ATG AAT TCT TAC GTG AGG TGT GCT AAA GAT AGT GGG (SEQ ID NO:110).
  • the PCR reaction was performed in a Perkin-Elmer DNA thermal cycler 480 and the amplification reaction consisted in 1 cycle: 94°C 3', 57°C 1 ', 72°C 3' and 30 cycles: 94°C 1', 57°C 1', 72°C 3'.
  • the amplicon obtained had a size of 1930 bp and was cloned into the EcoRI-BamHI site of pET30a E.coli expression vector (Novagen).
  • coli was inoculated at a 1 :50 dilution into fresh Luria broth plus kanamycin (30 ⁇ g/ml); the cells were incubated under shaking at 37°C until the culture reaches 0.6 OD 6 oo- The ceils were then induced adding IPTG from a 100mM stock to a final concentration of 1 mM. After 3 hours of induction the cells were centrifuged and a protein extraction step was carried out after an overnight standing of the cellular pellet at -20°C.
  • the protein extraction was performed in three steps in order to separate the soluble and the insoluble protein fractions: the first step involved the extraction of soluble proteins with a phosphate buffer (20mM NaH 2 P0 4 ; 0.5M NaCl pH 7.4) and sonication 4x1 min. at 250W. After centrifugation at 9000g for 30' at 4°C, the supernatant was kept as soluble protein fraction, while the pellet was resuspended in a urea buffer (8M urea; 100mM NaCl; 20mM Na 2 HP0 4 pH 7.0), sonicated 2x1 min at 250 W, heated at 70°C for 10' and then centrifuged at 9000g for 30' at 4°C.
  • a urea buffer 8M urea; 100mM NaCl; 20mM Na 2 HP0 4 pH 7.0
  • the remaining pellet was again solubilized in 8M urea, recentrifuged and the residual pellet (if present) was resuspended in SDS-PAGE loading buffer containing 0.1 % SDS.
  • the recombinant ORF1 protein was recovered mainly in the urea fraction and loaded onto a nickel column for purification (HI TRAP Chelating Pharmacia, #17-0409-01 , used according to the manufacturer's protocols). Using an imidazole concentration of
  • the pools have been obtained by eluting the column with linear gradient of 500mM
  • Antigen preparations were electrophoresed in 10% acrylamide slab gels using sodium dodecyl sulfate. The gel was run at 130V at room temperature until the tracking dye reached the bottom of the gel. The gel was then transferred to nitrocellulose paper. The electrophoretic blot was first soaked in PBS containing
  • casein a serum from a haemophilic, poly-transfused patient which was PCR-positive for SENV-A
  • SENV-A ORF1 (pORF1 ) as detected by this Western blot method were collected and were further used for ELISA assay development.
  • AAT TCT TAC TGT TGG TCG TCT TCT TCG ACG G (SEQ ID NO:112).
  • the PCR reaction was performed in a Perkin-Elmer DNA thermal cycler 480 and the amplification reaction consisted in 1 cycle: 94°C 3', 60°C 1 ', 72°C 3' and 30 cycles:
  • the amplicon obtained had a size of 1930 bp and was cloned into the EcoRI-BamHI site of pET30a E.coli expression vector (Novagen).
  • coli was inoculated at a 1 :50 dilution into fresh Luria broth plus kanamycin (30 ⁇ g/ml); the cells were incubated under shaking at 37°C until the culture reaches 0.6 OD 6 oo- The cells were then induced adding IPTG from a 100mM stock to a final concentration of 1 mM. After 3 hours of induction the cells were centrifuged and a protein extraction step was carried out after an overnight standing of the cellular pellet at -20°C.
  • the protein extraction was performed in three steps in order to separate the soluble and the insoluble protein fractions: the first step involved the extraction of soluble proteins with a phosphate buffer (20mM NaH 2 P0 4 ; 0.5M NaCl pH 7.4) and sonication 4x1 min. at 250W. After centrifugation at 9000g for 30' at 4°C, the supernatant was kept as soluble protein fraction, while the pellet was resuspended in a urea buffer (8M urea; 100mM NaCl; 20mM Na 2 HP0 pH 7.0), sonicated 2x1 min at 250 W, heated at 70°C for 10' and then centrifuged at 9000g for 30' at 4°C.
  • a urea buffer 8M urea; 100mM NaCl; 20mM Na 2 HP0 pH 7.0
  • the obtained pellet was again solubilized in 8M urea and repelleted.
  • the residual pellet (if present) was resuspended in SDS-PAGE loading buffer.
  • the recombinant ORF1 protein was recovered mainly in the urea fraction and loaded onto a nickel column for purification (HI TRAP Chelating Pharmacia, #17-0409-01 , used according to the manufacturer's protocols).
  • a nickel column for purification HI TRAP Chelating Pharmacia, #17-0409-01 , used according to the manufacturer's protocols.
  • a partially purified fusion protein of 24000 dalton molecular weight (SEQ ID NO:105 and SEQ ID NO:106).
  • the pools have been obtained by eluting the column with linear gradient of 500mM
  • Antigen preparations were electrophoresed in 10% acrylamide slab gels using sodium dodecyl sulfate. The gel was run at 130V at room temperature until the tracking dye reached the bottom of the gel. The gel was then transferred to nitrocellulose paper. The electrophoretic blot was first soaked in PBS containing
  • SENV-A SENV-A diluted 1 :100 in PBS containing 0.05% casein for 1 hour at 37°C.
  • the nitrocellulose sheet was washed three times in PBS and incubated with horseradish peroxidase-conjugate goat anti-human-lgG at a dilution of 1 :500 in PBS containing
  • PCR reaction was carried out in a volume of 100 ⁇ l using the Perkin-Elmer GeneAmp® kit containing 10mM Tris-HCl pH 8.3, 50 mM KCI, 1.5 mM MgCI 2 , 0.001 % gelatin, 0.2 mM of dNTP mixture and 40 pmoles of the forward primer: ATG GAT CCC CAA CAG TAG ATG AAC TCT ATC TTC AAA AAC C (SEQ ID NO:113)and 40 pmoles of the reverse primer: TAG AAT TCT GGA ACA TGT CAT ACT TTA CGT GAG GTG TGC (SEQ ID NO:114).
  • the PCR reaction was performed in a Perkin-Elmer DNA thermal cycler 480 and the amplification reaction consisted in 1 cycle: 94°C 3'.
  • E. coli XL1 Blue cells (Stratagene cat # 200249)
  • the pET30aORF1 plasmid was transferred to E. coli strain BL21 (DE3) for protein production.
  • the induction protocol was performed according to the pET system manual (Novagen): an overnight culture of recombinant E. coli was inoculated at a 1 :50 dilution into fresh Luria broth plus kanamycin (30 ⁇ g/ml); the cells were incubated under shaking at 37°C until the culture reaches 0.6 OD 60 o.
  • the cells were then induced adding IPTG from a 100mM stock to a final concentration of 1 mM. After 3 hours of induction the cells were centrifuged and a protein extraction step was carried out after an overnight standing of the cellular pellet at -20°C.
  • the protein extraction was performed in three steps in order to separate the soluble and the insoluble protein fractions: the first step involved the extraction of soluble proteins with a phosphate buffer (20mM NaH 2 P0 4 ; 0.5M NaCl pH 7.4) and sonication 4x1 min. at 250W.
  • the supernatant was kept as soluble protein fraction, while the pellet was resuspended in a urea buffer (8M urea; 100mM NaCl; 20mM Na 2 HP0 4 pH 7.0), sonicated 2x1 min at 250 W, heated at 70°C for 10' and then centrifuged at 9000g for 30' at 4°C. After resolubilization in 8M urea, the pellet fraction was again centrifuged and the residual pellet (if present) was resuspended in SDS-PAGE loading buffer.
  • a urea buffer 8M urea; 100mM NaCl; 20mM Na 2 HP0 4 pH 7.0
  • the recombinant ORF1 protein was recovered mainly in the urea fraction and loaded onto a nickel column for purification (HI TRAP Chelating Pharmacia, #17-0409-01 , used according to the manufacturer's protocols). Using an imidazole concentration of 500mM, a partially purified fusion protein of 15000 daltons of molecular weight has been obtained (SEQ ID NO: 107 and SEQ ID NO:108).
  • Antigen preparations were electrophoresed in 10% acrylamide slab gels using sodium dodecyl sulfate. The gel was run at 130V at room temperature until the tracking dye reached the bottom of the gel. The gel was then transferred to nitrocellulose paper. The electrophoretic blot was first soaked in PBS containing 0.05% casein for 20min and then incubated with serum n°2174 (SENV-A positive in PCR assay) diluted 1 :100 in PBS containing 0.05% casein for 1 hour at 37°C. The nitrocellulose sheet was washed three times in PBS and incubated with horseradish peroxidase-conjugate goat anti-human-lgG at a dilution of 1 :500 in PBS containing
  • SENV-D ORF2 cloned into the pGEM®-T Easy was amplified by PCR in order to add two unique restriction site sequences at the 5' and the 3' end which allowed the cloning of the sequence into a pET24a expression vector (Novagen).
  • the forward primer had a non-complementary EcoRl recognition sequence at the 5' end (GGG AAT TCG GGC TCT GGG CAA GGC TCT T, SEQ ID NO: 131), whilst the reverse primer had a non-complementary Hind III recognition sequence at the 5' end (TTC AAG CTT ACT GTT GGT CTT CTT CGA CGG CG, SEQ ID NO: 132).
  • PCR reaction was carried out in a volume of 100 ⁇ l using the Taq DNA Polymerase in storage buffer A (Promega,), Thermophilic DNA Polymerase 10X buffer magnesium free (Promega - it has a composition of 10 mM Tris-HCl pH 9 at 25°C, 50 mM KCI and 0.1% Triton® X-100), 1.5 mM magnesium chloride solution (Promega), 0.2 mM of dNTP mixture (Perkin-Elmer GeneAmp®kit) and 20 pmoles of both primers.
  • storage buffer A Promega
  • Thermophilic DNA Polymerase 10X buffer magnesium free Promega - it has a composition of 10 mM Tris-HCl pH 9 at 25°C, 50 mM KCI and 0.1% Triton® X-100
  • 1.5 mM magnesium chloride solution Promega
  • 0.2 mM of dNTP mixture Perkin-Elmer GeneAmp®kit
  • the PCR was performed for 35 cycles (94°C, 1'; 59°C, 1 '; 72°C, 1'; with additional 20' for the last cycle) and the amplification products measured 512 bp.
  • the PCR product was digested by restriction enzymes EcoRl and Hind III (New England Biolab) and ligated to pET24a expression vector (Novagen). SEQ ID NO: 133 is shown in table 4. TABLE 4
  • E.coli XL1 Blue strain competent cells (Stratagene, #200249)
  • the pET24a-ORF2 plasmid was transferred to E.coli BL21 (DE3) (F ompT gal dcm (DE3) .Novagen) competent cells for recombinant protein production.
  • the induction protocol of transformed E.coli BL21 (DE3) strain was performed according to the pET system manual (Novagen); an overnight culture of recombinant E. coli was inoculated at a 1 :50 diluition into fresh Luria-Bertani broth (Molecular Cloning - A Laboratory Manual - Sambrook.Fritch, Maniatis) plus kanamycin (30 ⁇ g/ml); the cells were incubated under shaking at 37°C until the culture reaches 0.7 OD 59 o-The cells were then induced adding IPTG from a 100 mM stock to a final concentration of 1 mM.
  • inclusion body form.
  • the recombinant ORF2 protein was recovered in the 8M urea fraction (buffer C: 50 mM TRIS-HCI; pH 9, 1 mM EDTA, 8M urea, 14 mM ⁇ OH, 100 mM NaCl).
  • E. coli BL21 pellet After 3 hours of induction the cells were centrifuged at 4000 rpm for 30' at 4°C and the supernatant was discarded. The weight of the E. coli BL21 pellet was determined and for each gram (wet weigh) of E. coli, 10 ml of STET buffer (Sucrose, 10mM Tris, 1 mM EDTA, 5% Triton-X) were added in order to resuspend the pellet; stirring overnight at 4°C the suspension of cells in buffer STET plus 130 ⁇ l of lisozyme (10mg/ml) for each gram (wet weigh) of E. coli pellet.
  • STET buffer Sucrose, 10mM Tris, 1 mM EDTA, 5% Triton-X
  • the pellet was resuspended in buffer A; stirring for three hours at 37°C the suspension of inclusion bodies in buffer A plus 10 ⁇ l of benzonase per ml of buffer A and MgCI 2 from a 100 mM stock solution to final concentration of 1 mM. After 3 hours of stirring the cell lysate was centrifuged at 14.000 rpm for 20' at 4°C; the supernatant was discarded and the pellet was resuspended in buffer A and centrifuged at 14.000 rpm for 20' at 4°C.
  • SENV-D ORF3 cloned into the pGEM®-T Easy was amplified by PCR in order to add two unique restriction site sequences at the 5' and the 3' end which allowed the cloning of the sequence into a pET24a expression vector (Novagen).
  • the forward primer had a non-complementary EcoRl recognition sequence at the 5' end (GGA ATT CAC GAT GAA CAG ATT GAT GTT CCA GAC TTT ACA GA, SEQ ID NO: 135), whilst the reverse primer had a non-complementary Hind III recognition sequence at the 5' end (CCC AAG CTT AAT GTA AAT TTG CTTT GGT TTT CTG GAG TTG G, SEQ ID NO: 136).
  • PCR reaction was carried out in a volume of 100 ⁇ l using the Taq DNA Polymerase in storage buffer A (Promega,), Thermophiiic DNA Polymerase 10X buffer magnesium free (Promega - it has a composition of 10 mM Tris-HCl pH 9 at 25°C, 50 mM KCI and 0.1 % Triton® X-100), 1.5 mM magnesium chloride solution (Promega), 0.2 mM of dNTP mixture (Perkin-Elmer GeneAmp®kit) and 20 pmoles of both primers.
  • the PCR was performed for 35 cycles (94°C, 1 "; 59°C, 1'; 72°C, 1 '; with additional 20' for the last cycle) and the amplification products measured 512 bp.
  • the PCR product was digested by restriction enzymes EcoRl and Hind III (New England Biolab) and ligated to pET24a expression vector (Novagen). SEQ ID NO: 137 is shown in table 7. TABLE 7
  • E.coli XL1 Blue strain competent cells (Stratagene, #200249)
  • the pET24a-ORF3 plasmid was transferred to E. coli BL21 (DE3) (F " ompT /?s ⁇ fSB(rB " mB “ ) gal dcm (DE3) , Novagen) competent cells for recombinant protein production.
  • the method used for the determination of anti SENV-A antibodies was an indirect sandwich immunoassay; plates were passively coated with the three recombinant SENV-A antigens (ORF1 , ORF2 and ORF3) using a carbonate buffer solution (2.93 g/l NaHC0 3 , 1.56 g/l Na 2 C0 3 , pH 9,6 + 0.2); 150 ⁇ l of the coating solution were added into the wells of the microplate and incubated overnight. After the removal of the coating solution, 300 ⁇ l of post-coating solution was dispensed (0.1 M Tris-HCl, 6.7 ml/l BSA 30%, pH 7.4). After 1-3 hours of incubation 300 ⁇ l of fixing solution (40 g/l PVP, 100 g/l sucrose) were added into the wells and further incubated 1 -3 hours at room temperature.
  • a carbonate buffer solution (2.93 g/l NaHC0 3 , 1.56 g/l Na 2 C0 3 , pH 9,6
  • the murine monoclonal antibody HP6017 anti-human IgG-HRP obtained from the murine monoclonal antibody
  • Trizma 84.4 ml/l HCI 1 N, 8 g/l NaCl, 0.16 g/l KCI, 100 g/l Sucrose, 2 ml/l Proclin 300,
  • the chromogen/substrate solution was prepared by mixing the solution of chromogen (Tetramethylbenzidine derivative in citrate buffer) with substrate (hydrogen peroxide in citrate buffer) in amounts 1 :1.
  • wash buffer (9 g/l NaCl, 0.01 g/l Tween 20) ranging from 0.3 to 0.37 ml.
  • 100 ⁇ l of chromogen/substrate were dispensed into the wells included the negative-control blank wells.
  • the microplate was incubated for 30 ' + 2 minutes at room temperature, away from direct or intense light.
  • HIV-HCV positive patients collected from Bioclinical Partners, MA
  • 68 samples were from colorectal diseases patients (diverticulitis or benign adenomas, from Bioclinical
  • the percentage of the SENV-A positive samples was the following: 3.2% for blood donors, 0.0% for other cancers, 2.0% for HIV-HCV positive patients, 3.0% for colorectal diseases. Then the percentage raised up to 8.3% for the hemophiliac patients, 11.6% for the cirrhosis patients and 33.3% for the colon cancer patients.
  • the amino acids for the peptide synthesis on cellulose purchased from Novabiochem (Bad Soden, Germany) and Bachem (Bubendoerf, Switzerland) were 9- fluorenylmethoxycarbonyl (Fmoc)-protected.
  • the following side-chain protecting groups were used: trityl for C, H, N and Q; t-butyl for D, E, S and T; t-butoxycarbonyl for K; and pentamethylchroman sulfonyl for R.
  • the cellulose membrane Wathman 540 paper (Maidstone, England) was used.
  • Dimethylformamide (DMF), diisopropylcarbodiimide (DCI), N-methylimidazole (NMI), N-methylpyrrolidone (NMP), methanol, diisopropylethylamine (DIPA) were purchased from Fluka (Buchs, Switzerland).
  • 2-(1 H-benzotriazole-1 -yl)-1 , 1 ,3,3- tertamethyluroniumtrafluoroborate (TBTU) was purchased from Novabiochem.
  • the dried paper sheet was chemically modified in order to introduce suitable anchor functions for the subsequent peptide synthesis.
  • the membrane was treated without shaking for 3 h with 12 ml of a 0.20 M Fmoc- ⁇ -alanine solution
  • the Fmoc protecting groups were cleaved by treatment with 20% piperidine in DMF for 20 minutes.
  • the membrane was washed with DMF (five times) and methanol (twice) and dried.
  • the membrane was washed four times with DMF and twice with methanol and dried.
  • ORF2 SENV-A antigen to be used as a positive control has been washed with 80% methanol to fix all the polypeptides. Then the membrane has been blocked with PBS buffer containing 0.5% Casein, 0.02% Proclin-300 and 0.05 g/ml Sucrose for 1 hour at room temperature and stored at -20°C before use.
  • the membrane has been put in a transparent bag and 200 ⁇ l chromogen/substrate
  • BM chemiluminescence Blotting substrate POD
  • Boehringer Mannheim BM chemiluminescence Blotting substrate
  • NDNTNTR SEQ ID NO : 143
  • ORF2 protein of SENV-A The common sequence among the peptides is NDNTNTR (SEQ ID NO : 143 ) corresponding to a linear epitope of ORF2 protein of SENV-A.
  • the same procedure has been used for a negative serum (negative control) and no signal was obtained.
  • each aminoacid of the mapped epitope is substituted by all 20-L aminoacids to provide detailed information on the peptide binding.
  • These peptides are synthesized on cellulose membrane as described in example 27 and can be incubated with the antibody in solution. Bound antibody is then detected on the cellulose membrane using standard immunoassay reaction (same procedure of example 27).
  • the T in position 4 can be substituted by: the whole set of aminoacids but K and F
  • the N in position 5 can be substituted by: A, F, G, K, L, M, N, F, R , S, T, Q, V, W, Y
  • the T in position 6 can be substituted by: A, C, D, E, F, R and S
  • the R in position 7 can be substituted by: D, E, K.
  • NKVIGFPMKGEEA SEQ ID NO : 144
  • the common sequence is QFRPAYYDV (SEQ ID NO : 148 )
  • KFDTRRGFYSS SEQ ID NO : 151
  • the common sequence is: FKFLFGG (SEQ ID NO : 156 )
  • the common sequence is: FYDTNFGNGKM (SEQ ID NO : 159 )
  • the major epitopes for antigen from ORF 1 for SENV-A are therefore:
  • NKVIGFPMKGEEA SEQ ID NO: 144
  • QFRPAYYDV (SEQ ID NO : 148 ) KFDTRRGFYSS (SEQ ID NO : 151 ) FKFLFGG (SEQ ID NO : 156 ) FYDTNFGNGKM (SEQ ID NO : 159 )
  • SRLRP SEQ ID NO : 165
  • the common sequence is LPTAN (SEQ ID NO : 171 ) .
  • SENV-D The major epitopes of SENV-D are, therefore, SRLRP (SEQ ID NO : 165 ) and
  • SENV-C and SENV-E were developed. First, all the available SENV sequences were aligned and specific primers and a specific probe for SENV-C and SENV-E were identified.
  • DNA extracted from 5 patients infected with SENV-A, 5 with SENV-B, 5 with SENV- C, 5 with SENV-D and 5 with SENV-E was amplified by PCR with primers SENV-C 5S (SEQ ID NO: 172) and primer L 2AS (SEQ ID NO: 71).
  • the PCR reactions were performed as described herein above and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 62 °C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • PCR reactions were again performed as described herein above and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 55 °C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • Figure 62 A shows the percentage of positive samples among healthy blood donors
  • IVDU intravenous drug users
  • SENV-B is highly prevalent in the healthy population and this suggests that it may not be pathogenic.
  • SENV-A is highly prevalent in IVDU and less in thalassemic patients. Without being bound by theory, one possible interpretation for the unusual distribution of SENV-A is that it may be more resistant to atmospheric agents than the other SENV subtypes.
  • Figure 62 B compares the SENVs prevalence among patients affected by alcoholic hepatitis, patients with chronic NANE hepatitis from US and patients with chronic
  • SENV-C and SENV-D may be responsible for at least some chronic NANE hepatitis in US.
  • SENV-C subtype appears to be completely absent in Japanese patients.
  • a large predominance of SENV-D can be detected.
  • One of the Japanese patients was also co-infected with SENV-A.
  • Figure 62 C compares the prevalence of SENVs among patients that had heart surgery without transfusions, patients that had heart surgery with transfusions and did not developed hepatitis and patients that developed hepatitis after one single transfusion.
  • SENV-D and SENV-C appear to be associated to hepatitis transmission while the other subtypes do not play any role in this pathology.
  • SENV-B appears to be quite prevalent in the normal population. Without being bound by theory, this suggests that SENV-B may not have a pathogenic role.
  • SENV-C were selectively found in a high percentage of NANE-hepatitis patients but not in patients affected by other diseases. The two viruses were also found in a low but significative percentages of patients infected with hepatitis C or hepatitis B, suggesting that the route of transmission of SENV-C and SENV-D is similar to the one of other hepatitis viruses.
  • SEQ ID NO: 177 relates to the prevalent SENV-C sequence obtained with primers C5S (SEQ ID NO: 172) and 2AS (SEQ ID NO: 71) which was detected in the sera of NANE patients and shows high similarity to the above-described SENV-C sequence (SEQ ID NO: 75). Although this sequence is similar to SENV-C, several mismatches have been identified with the canonic sequence and therefore its relation to SENV-C remains at this point only speculative.
  • lymphocytes In order to study whether SENV viruses can also be found in lymphocytes PCR reactions on DNA extracted from purified lymphocytes were performed. These lymphocytes were obtained from 4 patients which were intravenous drug users and HIV positive. These patients were identified as VIR 18, VIR 16, VIR 12 and VIR 10. Their sera were SENV positive.
  • primer NEW BCD 1 S (SEQ ID NO: 115) was used in combination with primer L 2AS (SEQ ID NO: 71).
  • the PCR reactions were performed on 1 ⁇ g of isolated lymphocyte DNA which was deluted in a 50 ⁇ l PCR reaction mixture as described before (see, inter alia, example 9).
  • the PCR reaction was performed in a DNA thermal cycler with mineral oil and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • VIR 10 resulted negative for the presence of all known SENV subtypes, suggesting the presence of a new viral isolate.
  • PCR reactions were performed with a 50- ⁇ l mixture containing template, as described herein above. It was performed in a DNA thermal cycler with mineral oil and consisted of a preheating at 94°C for 5 min, 45 cycles of 94°C for 1 min, 58°C for
  • SENV-F contains three open reading frames (ORF1 , ORF 2 and ORF 3) respectively of 758, 160 and 182 amino acids (SEQ ID NO: 180, SEQ ID NO: 181 and SEQ ID NO: 182).
  • the ORF 3 of SEN-F is much longer (185 amino acid) than the one of the other SENVs which, containing only 80 amino acid in average. This is due to the presence of a methionine upstream to the two consecutive methionines present in all the other isolates.
  • TTV is shown in Figure 64.
  • the identities of the entire genome of SENV-F with the one of SENV-A, B, C, D, E and TTV are 66.23, 62.54, 63.54,
  • SENV-F ORF1 , ORF2 and ORF3 (SEQ ID NO: 183, SEQ ID NO: 184 and SEQ ID NO:
  • SENV-F is an additional member of the SENV family and, within this family, it appears closely related to SENV-D.
  • SENV-G The second sequence was named SENV-G (SEQ ID NO 187).
  • SENV-G contains three open reading frames (ORF1 , ORF 2 and ORF 3), respectively, comprising 763, 146 and 82 amino acids (SEQ ID NO: 188, SEQ ID NO: 188, SEQ ID NO: 188, SEQ ID NO: 188, SEQ ID NO: 188, SEQ ID NO: 188, SEQ ID NO:
  • TTV is shown in Figure 64.
  • the identities of the entire genome of SENV-G with the one of SENV-A, B, C, D, E, F and TTV are 66.23, 62.54, 63.54,
  • SENV-G is an additional member of the SENV family and, within this family, it appears to be equally distant with all the other SENV members.
  • SENV-H contains three open reading frames (ORF1 , ORF 2 and ORF 3) of 762, 156 and 87 amino acids
  • TTV is shown in Figure 64. At the nucleotide level the identities of the entire genome of SENV-H with the one of SENV-A, B, C, D, E, F, G and TTV are respectively.
  • SENV-H ORF1 , ORF2 and ORF3 (SEQ ID NO: 200, SEQ ID NO: 201 and SEQ ID NO:
  • SENV family and, within this family, it appears to be closely related to SENV-C.
  • HAART Higly Active Antiretroviral Therapy
  • serum samples were collected at the time of the HAART entry and 4, 8, 16, 24, 30, 32 and 40 weeks later.
  • HIV viremia was monitored by an Amplicore HIV quantitative test (Roche Diagnostics) while SENV viremia was monitored by a PCR assay using primers NEW BCD 1S (SEQ ID NO: 115 in combination with primer L 2AS (SEQ ID NO: 71).
  • the PCR reactions were performed with a 50- ⁇ l mixture containing template comparison, 1/10 th of DNA extracted from 100 ul of serum from said patient employing PCR conditions as described herein above.
  • the reaction was performed in a DNA thermal cycler with mineral oil and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • Peripheral Blood Cells were isolated from of 4 SENV positive and 4 SENV negative patients by Ficoll density gradient. After the isolation the cells, they were repeatedly washed and the DNA from 2 x 10 6 cells was isolated using a QIAamp DNA Blood kit
  • PCR reactions were performed as described in example 31 , i.e. 1 ⁇ g of starting DNA obtained as described herein above was diluted in 50 ⁇ l PCR reaction buffer. The reaction was performed in a DNA thermal cycler with mineral oil and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • SENV positive patients harbored the virus.
  • the possibility that the virus detected in these cells is due to a residual blood contaminant is very unlikely since the cells have been extensively washed.
  • peripheral blood cells were isolated from a SENV positive patients as described in example 34.
  • Cells were cultured 5 ml at a staring density of 0.5 x 10 6 /ml of RPMI 1640 medium (Life technologies) supplemented with 10% fetal calf serum (FCS), in the presence of 1 ⁇ g/ml of Phytoagglutinin (PHA). Two hundred microliters of supernatant were carefully removed at days 1 , 2, 3, 4 and 5 of culturing.
  • DNA was extracted from the centrifuged supernatants aliquots and amplified using primers NEW BCD 1 S (SEQ ID NO: 1 15) in combination with primer L 2AS (SEQ ID NO: 71).
  • the PCR reactions were performed as described herein above. It was performed in a DNA thermal cycler with mineral oil and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and an incubation at 72°C for 7 min.
  • Figure 68 shows that already at day 1 of culturing the SENV virus was detectable in the cell supernatant but, more important, the level of the virus increased constantly in the supernatant during the following three days of culture.
  • the decrease of the virus level observed in the supernatant of day 5 of culture reflect, probably, the effect of dead cells and the subsequent release of protease and DNase.
  • these experiments demonstrate that the virus can be obtained in vitro by culturing cells from infected individuals. This rises the possibility of obtaining large quantities of virions that can be used as source of viral antigens.
  • RNA were retrotrascribed to single stranded DNA using the SUPERSCRIPT TM II Rnase H ' reverse transcriptase (Gibco BRL) using 500 ng of primer L3AS (SEQ ID 42) in a final volume of 20 ul.
  • Five ul of cDNA and 2.5 ul of RNA (as control) were amplified using primers NEW BCD 1S (SEQ ID NO: 115) in combination with primer L 2AS (SEQ ID NO: 71).
  • the PCR reactions were performed on 4 ⁇ l isolated cDNA whcih was diluted into 50 ml PCR-reaction buffer as described herein above.
  • PCR reaction was performed in a DNA thermal cycler with mineral oil and consisted of a preheating at 94°C for 5 min, 40 cycles of 94°C for 1 min, 54°C for 1 min, and
  • Figure 69 shows that all the cDNAs, but not the RNA could be specifically amplified with SENV primers, demonstrating that SEN virus can replicate in the liver, since
  • DNA viruses must have an RNA intermediate during replication.
  • Plasmids containing inserts corresponding to the SENV sequences have been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH (German Collection of Microorganisms and Cell Cultures), Mascheroder Weg 1 b, D-38124 Braunschweig, GERMANY.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • the deposition was carried out in accordance with the Budapest Treaty on July 28, 1999 (DSM 12941 , DSM 12942, DSM 12943, DSM 12944, DSM 12957, DSM 12945, DSM 12958, DSM 12946, DSM 12947, DSM 12950, DSM 12951 , DSM 12952, DSM 12953, DSM 12959, DSM 12954) and on October 13, 1999 (DSM 13092, DSM 13093, DSM 13094 DSM 13095, DSM 13096, DSM 13097, DSM 13098).
  • Duplicates of 22 "stubs" solidified agar, containing the bacterial colony in a glass tube
  • E. Coli bacteria XL1-blue (Sup E + , Lac “ , hsdR17, recA1 , F'proAB, Lac l a , Lac ZAM 1 s) transformed with pGEM-Teasy vector or in E. Coli TOP 10 transformed with pCR R 2.1 -TOPO vector, TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA) containing the different inserts (representing partial sequences of the different SENV subtypes) were deposited. The following stubs were deposited:
  • A1 (Deposit number: DSM 12941 ): The insert is a 527bp fragment of SEQ ID NO: 24 from base 1813 to base 2340 A2 (Deposit number: DSM 12942): The insert is a 1082 bp fragment corresponding to the most 5' sequence of SEQ ID NO: 24 (from base 1 to base 1082)
  • A3 (Deposit number: DSM 12943): The insert is a 1136 bp fragment corresponding to the most 3' sequence of SEQ ID NO: 24 (from base 2215 to base 3350)
  • A4 (Deposit number: DSM 12944): The insert is a 1353bp fragment spanning base 1008 to base 2340 of SEQ ID NO: 24
  • A5 (Deposit number: DSM 12957): The insert is a 2196 bp fragment of
  • B1 (Deposit number: DSM 12945): The insert is a 1798bp fragment corresponding to the most 5' sequence of SEQ ID NO: 34 (from base 1 to base 1798)
  • B2 (Deposit number: DSM 12958): The insert is a 1563bp fragment corresponding to the most 3' sequence of SEQ ID NO: 34 (from base 2056 to base 3619)
  • C1 (Deposit number: DSM 12947): The insert is a 1536bp fragment corresponding to the most 5' sequence of SEQ ID NO: 75 (from base 1 to base 1536)
  • C4 (Deposit number: DSM 13092): The insert is a 2285.bp fragment spanning base 576 to base 2861 of SEQ ID NO: 75 and containing the entire sequence encoding for ORF 1
  • D1 (Deposit number: DSM 12950): The insert is a 1798bp fragment corresponding to the most 5'sequence of of SEQ ID NO: 87 (from base 1 to base 1798)
  • D2 (Deposit number: DSM 12951 ): The insert is a 1714bp fragment corresponding to the most 3' sequence of of SEQ ID NO: 87 (from base 1550 to base 3264)
  • D3 (Deposit number: DSM 12952): The insert is a 2262bp fragment spanning base 584 to base 2846 of SEQ ID NO: 87 and containing the entire sequence encoding for ORF 1.
  • E1 (Deposit number: DSM 12953): The insert is a 890bp fragment corresponding to the most 5'sequence of SEQ ID NO: 118 (from base 175 to base 1065)
  • E2 (Deposit number: DSM 12959): The insert is a 2231 bp fragment corresponding to the most 3' sequence of SEQ ID NO: 118 (from base
  • E3 (Deposit number: DSM 12954): The insert is a 1358bp fragment corresponding to the most 5' sequence of SEQ ID NO: 118 (from base 1 to base 1358)
  • F1 (Deposit number: DSM 13093): The insert is a 1565bp fragment corresponding to the most 5'sequence of SEQ ID NO: 179 (from base 1 to base 1565)
  • F3 (Deposit number: DSM 13094): The insert is a 2054bp fragment corresponding to the most 3' sequence of SEQ ID NO: 179 (from base 1286 to base 3340)
  • G2 (Deposit number: DSM 13095): The insert is a 1453bp fragment corresponding to the most 5'sequence of SEQ ID NO: 187 (from base 1 to base 1453)
  • G3 (Deposit number: DSM 13096): The insert is a 1936bp fragment corresponding to the most 3' sequence of SEQ ID NO: 187 (from base 1313 to base 3249) SENV-H:
  • H2 (Deposit number: DSM 13097): The insert is a 1439bp fragment corresponding to the most 5'sequence of SEQ ID NO: 195 (from base 1 to base 1439)
  • H1 (Deposit number: DSM 13098): The insert is a 1957bp corresponding to the most 3' sequence of SEQ ID NO: 195 (from base 1275 to base
  • Maps of all deposited clones are shown in Figure 70.
  • SENV-G A method for selective amplification of and detection for SENV-G was developed. To this end, all the available SENV sequences were aligned and primers and probe(s) which are potentially specific for SENV-G were identified.
  • DNA extracted from 5 patients infected with SENV-A, 5 with SENV-B, 5 with SENV- C, 5 with SENV-D and 5 with SENV-E and 1 with SENV-G were amplified by PCR employing the primers VIR 17-1S (SEQ ID NO: 203) and primer SENVG-4AS (SEQ ID NO: 204).
  • the microorganism identified under I above was accompanied by
  • microorganism identified under I above wa ⁇ received bv this International Deposuan Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treatv was received by it on (date of recs pt of request for conversion)
  • the microorganism identified under I above was accompanied by

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EP99971855A 1998-11-10 1999-11-09 Feststellung von senv genotypen Withdrawn EP1137779A2 (de)

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ITMI982437 1998-11-10
ITMI982437 IT1303732B1 (it) 1998-11-10 1998-11-10 Molecole di acido nucleico che rappresentano un virus trasmissibileper via parenterale.
ITMI990923 1999-04-30
ITMI990923 IT1312552B1 (it) 1999-04-30 1999-04-30 Molecole di acido nucleico che rappresentano un virus trasmissibileper via parenterale
EP99830298 1999-05-14
EP99830298 1999-05-14
EP99113932 1999-07-16
EP99113932 1999-07-16
EP99971855A EP1137779A2 (de) 1998-11-10 1999-11-09 Feststellung von senv genotypen
PCT/EP1999/008566 WO2000028039A2 (en) 1998-11-10 1999-11-09 Identification of senv genotypes

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US20110318363A1 (en) 2010-06-23 2011-12-29 Deutsches Krebsforschungszentrum Specific TT virus sequences and chimeric TT virus host cell DNA molecules for use in diagnosis, prevention and treatment of cancer and autoimmunity
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