Classifications

• • 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
• • 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
  • C12Q1/702—Specific hybridization probes for retroviruses
  • C12Q1/703—Viruses associated with AIDS
• • 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
  • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16511—Roseolovirus, e.g. human herpesvirus 6, 7
  • C12N2710/16522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to fragments of nucleic acids derived from the genome of the virus.
• HHV6 to vectors containing said fragments and to their use in the diagnosis of infections involving this virus.
• the HHV ⁇ viruses which are double-stranded DNA viruses, classified in the family of Herpes viruses, have been isolated from lymphocytes of patients suffering from AIDS or having lymphoproliferative disorders. These viruses are also considered to be the causative agent of sudden rash.
• Different strains of these viruses are known; the HBLV strain, which specifically infects B lymphocytes, has been described by SALAHUDIN et al. (Science 234, 396, 1986), and in PCT application WO 88/01387; two other strains have been described by the inventors: the 39 TAN strain (AGUT et al., Res. Virol. 140, 23, 1989) and the SIE strain. which infects T lymphocytes (AGUT et al., Lancet i, 712, 1988).
• the genome of the SIE strain about 160 kb in size, consists of double-stranded DNA in essentially linear form; its GC content is between 38 and 40%, and it has repetitive terminal sequences.
• the present invention aims to provide nucleic acid reagents for the specific diagnosis of HHV6 / SIE infections, and also nucleic acid reagents for the general diagnosis of infections involving any strain of HHV ⁇ .
• the inventors have selected, cloned and characterized three fragments of the HHV6 / SIE viral genome.
• the present invention relates to fragments of the genomic DNA of the HHV6 / SIE virus, characterized in that they are obtained by digestion with an appropriate restriction enzyme.
• one of said double-stranded DNA fragments is characterized in that it is obtained by digestion of the genomic DNA of the HHV6 / SIE virus with the enzyme Bam HI, in that its size is 5 kb and in that it contains an EcoRI restriction site at 1.8 kb from one of its ends.
• one of said double-stranded DNA fragments is characterized in that it is obtained by digestion of the genomic DNA of the HHV6 / SIE virus with the enzyme Bam HI, in that its size is 850 bp, and in that it contains the following sequence:
• the present invention also encompasses synthetic, semi-synthetic or genetically engineered nucleotide sequences derived from DNA fragments as defined above, whether they are double-stranded DNA sequences containing all or part of the sequence of said fragments, or single-stranded DNA or RNA complementary to all or part of one or the other strand of said fragments.
• a preferred embodiment of the present invention includes in particular the following oligonucleotide sequences:
• Another preferred embodiment of the present invention comprises nucleotide sequences obtained by the PCR amplification method, using as oligonucleotide sequences (II) and
• Another preferred embodiment of the present invention comprises recombinant DNA sequences, containing all or part of the sequence of one of the fragments of the HHV6 / SIE genome in accordance with the invention, such as in particular:
• the present invention also relates to clones of transformed eukaryotic or prokaryotic cells, containing at least one recombinant DNA sequence as defined above.
• Clones of the DH5 strain of Escherichia coli, transformed by the plasmids mentioned above were the subject of a deposit with the National Collection of Cultures of Microorganisms (CNCM), on July 19, 1989: -
• the clone transformed by the plasmid pHC-5 carries the deposit number 1-895.
• the present invention also relates to diagnostic reagents, characterized in that they contain at least one of the nucleotide sequences derived from the fragments of the genome of the HHV ⁇ / SIE virus in accordance with the invention, as defined above, associated with at least one suitable detection means.
• Said reagents can be used in a very large number of diagnostic techniques based on the detection of nucleic acids by hybridization and / or amplification; in particular, reagents derived from the 850 bp Bam HI fragment, corresponding to a conserved sequence, can allow the detection of all strains of HHV ⁇ .
• the present invention therefore relates to a diagnostic method allowing either the detection of the HHV6 / SIE virus, or more generally, the detection of the HHV6 virus in a biological sample, characterized in that it comprises a step in which the 'the biological sample is brought into contact with a reagent according to the invention, and a step in which a specific interaction is detected between said reagent and one " or more DNA sequences of said virus possibly present in the biological sample
• the detection is carried out by the PCR amplification method (chain amplification by Taq polymerase) and oligonucleotide sequences derived from fragments of oligonucleotide sequences are used as primers and optionally as probes. DNA defined above.
• nucleotide sequences (II) and (III), or the nucleotide sequences (III) and (IV) are used as primers. ), and possibly as probe the nucleotide sequence (IV).
• the present invention further relates to a kit for the diagnosis of HHV ⁇ infections and / or
• HHV6 / SIE characterized in that it comprises at least one reagent according to the invention, associated with means for implementing a method as described above.
• the virus has spread to peripheral blood lymphocytes.
• the isolation and spread of this strain are described in the publication by AGUT et al. (Lancet i, 712, 1988).
• the Bam HI enzyme The fragments resulting from digestion are separated on a 10-30% sucrose gradient. Fragments of size less than 15 kb are inserted at the Bam HI site of the multifunctional plasmid PTZ 19R, and introduced into the bacteria E.coli DH5.
• the genomic library thus obtained is screened with viral DNA isolated from the purified virion on a 10-60% sucrose gradient and labeled with CTP ⁇ P.
• FIG. 1 represents the restriction map of the plasmid pHC 5.
• the hatched area indicates the location of the 850 bp insert.
• This insert contains no restriction site for the enzymes Hind III, Kpm I, Sacl, Smal. This insert has been partially sequenced.
• the sequence corresponding to the first 310 bases is represented by formula (I). - a clone containing the plasmid pHC 147 which comprises an insert of 5 kb of the genomic DNA of HHV ⁇ / SIE. This clone was deposited on July 19, 1989 with the CNCM under the access number 1-896.
• FIG. 2 represents the restriction map of the plasmid pHC 147.
• the hatched area indicates the location of the 5 kb insert.
• This insert " carries a restriction site
• EcoRI at 1.8 kb from one of its ends. It does not contain any site for the enzymes Sma I, Sal I, Sac I, Kpm I, Xba I.
• Example 2 The procedure is identical to that followed in Example 1 except that the DNA extracted from the PBL cells is digested with the enzyme Cla I, and that the fragments obtained are inserted at the Cla I site of the plasmid pBR 322.
• a recombinant clone was selected from the genomic library obtained.
• This clone contains the plasmid pHC-44 which carries a 3.9 kb insert of the genomic DNA of HHV6 / SIE. It was deposited on July 19, 1989 with the CNCM, under the access number 1-895.
• FIG. 3 represents the restriction map of the plasmid pHC-44.
• the hatched area indicates the location of the 3.9 kb insert.
• This insert carries a Pst I site at 54 bp from one of its ends. It does not contain any Hind III site.
• Synthetic oligonucleotides were obtained from the sequence (I).
• the first (A) consists of the sequence (II) which corresponds to the first 20 bases of the sequence (I).
• the second (B) consists of the sequence (III), complementary to bases 227 to 249 of the sequence (I).
• the third (S) consists of the sequence (IV), and corresponds to bases 79 to 91 of the sequence (I).
• oligonucleotides are used in a chain amplification reaction with Taq polymerase.
• the reaction medium contains, for a total volume of 100 ⁇ l: 0.1 to 0.5 ⁇ M of each of the two primers
• the amplification products are analyzed on an agarose gel containing 1 g / ml of ethidiu bromide and can be detected by measuring the fluorescence at 254 nm.
• the specificity of the amplification reaction is demonstrated by detection of the products thereof by the "Southern blotting" technique: the amplification products are transferred from an agarose gel onto a nylon membrane ( Zeta Probe); detection is carried out using the oligonucleotide S labeled with 32 p> as probe
• oligonucleotides A and B are used as primers, a fragment of 249 bp is detected, after 30 amplification cycles; when oligonucleotides B and S are used as primers, a fragment of 170 bp is detected, after 30 amplification cycles.
• the sensitivity of the detection was determined by using as amplification sample either a plasmid DNA, into which is inserted a fragment of the genome of the HHV6 / SIE virus containing the sought sequence, or the total DNA of PBL cells infected with HHV6; in the first case, the lower detection limit corresponds to 0.3 fg of DNA; in the second, it corresponds to 5 fg of DNA.

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• 1989
  • 1989-08-18 FR FR8911016A patent/FR2651007B1/fr not_active Expired - Lifetime
• 1990
  • 1990-08-17 EP EP95112386A patent/EP0688874A2/fr not_active Withdrawn
  • 1990-08-17 JP JP2512026A patent/JPH04504803A/ja active Pending
  • 1990-08-17 CA CA002039159A patent/CA2039159A1/fr not_active Abandoned
  • 1990-08-17 EP EP90912921A patent/EP0440772B1/fr not_active Expired - Lifetime
  • 1990-08-17 AT AT90912921T patent/ATE135404T1/de not_active IP Right Cessation
  • 1990-08-17 WO PCT/FR1990/000618 patent/WO1991002794A1/fr not_active Ceased
  • 1990-08-17 DE DE69025907T patent/DE69025907T2/de not_active Expired - Fee Related
• 1993
  • 1993-09-07 US US08/161,339 patent/US5545520A/en not_active Expired - Fee Related
• 1995
  • 1995-06-07 US US08/478,141 patent/US5698392A/en not_active Expired - Fee Related

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