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WO2002000884A2 - Sequence de nucleotides du genome influenza a/udorn/72 (h3n2) - Google Patents

Sequence de nucleotides du genome influenza a/udorn/72 (h3n2) Download PDF

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WO2002000884A2
WO2002000884A2 PCT/US2001/019826 US0119826W WO0200884A2 WO 2002000884 A2 WO2002000884 A2 WO 2002000884A2 US 0119826 W US0119826 W US 0119826W WO 0200884 A2 WO0200884 A2 WO 0200884A2
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seq
sequence
influenza
virus
amino acid
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PCT/US2001/019826
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WO2002000884A3 (fr
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Jose M. Galarza
Theresa E. Latham
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American Cyanamid Company
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Priority to JP2002506199A priority Critical patent/JP2004514415A/ja
Priority to EP01948565A priority patent/EP1292614A2/fr
Priority to IL15300501A priority patent/IL153005A0/xx
Priority to AU2001270033A priority patent/AU2001270033A1/en
Priority to BR0111819-6A priority patent/BR0111819A/pt
Priority to MXPA02012255A priority patent/MXPA02012255A/es
Priority to CA002410284A priority patent/CA2410284A1/fr
Publication of WO2002000884A2 publication Critical patent/WO2002000884A2/fr
Publication of WO2002000884A3 publication Critical patent/WO2002000884A3/fr

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • NUCELOTIDE SEQUENCE OF INFLUENZA A/Udorn/72 (H3N2) GENOME
  • This invention relates to the nucleotide sequence of each of the segments of the Influenza virus strain A/Udorn/72 (H3N2) .
  • influenza viruses consist of subtypes designated A, B and C.
  • Influenza A viruses possess a single negative strand RNA genome bf eight segments/ which encodes 10 polypeptides (proteins) that are required for the life cycle of the virus.
  • the order of the proteins is as follows:
  • RNA segments of the influenza subtype A complete genome is encapsidated with multiple subunits of the nucleocapsid protein (NP) and associated with a few molecules of the tri eric polymerase (PB1, PB2 and PA subunits) , thereby forming the ribonucleoprotein complex (RNP)
  • NP nucleocapsid protein
  • PB1, PB2 and PA subunits tri eric polymerase
  • the NP protein is a structural and transcription/replication regulatory protein. Surrounding these structures is a layer of the matrix protein (Ml) , which is the major structural component of the virion and appears to serve as a nexus between the core and the viral envelope.
  • Ml matrix protein
  • This host cell-derived envelope is studded with the two major virally encoded surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) , which are antigenic determinants, and a much smaller amount of a nonglycosylated small protein M2 (Lamb 1989; Lamb, R.A., et al., Cell, 40, 627-633 (1985)).
  • the M2 protein is a spliced gene product from segment #7 (which also encodes the Ml protein) which has ion channel activity; spliced genes are only present in infected cells.
  • the nonstructural NS2 protein is a spliced gene product from segment #8
  • HA glycoprotein is cleaved by a protease to form HA1 and HA2.
  • Influenza viral infection is initiated by the attachment of the surface hemagglutinin to a sialic acid-containing cellular receptor. This first virus-cell interaction induces the uptake of the viral particle into the cell by receptor-mediated endocytosis.
  • the HA Under the low pH conditions of the endosome, the HA undergoes a conformational change that facilitates the interaction of the hydrophobic NH 2 terminal domain of HA2 and the endosomal membrane, resulting in membrane fusion and subsequent release of the core RNPs and matrix protein (Ml) into the cytosol. Disassociation of the RNPs and matrix proteins occurs in the cytosol before the RNPs are translocated to the nucleus where transcription and replication of the complete genome take place (Martin, K., and Helenius, A., Cell, 67, 117-130 (1991); Shapiro, G.I., et al . , J. Virology, 61, 764-773 (1987)) .
  • Virus assembly is assumed to begin via some sort of interaction between the cytoplasmic and/or transmembrane domains of the four virally encoded structural proteins : the membrane anchored proteins (HA, NA and M2) , and the underlying matrix protein (Ml) , which in turn maintains a close association with the RNPs (Garoff, H., et al . , Microbiology and
  • influenza A virus subtypes and antigenic variants that continuously evolve by mutations introduced by the RNA polymerase during the replication of the viral genome. Mutations also occur by genetic reassortment between the segments of two different subtypes when they happen to infect the same cellular host. This biological characteristic of influenza A virus gives rise to new virus strains, which necessitates periodic updating of the immunogenic compositions against influenza. Therefore, the availability of a complete nucleotide sequence and a full length clone of one particular strain of influenza A virus is essential for the development of new and innovative immunogenic compositions against influenza. Thus, there is a need to determine the complete nucleotide sequence of an influenza A virus and to construct a full length clone containing that sequence .
  • influenza A strain designated Influenza A/Udorn/72 was first isolated in 1972.
  • the nucleotide sequences of the polymerase genes PB2, PB1 and PA have not been sequenced.
  • the nucleotide sequences of the segments encoding HA, NP, Ml, M2, NS1 and NS2 have been sequenced.
  • those sequences were obtained a number of years ago using instrumentation which may not have provided accurate sequences.
  • nucleic acid molecules constituting the nucleotide sequences of all of the segments of the Influenza A/Udorn/72 (H3N2) strain which together comprise the complete nucleotide sequence of that strain, as well as their spliced messages. All of these nucleotide sequences are presented in positive strand, antigenomic message sense.
  • the complete nucleotide sequence consists of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13 and SEQ ID NO: 17, and biological equivalents thereof .
  • the complete nucleotide sequence includes a variant of the HA sequence, designated HA (PI) (SEQ ID NO:21), and consists of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17 and SEQ ID NO:21, and biological equivalents thereof.
  • HA PI
  • these isolated nucleic acid molecules are directed to individual isolated influenza A virus nucleic acid molecules which encode individual proteins and are selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 21 (the variant of SEQ ID NO: 7) , and biological equivalents thereof.
  • these individual isolated nucleic acid molecules encode proteins having amino acid sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and biological equivalents thereof.
  • there are provided individual isolated influenza A virus amino acid sequences where said sequences are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and biological equivalents thereof.
  • an individual influenza A virus amino acid sequence where said sequence has the sequence of SEQ ID NO: 8 or SEQ ID NO: 12.
  • an isolated nucleic acid molecule having the nucleotide sequence of a segment of the Influenza A/Udorn/72 (H3N2) strain is used: (1) to design polymerase chain reaction (PCR) primers for use in a PCR assay to detect. the presence of the corresponding virus segment in a sample; or (2) to design and select peptides for use in an ELISA to detect the presence of the corresponding protein produced by that segment in a sample.
  • PCR polymerase chain reaction
  • the Influenza A/Udorn/72 (H3N2) strain was grown in and purified from Madin-Darby canine kidney (MDCK) cells .
  • the viral RNA was extracted from purified virions and amplified by RT-PCR using termini-specific primers. Gel purified genes were cloned into pGe T vectors (Pro ega) and sequenced using multiple sets of primers. The sequences of the termini were determined by 3' and 5" ligation and sequencing of the RT-PCR fragments crossing the. junction (Galarza, J.M., et al . , J.
  • the M2 and NS2 genes are spliced products of the mRNA of segments seven and eight, respectively. These genes were recovered from mRNA purified from MDCK-Influenza A/Udorn-infected cells by RT-PCR using oligo-dT and gene-specific primers. The RT-PCR products were gel purified and cloned into pGemT vectors . All gene sequences were determined by using a fluorescence dye terminator with AmpliTaq DNA polymerase (Perkin-Elmer) and an Applied Biosystems ABI 377 DNA sequencer. This procedure was repeated with multiple clones for each fragment; the sequences obtained were consistent.
  • H3N2 The complete genome of the Influenza A/Udorn/72 (H3N2) strain totaled 13628 nucleotides. This is the first description of the complete nucleotide sequence of the Influenza A/Udorn/72 (H3N2) strain.
  • the eight segments were sequenced in their coding regions, and their 5' and 3' non-coding regions, including regulatory sequences such as promoters, enhancers and polyadenylation signals.
  • the segments, their number of nucleotides, their isolated nucleic acid molecule sequences (shown in positive strand, antigenomic, message sense, that is, in 5' to 3' orientation), their coding regions and amino acid translations are as follows: Segment #1: 2341 nucleotides, SEQ ID NO:l, coding region nucleotides 28-2304, encodes PB2, 759 amino acids (SEQ ID NO: 2) .
  • Segment #2 2341 nucleotides, SEQ ID NO: 3, coding region nucleotides 25-2295, encodes PBl, 757 amino acids (SEQ ID NO: 4) .
  • Segment #3 2233 nucleotides, SEQ ID NO: 5, coding region nucleotides 25-2172, encodes PA, 716 amino acids (SEQ ID NO: 6) .
  • Segment #4 1765 nucleotides, SEQ ID NO: 7, coding region nucleotides 30-1727, encodes HA, 566 amino acids (SEQ ID NO: 8) .
  • SEQ ID NO:7 and SEQ ID NO: 8 differ from the published Yuferov et al. nucleotide and amino acid sequences for HA in several respects .
  • SEQ ID NO: 9 and SEQ ID NO: 10 are identical to the published Buckler-White et al. nucleotide and amino acid sequences for NP.
  • Segment #6 1466 nucleotides, SEQ ID NO: 11, coding region nucleotides 20-1426, encodes NA, 469 amino acids (SEQ ID NO: 12).
  • SEQ ID NO: 11 and SEQ ID NO: 12 differ from the published Markoff et al. nucleotide and amino acid sequences for NA in several respects.
  • Segment #7 1027 nucleotides, SEQ ID NO:13, coding region nucleotides 26-781, encodes Ml, 252 amino acids (SEQ ID NO: 14) .
  • SEQ ID NO: 13 and SEQ ID NO: 14 are identical to the published Lamb Virology 1981 nucleotide and amino acid sequences for Ml.
  • Segment #7, spliced product 322 nucleotides, SEQ ID NO: 15, coding region nucleotides 26-316, encodes M2, 97 amino acids (SEQ ID NO: 16) [first spliced amino acid at residue 10, nucleotides 53-55] .
  • SEQ ID NO: 15 and SEQ ID NO: 16 are identical to the published Lamb PNAS 1981 nucleotide and amino acid sequences for M2.
  • SEQ ID NO: 17 and SEQ ID NO: 18 are identical to the published Lamb et al . 1980 nucleotide and amino acid sequences for NS1.
  • Segment #8 spliced product 402 nucleotides, SEQ ID NO: 19, coding region nucleotides 27-389, encodes NS2, 121 amino acids (SEQ ID NO: 20) [first spliced amino acid at residue 11, nucleotides 57-59] .
  • SEQ ID NO: 19 and SEQ ID NO: 20 are identical to the published Lamb et al. 1980 nucleotide and amino acid sequences for NS2.
  • HA is also an alternate HA sequence, designated HA (PI) : Segment #4: 1764 nucleotides, SEQ ID NO: 21, coding region nucleotides 30-1727, encodes HA, 566 amino acids (SEQ ID NO:22).
  • the HA (PI) sequence may incorporate minor mutations from the Influenza A/Udorn/72 (H3N2) strain which occurred over time.
  • the HA (PI) sequence (SEQ ID NO:21) has 1764 nucleotides, which is one fewer than in the HA sequence (SEQ ID NO: 7), due to a one nucleotide deletion in the non-coding region of segment #4 (position 1756) .
  • the two HA sequences of this invention differ in their nucleotide and amino acid sequences as follows :
  • the HA (PI) sequence also differs slightly from the Yuferov et al. HA nucleotide sequence as follows :
  • nucleic acid sequences of the mRNAs of the segments which are in genome, negative sense (that is, in 3 ' to 5' orientation) , are the complements of the positive strand, antigenomic, message sense sequences set forth above.
  • the present invention further comprises isolated nucleic acid molecules containing individual influenza A virus nucleotide sequences which, by virtue of the redundancy of the genetic code, are biologically equivalent to those sequences which encode the viral proteins, that is, these other nucleotide sequences are characterized by nucleotide sequences which differ from those set forth herein, but which encode a protein having the same amino acid sequence as that encoded by any of the nucleotide sequences in SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID N0:7, SEQ ID NO: 11 and SEQ ID NO:21.
  • the invention contemplates those nucleotide sequences which are sufficiently duplicative of any of the sequences of SEQ ID N0:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 21, so as to permit hybridization therewith under standard high stringency Southern hybridization conditions, such as those described in Sambrook et al. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) .
  • This invention also comprises nucleotide sequences which encode amino acid sequences which differ from those of the Influenza A/Udorn/72 (H3N2) viral proteins, but which are biologically equivalent to those described for one of these viral proteins (SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6).
  • amino acid sequences may be said to be biologically equivalent to any of the viral proteins if their sequences differ only by minor deletions from, insertions into or substitutions to the viral protein sequences, such that the tertiary configurations of the sequences are essentially unchanged from those of the viral proteins.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged (acidic) residue for another such as aspartic acid for glutamic acid, or one positively charged (basic) residue for another, such as lysine for arginine or histidine, as well as changes based on similarities of residues in their hydropathic index, can also be expected to produce a biologically equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal or C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
  • This invention further comprises the isolated amino acid sequences comprising the individual influenza A virus amino acid sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4 and SEQ ID NO: 6.
  • This invention still further comprises the individual isolated amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 12.
  • influenza virus The fundamental biological properties of the Influenza A/Udorn/72 (H3N2) virus depend on its genome, which consists of eight segments.
  • the segments of influenza virus consists of a single strand of negative polarity RNA, and each viral strain has its own specific nucleotide sequence.
  • Complete differential identification between viral strains is, therefore, necessary to determine the nucleotide sequence of the virus. Since the entire specific nucleotide sequence, consisting of 13,628 bases, has been clearly determined as described herein, the virus can be identified at the genetic level, and the identification technique can therefore provide an absolute determination.
  • sequences described herein are useful to provide an identification method comprising detecting a part of the nucleotide sequence of influenza viral antigenomic DNA using the polymerase chain reaction (PCR) method, as well as to generate peptides to detect an antigen produced by that DNA using an ELISA.
  • PCR polymerase chain reaction
  • mutations are introduced where desired into one or more of the segments whose sequences are set forth herein.
  • One or more mutations are introduced by using site-directed mutagenesis.
  • a mutant influenza virus cannot be directly generated from viral RNA, because neither the genomic viral RNA nor the antigenomic cDNA can serve as a direct template for synthesis. Instead, the viral RNA, after its encapsidation by NP, must be transcribed into positive-sense mRNA by the viral RNA polymerase complex.
  • Palese et al. (U.S. Patent 5,166,057, which is hereby incorporated by reference) described a reverse genetics helper virus-dependent system for the "rescue" of an influenza A virus segment. Briefly, a ribonucleoprotein (RNP) complex is generated by in vitro synthesis in the presence of the three polymerase proteins and NP. The RNP complex is then used to transfect eukaryotic cells. Subsequent infection with influenza A helper virus results in the generation of viruses possessing a gene derived from the cloned cDNA segment. A selection method is then used to separate the desired transfectants from the larger number of helper viruses .
  • RNP ribonucleoprotein
  • a different system is used if an entire mutant influenza A strain is to be rescued.
  • Neumann et al. Using the system described by Neumann et al. (Neumann, G., et al., Proc. Natl. Acad. Sci. USA, 96, 9345-9350 (1999), which is hereby incorporated by reference) , a modified Influenza virus strain A/Udorn/72 (H3N2) is generated entirely from cloned cDNAs .
  • This plasmid-based system does not require the use of helper virus infection.
  • a cell line such as human embryonic kidney cell line 293T, is transfected with eight plasmids, each encoding a viral RNA segment of the strain and flanked by a suitable RNA polymerase promoter and terminator, together with another four plasmids encoding the viral NP, PB2, PBl and PA proteins (which serve to synthesize RNPs intracellularly in vivo) .
  • Yields are increased substantially by the addition of five further plasmids expressing the viral structural proteins HA, NA, Ml, M2 and NS2.
  • only eight plasmids are required (Hoffmann, E., et al., Proc. Natl. Acad. Sci.
  • Each plasmid contains two promoters, a human RNA polymerase I (pol I) promoter and a human RNA polymerase II (pol II) promoter.
  • pol I human RNA polymerase I
  • poly II human RNA polymerase II
  • the human pol I and II promoters each transcribe the plasmid template. This results in the synthesis of both viral mRNAs and vRNAs, ultimately leading to the generation of infectious influenza A virus. Because helper virus is not required in any of the 8, 12 or 17 plasmid transfection systems, the transfectant viruses are recovered without plaque purification.
  • This system facilitates the production of live attenuated influenza A viruses for use in combating epidemics involving new HA or NA subtypes, where the corresponding segment of the Influenza A/Udorn/72 (H3N2) strain is mutated to match the sequence of the new subtypes.
  • Predetermined mutations to the segment encoding HA or NA in the Influenza A/Udorn/72 (H3N2) strain which correspond to the sequence of a new circulating subtype are made by using site-directed mutagenesis. Such mutations are introduced by standard recombinant DNA methods into a DNA copy of the viral genome .
  • reassortant viruses are generated by substituting for the segment encoding the HA or NA of the Influenza A/Udorn/72 (H3N2) strain (or mutant thereof) the corresponding segment from a different influenza virus strain.
  • an isolated nucleic acid molecule having the nucleotide sequence of a segment of the Influenza A/Udorn/72 (H3N2) strain is used to generate oligonucleotide probes (from either positive strand antigenomic message sense or negative strand complementary genomic sense) and to express peptides (from positive strand antigenomic message sense only) , which are used to detect the presence of a segment of that influenza A strain (or a mutant thereof) in samples of body fluids and tissues.
  • the nucleotide sequences are used to design highly specific and sensitive diagnostic tests to detect the presence of the viral segment in a sample.
  • PCR primers are synthesized with sequences based on the viral sequences described herein.
  • test sample is subjected to reverse transcription of RNA, followed by PCR amplification of selected cDNA regions corresponding to the nucleotide sequence described herein which have nucleotides which are distinct for a defined segment of that viral strain. Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific nucleotide probes.
  • ELISA tests are ⁇ used to detect the presence of a protein produced by a viral segment.
  • Peptides are designed and selected to contain one or more distinct residues based on the viral sequences described herein. These peptides are then coupled to a hapten (e.g., keyhole limpet hemocyanin (KLH) and used to immunize animals (e.g., rabbits) for the production of monospecific polyclonal antibody.
  • KLH keyhole limpet hemocyanin
  • a selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies can then be used in a "capture ELISA" to detect a protein produced by that viral segment.
  • Influenza A/Udorn/72 was grown in Madin-Darby canine kidney (MDCK) cells and concentrated from clarified culture supernatant.
  • Genomic viral RNA was prepared from lOO ⁇ l of virus stock using a RNAeasy extraction kit (Qiagen) .
  • RNA-pri er mixture was heated at 70 °C for 10 minutes and quickly chilled on ice.
  • the following reagents were added to this tube: 4 ⁇ l of 5X first strand buffer (250mM Tris-HCl pH 8.3, 375mM KCl, 15mM MgCl 2 ) , 2 ⁇ l of 0.1M DTT, and I ⁇ l of dNTP mix (10 mM each dATP, dTTP, dGTP, dCTP) and incubated at 42 °C for two minutes . Subsequently, l ⁇ l of Superscript II (Gibco) reverse transcriptase was added and re- incubated at 42 °C for an additional 50 minutes. The RT reaction was stopped by incubation at 70 °C for 15 minutes.
  • 5X first strand buffer 250mM Tris-HCl pH 8.3, 375mM KCl, 15mM MgCl 2
  • I ⁇ l of dNTP mix 10 mM each dATP, dTTP, dGTP, dCTP
  • PCR reactions were set up by adding 2 ⁇ l of the previous RT to a mixture of the following reagents: 5OmM Tris-HCl (pH 8.0), 1.5mM gCl 2 , 50mM KCl, l ⁇ M each primer, 0.2mM each dNTP and 2 units of Taq polymerase.
  • DNA was amplified in a Perkin-Elmer 9600 PCR machine as follows: One cycle of denaturation at 94 °C for two minutes, followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 42 °C for 30 seconds, and • elongation at 72 °C for one minute. This was followed by one cycle of elongation at 72 °C for seven minutes.
  • PCR-amplified DNA was analyzed on a 1% agarose gel and purified using a Qiagen purification kit.
  • plasmid pGEM-T containing the corresponding DNA segment generated by RT-PCR was added to a 0.2ml tube containing 3.2pmol of sequencing primer and 8 ⁇ l of Terminator ready reaction mix (Perkin-Elmer) and placed into a Perkin- Elmer 9600 PCR machine.
  • the reaction was amplified by 25 cycles at 96°C for 10 seconds, 50°C for five seconds, and 60°C for four minutes.
  • the products were then purified over a G50 Sephadex spin column, lyophilized, and resuspended in 3 ⁇ l of 25mM EDTA (pH8.0) with 50 mg/ml Blue dextran loading dye.
  • the samples were run on an ABI 377 automated sequencer and the sequence analyzed using Sequencher (sequence analysis program from Gene Codes Corporation) .
  • RNA 5' and 3 ' termini were ligated with 50U of T4 RNA- ligase (Pharmacia Biotech.) at 37°C for one hour, extracted with phenol-chloroform and precipitated with ethanol .
  • the primer sequence differs from the PB2 gene sequence as follows:
  • the primer sequence differs from the PBl gene sequence as follows: Position PBl Gene 1203 F
  • Primer F/R Primer sequence bp spanned • RT Primer
  • the primer sequence differs from the PA gene sequence as follows :
  • the primer sequence differs from the PA gene sequence as follows :
  • the primer sequence differs from the PA gene sequence as follows:
  • a PCR assay is used to detect the presence of a segment of the Influenza A/Udorn/72 (H3N2) strain.
  • PCR primers are designed and selected based on homologies to the viral sequences described herein.
  • the assay is conducted by subjecting the sample to reverse transcription of RNA, followed by PCR amplification of selected cDNA regions corresponding to the specific nucleotide sequence described herein. Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific nucleotide probes.
  • An ELISA test is used to detect the presence of an antigen produced by a segment of the Influenza A/Udorn/72 (H3N2) strain. Peptides are designed and selected based on homologies to the viral sequences described herein. These peptides are then coupled to KLH and used to immunize rabbits for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies is then used in a "capture ELISA" to detect the presence of a protein produced by that viral segment.

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Abstract

L'invention concerne la séquence complète des nucléotides de chacun des huit segments de la souche A/Udorn/72 (H3N2) du virus Influenza A à brin négatif, ladite séquence produisant la séquence complète du génome dans un sens de message antigénomique. La séquence est utilisée au cours de diagnostics et peut être modifiée par mutation pour engendrer de nouvelles souches variantes d'Influenza A.
PCT/US2001/019826 2000-06-23 2001-06-21 Sequence de nucleotides du genome influenza a/udorn/72 (h3n2) WO2002000884A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2002506199A JP2004514415A (ja) 2000-06-23 2001-06-21 インフルエンザＡ／Ｕｄｏｒｎ／７２（Ｈ３Ｎ２）ゲノムのヌクレオチド配列
EP01948565A EP1292614A2 (fr) 2000-06-23 2001-06-21 Sequence de nucleotides du genome influenza a/udorn/72 (h3n2)
IL15300501A IL153005A0 (en) 2000-06-23 2001-06-21 Nucleotide sequence of influenza a/udorn/72 (h3n2) genome
AU2001270033A AU2001270033A1 (en) 2000-06-23 2001-06-21 Nucleotide sequence of influenza A/Udorn/72 (H3N2) genome
BR0111819-6A BR0111819A (pt) 2000-06-23 2001-06-21 Moléculas de ácido nucléico isoladas, e, seq ência de aminoácido isolada
MXPA02012255A MXPA02012255A (es) 2000-06-23 2001-06-21 Secuencia de nucleotidos del genoma a/udorn/72(hen2) de influenza.
CA002410284A CA2410284A1 (fr) 2000-06-23 2001-06-21 Sequence de nucleotides du genome influenza a/udorn/72 (h3n2)

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US21365000P 2000-06-23 2000-06-23
US60/213,650 2000-06-23

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WO2002000884A2 true WO2002000884A2 (fr) 2002-01-03
WO2002000884A3 WO2002000884A3 (fr) 2002-08-15

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WO2008128176A1 (fr) * 2007-04-12 2008-10-23 Nucleonics, Inc. Polynucléotides de l'influenza, constructions d'expression, compositions et procédés d'utilisation
KR100913425B1 (ko) 2007-05-14 2009-08-21 녹십자수의약품(주) 인플루엔자 바이러스 및 그를 포함하는 면역원성 조성물
WO2010069600A3 (fr) * 2008-12-19 2010-08-05 European Molecular Biology Laboratory (Embl) Fragments de polypeptide possédant une activité d'endonucléase et leur utilisation
US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
WO2012009373A2 (fr) 2010-07-12 2012-01-19 Gen-Probe Incorporated Compositions et dosages pour détecter le virus h1n1 de la grippe a porcine, le virus h1 de la grippe a saisonnière et les acides nucléiques du virus h3 de la grippe a saisonnière
US8124335B2 (en) 2005-05-06 2012-02-28 Gen-Probe Incorporated Compositions and assays to detect influenza virus A and B nucleic acids
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria

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US8013142B2 (en) 2003-09-11 2011-09-06 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
WO2006102461A3 (fr) * 2005-03-22 2007-07-26 Massachusetts Inst Technology Therapeutique antigrippale
US8338095B2 (en) 2005-05-06 2012-12-25 Gen-Probe Incorporated Compositions and assays to detect influenza virus A and B nucleic acids
US8124335B2 (en) 2005-05-06 2012-02-28 Gen-Probe Incorporated Compositions and assays to detect influenza virus A and B nucleic acids
EP2471805A2 (fr) 2005-05-06 2012-07-04 Gen-Probe Incorporated Compositions et procédés pour la détection spécifique d'acide nucléique des virus Influenza A ou B
WO2008128176A1 (fr) * 2007-04-12 2008-10-23 Nucleonics, Inc. Polynucléotides de l'influenza, constructions d'expression, compositions et procédés d'utilisation
KR100913425B1 (ko) 2007-05-14 2009-08-21 녹십자수의약품(주) 인플루엔자 바이러스 및 그를 포함하는 면역원성 조성물
EP2623590A3 (fr) * 2008-12-19 2013-11-13 European Molecular Biology Laboratory (EMBL) Fragments de polypeptide possédant une activité endonucléase et leur utilisation
WO2010069600A3 (fr) * 2008-12-19 2010-08-05 European Molecular Biology Laboratory (Embl) Fragments de polypeptide possédant une activité d'endonucléase et leur utilisation
AU2009328502B2 (en) * 2008-12-19 2015-02-05 Centre National De La Recherche Scientifique, Direction De La Politique Industrielle Polypeptide fragments comprising endonuclease activity and their use
KR20160113335A (ko) * 2008-12-19 2016-09-28 유럽피안 몰레큘러 바이올로지 래보러토리 엔도뉴클레아제 활성을 포함하는 폴리펩티드 단편 및 이의 용도
KR20170061193A (ko) * 2008-12-19 2017-06-02 유럽피안 몰레큘러 바이올로지 래보러토리 엔도뉴클레아제 활성을 포함하는 폴리펩티드 단편 및 이의 용도
US9890368B2 (en) 2008-12-19 2018-02-13 European Molecular Biology Laboratory (Embl) Polypeptide fragments comprising endonuclease activity and their use
WO2012009373A2 (fr) 2010-07-12 2012-01-19 Gen-Probe Incorporated Compositions et dosages pour détecter le virus h1n1 de la grippe a porcine, le virus h1 de la grippe a saisonnière et les acides nucléiques du virus h3 de la grippe a saisonnière
EP3301195A1 (fr) 2010-07-12 2018-04-04 Gen-Probe Incorporated Compositions et tests permettant de détecter l'acide nucléique du virus de la grippe a porcine h1n1
EP3305920A1 (fr) 2010-07-12 2018-04-11 Gen-Probe Incorporated Compositions et tests permettant de détecter l'acide nucléique du virus de la grippe a saisonnière h3
EP3674423A1 (fr) 2010-07-12 2020-07-01 Gen-Probe Incorporated Compositions et tests permettant de détecter l'acide nucléique du virus de la grippe a saisonnière h3

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CA2410284A1 (fr) 2002-01-03
BR0111819A (pt) 2004-01-06
AU2001270033A1 (en) 2002-01-08
CN1437612A (zh) 2003-08-20
JP2004514415A (ja) 2004-05-20
WO2002000884A3 (fr) 2002-08-15
EP1292614A2 (fr) 2003-03-19
KR20030013451A (ko) 2003-02-14
MXPA02012255A (es) 2003-04-25
IL153005A0 (en) 2003-06-24

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