WO2009102459A2 - Analyse génétique pour identifier des mutants d’échappement du virus de la maladie de newcastle - Google Patents

Analyse génétique pour identifier des mutants d’échappement du virus de la maladie de newcastle Download PDF

Info

Publication number
WO2009102459A2
WO2009102459A2 PCT/US2009/000907 US2009000907W WO2009102459A2 WO 2009102459 A2 WO2009102459 A2 WO 2009102459A2 US 2009000907 W US2009000907 W US 2009000907W WO 2009102459 A2 WO2009102459 A2 WO 2009102459A2
Authority
WO
WIPO (PCT)
Prior art keywords
ndv
wild
strain
genome
escape
Prior art date
Application number
PCT/US2009/000907
Other languages
English (en)
Other versions
WO2009102459A3 (fr
Inventor
Harmen Jacob Geerligs
Cindy Aleida Maria Meinders
Original Assignee
Wyeth
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Wyeth filed Critical Wyeth
Publication of WO2009102459A2 publication Critical patent/WO2009102459A2/fr
Publication of WO2009102459A3 publication Critical patent/WO2009102459A3/fr

Links

Classifications

    • 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
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]

Definitions

  • Newcastle Disease is a serious disease of poultry caused by Newcastle Disease Virus (NDV).
  • Vaccines have been developed to protect birds from NDV, including vaccines referred to as "escape mutant” vaccines (also known as “Marker” vaccines). (See U.S. Patent No. 6,833,133).
  • NDV escape mutants are mutant strains of NDV which are distinguishable from wild-type strains of NDV and from other vaccine strains of NDV on the basis of their inability to be recognized by an antibody that ordinarily binds to one or more epitopes on wild- type NDV strains.
  • NDV escape mutant An example of an NDV escape mutant is disclosed in U.S. Patent No. 6,833,133. This NDV escape mutant is designated P13.
  • the P13 mutant was generated by selecting for mutant strains of NDV that escape recognition and neutralization by the antibody known as mAb54.
  • mAb54 is a monoclonal antibody that recognizes an epitope on the wild-type NDV F glycoprotein.
  • the F glycoprotein is one of four major proteins of NDV and is located as spikes on the surface of NDV virions. (See Avery and Niven, Infect. Immun. 26:795-801 (1979)).
  • NDV escape mutants are useful in the poultry industry because they allow workers to distinguish birds that have been vaccinated against NDV from birds that are infected with wild-type strains of NDV.
  • antibody-based assays can be used to distinguish NDV escape mutants from wild-type strains
  • alternative methods for distinguishing NDV escape mutants from wild-type strains would be useful.
  • the use of antibodies and related reagents and equipment e.g., secondary antibodies, detectable probes, fixing agents, microscopic detection equipment, etc.
  • a rapid and easily analyzable method would be preferred.
  • no such alternative methods have been available.
  • the present invention satisfies the aforementioned need in the art by providing alternative methods for distinguishing NDV escape mutants from wild-type NDV strains.
  • the present invention provides methods that are based not on antibody detection but on the detection of nucleotide changes at the genomic level.
  • the present invention is based on the discovery that mutations which allow NDV escape mutants to escape recognition by neutralizing antibodies also result in the creation and/or elimination of restriction enzyme cleavage sites at the genomic level.
  • the present invention takes advantage of these unique restriction enzyme cleavage sites (also referred to herein as "URECS") by providing a restriction fragment length polymorphism (RFLP) assay that quickly and conveniently distinguishes NDV escape mutants from wild-type strains.
  • URECS unique restriction enzyme cleavage sites
  • RT-PCR/RFLP assays for distinguishing NDV strains have been mentioned in the art (see, e.g., Kou et al. (1999) J. Vet. Med. Sci. 611 191-1195; Pham et a/. (2004) Arch. Virol. 749:1559-1569; Ujvari et a/. (2006) J. Virol. Methods 737:115-121), such assays are not suitable for addressing the problem solved by the present invention. That is, none of the previously mentioned RT-PCR/RFLP assays can be used to identify NDV escape mutants and/or distinguish NDV escape mutants from wild-type strains.
  • none of the previously mentioned RT-PCR/RFLP assays utilize restriction enzymes that recognize specific cleavage sites which are caused by mutations that render NDV escape mutants capable of escaping detection by NDV-specific antibodies.
  • the methods of the present invention provide a direct correlation between unique restriction fragment patterns and the escape mutant phenotype. This direct correlation is possible only because, unlike the assays of the prior art, the assays of the present invention begin with a detailed analysis of the genome of NDV escape mutants.
  • the present invention provides a method of distinguishing an NDV escape mutant from a wild-type NDV strain by first amplifying a portion of the genome of a candidate NDV escape mutant.
  • the candidate NDV escape mutant may be, e.g., an NDV strain that is obtained from a poultry animal suspected of being vaccinated with an NDV escape mutant vaccine strain.
  • the amplified portion is carefully selected to comprise the nucleotides which are responsible for the escape mutant phenotype.
  • the amplified portion contains or lacks (as the case may be) a URECS.
  • the method of the invention next comprises contacting the amplified portion with a restriction enzyme that recognizes the URECS, thereby forming a cleavage reaction mixture.
  • the cleavage reaction mixture is then subjected to size separation to obtain a restriction fragment pattern of the candidate NDV strain.
  • the candidate NDV strain is identified as an escape mutant if the restriction fragment pattern of the candidate NDV strain is different from the restriction fragment pattern obtained from a wild-type NDV strain subjected to the same (or substantially the same) amplification/cleavage/separation steps.
  • the candidate NDV strain can be identified as an escape mutant if the restriction fragment pattern of the candidate NDV strain is the same as the restriction fragment pattern obtained from a known NDV escape mutant subjected to the same (or substantially the same) amplification/cleavage/separation steps.
  • the present invention provides a rapid, convenient and inexpensive method for identifying NDV escape mutants.
  • the present invention therefore provides workers in the poultry industry with, inter alia, an important new tool for distinguishing vaccinated birds from infected birds.
  • Figure 1A-1 B is the nucleotide sequence of the F gene of the Wiltenburg strain of Newcastle Disease Virus (SEQ ID NO:1 ), which, for purposes of the present invention, is an example of a "wild-type" strain.
  • the boxed nucleotides in Figure 1A represent the codon for SeM 57 of the wild-type F protein.
  • Figure 2 is a photograph of an agarose gel depicting the restriction fragment patterns of a wild-type NDV strain (designated "NDW”) and an NDV escape mutant (designated P13) subjected to the RT-PCR/RFLP assay of the present invention, along with controls.
  • NDV wild-type NDV strain
  • P13 NDV escape mutant
  • Figure 3 is a photograph of an agarose gel depicting the restriction fragment patterns of five wild-type NDV strains and an NDV escape mutant (designated P13) subjected to the RT-PCR/RFLP assay of the present invention, along with controls.
  • the present invention provides methods for distinguishing Newcastle Disease Virus (NDV) escape mutants from wild-type NDV strains.
  • NDV Newcastle Disease Virus
  • NDV escape mutant means any NDV that is not recognized by the monoclonal antibody designated mAb54. (See U.S. Patent No. 6,833,133; See also, Collins et a/., Arch. Virol. 704:53-61 (1989)). Also included within the definition of "NDV escape mutant” is any NDV that is not recognized by an antibody that recognizes the same epitope as mAb54. NDV escape mutants may be obtained using standard methods in the art. (See, e.g., U.S. Patent No. 6,833,133).
  • NDV escape mutants can be obtained by contacting a population of wild- type NDV viruses with mAb54 (or an antibody that recognizes the same epitope as mAb54) and selecting for mutants that are able to propagate in the presence of this ordinarily growth- inhibiting antibody.
  • Exemplary NDV escape mutants include the mutant designated P13 which was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), of the lnstitut Pasteur, Paris, France, 25 Rue de Do Budapest Roue, F-75724, under accession number I- 2928 on August 29, 2002. (See U.S. " Patent No. 6,833,133). The generation and isolation of P13, along with additional escape mutants designated P7, P8, P12, P14, P15, P16 and P17, is described in Example 1 herein below.
  • wild-type NDV means any NDV that is not an escape mutant.
  • NDV strains even though they may contain one or more genotypic or phenotypic alterations, are still considered "wild-type NDV strains" so long as they are recognized by mAb54 or by an antibody that recognizes the same epitope as mAb54.
  • Wild-type NDV strains under this definition may be virulent, avirulent or attenuated.
  • Exemplary wild-type NDV strains include NDV field isolates, e.g., NDV isolates obtained from naturally infected animals. Wild-type NDV strains also include, e.g., NDV strains found in commercially available vaccines.
  • the Wiltenburg strain also called “NDW”
  • Hertz also referred to as Herts 33
  • LaSota Ulster 2C, Queensland V4, Hitchner B1 , F, H, Mukteswar, Roakin, Beaudette C, GB Texas, NY Parrot 70181 1972, lentil, Milano, Miyader
  • NDV strains that would be regarded as wild-type in the context of the present invention will be known and readily available to persons of ordinary skill in the art.
  • Recombinant NDV strains created by plasmid-based (reverse genetics) methods are also regarded as wild- type strains if they are not recognized by mAb54 or by an antibody that recognizes the same epitope as mAb54.
  • the methods of the present invention take advantage of the genetic changes that allow NDV escape mutants to escape recognition by mAb54.
  • NDV escape mutants contain mutations that create and/or destroy restriction enzyme cleavage sites within their genomes. Such mutations may be found, e.g., within the F gene.
  • the "F gene” refers to a polynucleotide sequence that encodes the fusion protein (also referred to as the 11 F protein" or "F glycoprotein”) of NDV.
  • URECS can be identified by first comparing the nucleotide sequence of the F gene of an NDV escape mutant to that of a wild-type NDV strain, thereby identifying any nucleotide changes that exist in the escape mutant's genome. When more than one nucleotide change is identified in the genome of an NDV escape mutant relative to a wild-type strain, one can easily determine which of them, individually or collectively, are responsible for the escape mutant phenotype.
  • nucleotide changes and combinations thereof can be created in the genome of wild-type strains, and the resulting mutant viruses can be assayed for the escape mutant phenotype (i.e., the inability to be recognized by mAb54 or its equivalent).
  • the mutated nucleotide sequence of the escape mutant can be compared to that of a wild-type strain in order to determine if any restriction enzyme cleavage sites are present in the escape mutant genome but not in the wild-type genome, and vice versa. Such comparisons can be carried out, e.g., manually, or by using any number of commercially- and freely-available sequence analysis software programs. Any restriction enzyme cleavage sites that are present at a position within the escape mutant genome but are not present at the corresponding position in the wild-type genome, or vice versa, are "unique" and are therefore regarded, for purposes of this disclosure, as URECS.
  • the methods of the present invention comprise molecular assays involving restriction enzymes that recognize URECS.
  • a polynucleotide molecule corresponding to a region of an NDV genome known to contain a URECS is first obtained from a candidate NDV strain (e.g., an NDV strain that may or may not be an escape mutant). The polynucleotide molecule is then contacted with an enzyme that cleaves the URECS.
  • cleavage of the polynucleotide by the restriction enzyme will identify the candidate NDV strain as an escape mutant, and absence of cleavage will identify the candidate NDV strain as a non- escape mutant (also referred to herein as a "wild-type" NDV strain).
  • the URECS is one that is absent in the genome of an escape mutant but present at the corresponding position in the genome of a wild-type strain
  • cleavage of the polynucleotide by the restriction enzyme will identify the candidate NDV strain as a wild-type strain, and absence of cleavage will identify the candidate NDV strain as an escape mutant.
  • the polynucleotide molecule referred to above can be obtained from a candidate NDV strain, or from a biological sample (e.g., a tissue sample obtained from an animal), using routine molecular biological techniques.
  • the polynucleotide molecule is obtained by amplifying a portion of the genome of a candidate NDV strain.
  • viral RNA can be obtained from the candidate NDV strain, or from a biological sample, and subjected to a reverse-transcriptase-polymerase chain reaction (RT-PCR).
  • the RT-PCR is carried out with primers that flank a portion of the NDV genome (e.g., a portion of the F gene) which is known to contain a URECS, thereby producing an amplified polynucleotide molecule of known size.
  • An amplified polynucleotide molecule thus produced may be referred to herein as an "amplified portion of the genome of a candidate NDV strain," or simply an "amplified portion.”
  • a convenient method for determining whether an amplified portion of the genome of a candidate NDV strain contains or does not contain a URECS is by contacting the amplified portion with an enzyme that recognizes the URECS.
  • the resulting mixture is referred to as a cleavage reaction mixture.
  • the cleavage reaction mixture is incubated under appropriate conditions which facilitate cleavage by the restriction enzyme. Such conditions (e.g., time of incubation, temperature of incubation, buffers, etc.) will be known to persons of ordinary skill in the art and can be obtained, e.g., from any manufacturer or supplier of restriction enzymes.
  • the cleavage reaction mixture can then be subjected to size separation to obtain a restriction fragment pattern.
  • the cleavage reaction mixture (or the nucleic acid isolated therefrom) can be subjected to gel (e.g., polyacrylamide, agarose, etc.) electrophoresis and visualization of the nucleic acid bands within the gel.
  • gel e.g., polyacrylamide, agarose, etc.
  • the pattern of nucleic acid bands visualized within the gel is the restriction fragment pattern.
  • the restriction fragment pattern produced from the amplified portion of the candidate NDV strain may be compared to the restriction fragment pattern obtained from the corresponding amplified portion of either a wild-type NDV strain and/or a known escape mutant (subjected to the same or substantially similar restriction enzyme cleavage conditions and size separation methods). It can be concluded that the candidate NDV strain is an NDV escape mutant if either: (a) the restriction fragment pattern obtained from the candidate NDV strain is different from the restriction fragment pattern obtained from the wild-type NDV strain; or (b) the restriction fragment pattern obtained from the candidate NDV strain is the same as the restriction fragment pattern obtained from a known escape mutant.
  • the methods of the present invention are useful, inter alia, for determining if a poultry animal has been vaccinated with an NDV escape mutant vaccine strain. For example, in populations of chickens, it is often important to distinguish vaccinated animals from infected animals (e.g., animals that are infected with a wild-type NDV strain). Animals that have been vaccinated with an NDV escape mutant can be distinguished from infected animals using, e.g., antibodies that recognize wild-type NDV strains but not escape mutants. (See, e.g., U.S. Patent No. 6,833,133). The methods of the present invention provide an additional, or alternative, method for identifying vaccinated animals.
  • a biological sample is removed from a poultry animal and viral RNA can be isolated therefrom.
  • a portion of the NDV genome is amplified using RT-PCR.
  • the amplified portion will include a region that is known to contain or lack a URECS.
  • the amplified portion is then treated with a restriction enzyme that cleaves the URECS, and the enzyme-treated nucleic acid is subjected to size separation to obtain a restriction fragment pattern. If the restriction fragment pattern is the same as that which is obtained from an NDV escape mutant, it can be concluded that the animal was vaccinated with an NDV escape mutant (vaccine strain).
  • restriction fragment pattern is different from that which is obtained from an NDV escape mutant (e.g., a restriction fragment pattern indicative of a wild-type strain)
  • a restriction fragment pattern indicative of a wild-type strain e.g., a restriction fragment pattern indicative of a wild-type strain
  • nucleotide sequence of an exemplary wild-type NDV F gene is depicted in Figures 1A and 1 B (SEQ ID NO:1 ).
  • Exemplary NDV escape mutants include the strains designated P12, P13, P14, P15 and P16, as described in Example 1 , herein. These escape mutants are distinguishable from wild-type NDV strains at the genomic level by the presence of nucleotide changes in the codon that encodes the Serine at position 157 ("Ser157”) of the wild- type F protein.
  • Serine at position 157 Serine at position 157
  • Wild-type NDV F protein amino acid sequences are known in the art and can be obtained, e.g., from NCBI under accession numbers BAA00173, CAA78095, ABK63993, etc.).
  • the codon encoding SeM 57 of the F protein is nucleotides 469-471 of SEQ ID NO:1 and is identified by a box in Figure 1A. Based on the structural configuration of the F protein in its natural context (i.e., exposed on the surface of the NDV particle), it is likely that mutations that result in changes in other amino acids in the vicinity of Ser157 would also abolish mAb54 binding and thereby create additional NDV escape mutants.
  • mutations in the codons encoding e.g., Leu154 (encoded by nucleotides 460-462 of SEQ ID NO:1 ), Lys155 (encoded by nucleotides 463-465 of SEQ ID NO:1 ), Glu156 (encoded by nucleotides 466-468 of SEQ ID NO:1 ), Ile158 (encoded by nucleotides 472-474 of SEQ ID NO:1), Ala159 (encoded by nucleotides 475-477 of SEQ ID NO:1 ), or Ala160 (encoded by nucleotides 478-480 of SEQ ID NO:1) of the F protein would likely produce additional NDV escape mutants.
  • a method of distinguishing an NDV escape mutant from a wild-type NDV strain can be performed by first amplifying a portion of the genome of a candidate NDV strain.
  • the amplified portion preferably comprises the region corresponding to the 462 nd through the 478 th nucleotide of the F gene (SEQ ID NO:1 ).
  • the amplified portion may comprise the 450 th , 425 th , 400 th , 375 th , 350 th , 325 th , 300 th , 375 th , 350 th , 325 th , 300 th , 275 th , 250 th , 225 th or 200 th nucleotide of the F gene (SEQ ID NO: 1 ) through the 480 th , 500 th , 525 th , 550 th , 575 th , 600 th , 625 th , 650 th , 675 th , or 700 th nucleotide of the F gene (SEQ ID NO:1 ).
  • Other suitable regions would be appreciated by those of ordinary skill in the art in view of the teachings of the present disclosure.
  • the amplified portion of the NDV genome can be produced, e.g., by a PCR (e.g., RT-PCR) using a forward and a reverse primer.
  • the forward and reverse primers comprise nucleotide sequences that correspond to, or are complementary to, sequences of the NDV genome that flank the region of the NDV genome containing a URECS.
  • the forward and reverse primers will contain sequences that correspond to, or are complementary to, sequences of the NDV genome located upstream and downstream, respectively, from nucleotides 469-471 of SEQ ID NO:1 (which encode SeM 57 of the F protein).
  • the forward primer used in the context of the present invention is one that is able to hybridize to a segment of a cDNA copy of the NDV genome located between the 50 th and 470 th nucleotides of the F gene (SEQ ID NO:1 ).
  • the reverse primer used in the context of the present invention is one that is able to hybridize to a segment of a cDNA copy of the NDV genome located between the 476 th and 800 th nucleotides of the F gene (SEQ ID NO:1 ).
  • the forward and reverse primers may be of any length suitable for use in a PCR.
  • the forward and reverse primers may be about 5, 10, 15, 20, 25 or 30 nucleotides in length.
  • the expression "able to hybridize” means that the primer in question is able to bind to a substantially complementary nucleic acid sequence at a temperature of about 50 0 C to about 6O 0 C in a solution comprising about 1.2 mM MgSO 4 .
  • Forward and reverse primers can be designed using the sequence of a wild-type NDV F gene (e.g., SEQ ID NO:1 ).
  • suitable primers can be designed "manually,” e.g., by analyzing various regions of the target sequence and selecting regions which would result in an amplified segment of desired length (e.g., about 25 to 3000 nucleotides in length) and which would permit hybridization to a complementary nucleotide at a temperature suitable for a PCR (e.g., about 40 0 C to about 70 0 C).
  • desired length e.g., about 25 to 3000 nucleotides in length
  • a complementary nucleotide at a temperature suitable for a PCR e.g., about 40 0 C to about 70 0 C.
  • numerous automated, computer- based methods are available for selecting the appropriate forward and reverse primers for use with the methods of the present invention.
  • An example of one such computer-based method is the LaserGene computer program available from DNAstar (Madison Wl) (see Example 3, herein).
  • the amplified portion of the NDV genome produced in accordance with the above-described methods is contacted with a restriction enzyme to produce a cleavage reaction mixture.
  • the restriction enzyme is one which recognizes a URECS, i.e., a restriction enzyme cleavage site that is either: (i) present at one or more positions in the genome of an NDV escape mutant but absent at the corresponding positions in the genome of a wild-type NDV strain; or (ii) absent at one or more positions in the genome of an NDV escape mutant but present at the corresponding positions in the genome of a wild-type NDV strain.
  • the URECS is preferably located within a region of the NDV genome that corresponds to the amplified portion of the candidate NDV strain.
  • the URECS is a restriction enzyme cleavage site that is created or destroyed in an NDV escape mutant due to one or more nucleotide changes in the codon encoding, e.g., Ser157 of the NDV F protein (i.e., nucleotides 469-471 of SEQ ID NO:1 ).
  • Ser157 of the NDV F protein i.e., nucleotides 469-471 of SEQ ID NO:1
  • the nucleotide sequence of a wild-type NDV F gene from nucleotides 462 through 478 of SEQ ID NO:1 is: 5'- T AAA GAG AG * C ATT GCT G --3 1 (SEQ ID NO:45).
  • the underlined sequence (AGC) is the codon for Ser157.
  • the asterisk ( * ) between the third G and the first C is the cleavage site for the restriction enzyme BsrDI.
  • an escape mutant e.g., P13
  • the F gene sequence may be mutated to : 5'- T AAA GAG AG#A ATT GCT G -3' (SEQ ID NO:46).
  • This change will eliminate the BsrDI cleavage site and at the same time create a new Tsp509l site (designated by the pound sign (#) between the third G and the sixth A). Accordingly, both BsrDI and Tsp509l are URECS for purposes of the present invention.
  • Ser157 appears to be at least one of the amino acids of the NDV F protein that is critical for recognition of wild-type NDV strains by mAb54. Mutation of SeM 57 to any of the other amino acids would therefore likely disrupt or abolish mAb54 binding, thereby creating additional NDV escape mutants. It therefore follows that additional URECS in NDV escape mutants would be created by, e.g., mutating the Ser157 codon (AGC) to the codon encoding any other amino acid. Examples of such additional URECS are set forth in Table 1.
  • Any of the restriction enzyme sites that are created or destroyed by the mutations depicted in Table 1 are URECS that can be used in the context of the present invention to distinguish NDV escape mutants from wild-type strains.
  • kits may comprise, e.g., one or more containers containing one or more components for carrying out the RT- PCR/RFLP assays of the present invention.
  • the kits of the present invention may comprise, e.g., one or more of the following components: a forward primer (e.g., any of the forward primers described elsewhere herein), a reverse primer (e.g., any of the reverse primers described elsewhere herein), a reverse transcriptase (e.g., a thermostable reverse transcriptase), a DNA polymerase (e.g., a thermostable DNA polymerase), restriction enzyme (e.g., a restriction enzyme that recognizes a URECS listed in Table 1), one or more buffers for carrying out reverse transcription and/or DNA polymerization, and/or restriction digestion.
  • a forward primer e.g., any of the forward primers described elsewhere herein
  • a reverse primer e.g., any of the reverse primers described elsewhere herein
  • kits may contain a wild-type NDV strain (e.g., NDW, Hertz, LaSota, Ulster 2C, etc.) and/or an NDW escape mutant (e.g., P12, P13, P14, P15, P16, etc.).
  • a wild-type NDV strain e.g., NDW, Hertz, LaSota, Ulster 2C, etc.
  • an NDW escape mutant e.g., P12, P13, P14, P15, P16, etc.
  • mAb54 is a monoclonal antibody that is known to recognize a conserved epitope on the F protein of wild-type NDV strains. (See U.S. Patent No. 6,833,133). Mutant strains of NDV that are not recognized by mAb54 ⁇ i.e., "escape mutants") can be used as vaccine strains which permit the distinction between vaccinated animals and those that are infected with virulent or wild-type forms of NDV.
  • Escape mutants were generated by mixing mAb54 with the NDV Wiltenburg strain (also referred to as the "NDW" strain; see U.S. Patent No. 5,149,530).
  • the antibody-treated viruses were then grown in either chicken embryonic fibroblast (CEF) or African Green Monkey Kidney (Vera) cells in the presence of trypsin and an agar overlay containing mAb54.
  • CEF chicken embryonic fibroblast
  • Vera African Green Monkey Kidney
  • the plaques obtained by this method were picked and the escape mutant viruses were purified by two further rounds of plating and plaque purification.
  • Mother virus stocks of the escape mutants were produced in embryonated fowls eggs after the third plaque purification.
  • a total of eight escape mutants were produced by this process (two using CEF cells and six using Vera cells). These escape mutants were designated P7, P8, P12
  • mAb54 did not bind any of the escape mutants; however, all escape mutants were recognized by the anti-hemagglutinin-neuraminidase antibody (mAb85) and by the polyclonal NDV antiserum.
  • the wild-type NDW strain was recognized by both monoclonal antibodies and by the polyclonal antiserum.
  • the P13 escape mutant was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), of the lnstitut Pasteur, Paris, France, 25 Rue de Doorganismes, F- 75724, under accession number I-2928 on August 29, 2002. (See U.S. Patent No. 6,833,133). The P13 escape mutant has been shown to be an effective vaccine strain against NDV infection in poultry. (See U.S. Patent No. 6,833,133).
  • RNA was extracted from the escape mutants and from wild-type strains (Ulster and NDW) using a QIAMP viral RNA mini kit (Qiagen Inc., Valencia, CA, USA).
  • the first strand was synthesized as follows: RNA (2.5 ⁇ L) and 6.5 ⁇ L of dH 2 O were added to 1.0 ⁇ L of forward primer (either Primer No. 7, 23 or 35 from Table 3).
  • RNAsin 0.5 ⁇ L
  • 5X concentrated reverse transcriptase buffer 4.0 //L
  • dH 2 O 4.0 ⁇ L
  • 40 mM dNTPs 1.0 ⁇ L
  • M-MLV reverse transcriptase 0.5 ⁇ L
  • PCR reaction mix comprised 25 ⁇ L PCR Ready Mix x 2 (Applied Biosystems, Foster City, CA, USA), 18 ⁇ L dH2O and 1 ⁇ L (50 pmol) of both the forward and reverse primer.
  • the components were well mixed, spun briefly and 5 ⁇ L of cDNA added before thermal cycling. Cycling parameters were 94 0 C for 10 minutes (one cycle), 94°C for 1 minute/50°C for 1 minute/72°C for 3 minutes (29 cycles), and 72°C for 5 minutes (one cycle). Control "no template 1 controls were included in each experiment to provide evidence, if any, of contamination.
  • reaction mixes were electrophoresed on an agarose gel containing ethidium bromide and visualized using an UV transilluminator.
  • Product size was estimated by comparing with standard marker DNA (pGEM) and the fragment was purified if it was of the predicted size.
  • DNA fragments were excised from the gel and purified using the QiaQuick gel extraction kit (Qiagen Inc., Valencia, CA, USA). Following fragment purification, DNA was again electrophoresed in an agarose gel and an estimate was made of the volume required for sequencing.
  • pGEM standard marker DNA
  • the sequencing reaction products were precipitated by adding 1 ⁇ L of 25mM glycogen and 52 ⁇ L of 2M sodium acetate pH 4.5. The mix was vortexed, left for 10 minutes and then centrifuged at 13,000 rpm for 30 minutes. The liquid was aspirated off leaving behind a pellet that was rinsed by the addition of 150 ⁇ L of 80% ethanol. Following centrifugation at 13,000 for 10 minutes, the alcohol was removed and the sample centrifuged again before removing any remaining alcohol.
  • the pellet was dried by heating on a block at 95°C for 2 minutes, resuspended in 15 ⁇ l_ TSR, vortexed and then centrifuged (pulse) before heating again at 95 0 C for 2 minutes and chilling on ice. Following an additional vortex and spin, samples were transferred to ABI tubes and then into the genetic analyzer (ABI PRISMTM 310 genetic analyzer). Sequences were aligned and compared with one another.
  • F gene The complete nucleotide sequence of the fusion protein-encoding gene ("F gene") of wild-type NDV strain (“NDW”) is set forth as SEQ ID NO:1 ( Figures 1A-1 B). It was observed that the nucleotide sequence of the F gene of the wild-type NDV strain (“NDW”) is identical to the sequences of the F gene of the escape mutants, except for a single nucleotide change.
  • escape mutants of NDV can be distinguished from wild-type strains on the basis of their inability to be recognized by mAb54. Nonetheless, the present inventors sought to establish a more sensitive, rapid, and less expensive way of identifying NDV escape mutants.
  • a molecular assay was established based on the nucleotide differences discovered in the genomes of the P12, P13, P14, P15 and P16 escape mutants relative to wild- type NDV strains (see Example 2, above). More particularly, a reverse transcription-polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) assay was established. This assay takes advantage of the fact that, in escape mutants, the nucleotide sequence in the region of nucleotides 471-475 of the F gene is different from the corresponding sequence in wild-type NDV strains.
  • RT-PCR/RFLP reverse transcription-polymerase chain reaction/restriction fragment length polymorphism
  • nucleotide sequence of the F gene of the NDW Ulster strain (a "wild-type” strain, as used herein) between nucleotides 462 and 478 was compared to the corresponding sequence in the F gene of P13. (See first two rows in Figure 1A).
  • sequence in this region is: [0056] 5' ⁇ T AAA GAG AG * C ATT GCT G -3 1 (SEQ ID NO:45). This sequence includes the cleavage site for the restriction enzyme BsrDI between the G and the C (indicated with an asterisk ( * ) in the foregoing sequence).
  • the LaserGene computer program (DNAstar, Madison Wl) was used to identify primer sets to amplify a region of the F gene that includes nucleotides 462-478. From this program, the primer set shown in Table 4 was selected for RT-PCR amplification.
  • Position refers to the nucleotide position of the primer within the F gene.
  • RT-PCR was carried out using the Superscript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA).
  • the final reaction mix contained 0.2 mM of each dNTP, 1.2 mM MgSO 4 and 0.2 ⁇ M of each primer.
  • the cycling conditions were as follows: 30 minutes 57 0 C (cDNA synthesis); 10 minutes 95 0 C (pre-denaturation); 30 cycles of 30 seconds 95 0 C (denaturation); 30 seconds 57 0 C (annealing); 45 seconds 72 0 C (extension); 10 minutes 72 0 C (final extension) and 5 minutes 4 0 C (reaction stop).
  • the PCR products were stored at -2O 0 C until purification, gel-electrophoresis and RFLP.
  • the P13 escape mutant is easily distinguished from the other NDV strains in the RT-PCR/RFLP assay based on the presence of a unique restriction pattern that includes a 359 bp band and an 80 bp band.
  • the "wild-type" strains when subjected to the same assay, produced a band of 439 bp, and the 80 bp band was absent.
  • Example 3 the RT-PCR/RFLP assay of Example 3 is used to distinguish chickens that have been vaccinated with the P13 NDV escape mutant vaccine strain from chickens that are vaccinated with standard vaccine strains or that harbor virulent NDV strains.
  • RNA-containing tissue samples is obtained from a chicken believed to have been vaccinated with the P13 NDV escape mutant.
  • Viral RNA is extracted from the tissue sample using a standard RNA purification method or a commercially available RNA purification kit.
  • the purified RNA is subjected to RT-PCR using the selected primer set shown in Table 4 (SEQ ID NOs. 11 and 12). Standard RT-PCR conditions are used, for example, those described in Example 3.
  • the PCR product is digested with Tsp509l at 65°C overnight. The restriction enzyme reaction is then subjected to agarose gel electrophoresis, and the restriction fragment bands are analyzed.
  • Appropriate controls may be included on the gel such as Tsp509l-treated PCR product from the P13 virus as a positive control, and Tsp509l-treated PCR product from one or more wild-type strains (such as those used in Example 3) as negative controls.
  • the Tsp509l-treated PCR product obtained from viral material of the chicken tissue sample shows the restriction fragment pattern depicted in Figures 2 and 3 for P13 (i.e., a 359 bp band and an 80 bp band), then it can be concluded that the chicken was vaccinated with the P13 escape mutant.
  • the restriction fragment pattern depicted in Figures 2 and 3 for the wild-type strains is observed ⁇ i.e., a 439 bp and no 80 bp band), then it must be concluded that the chicken was exposed to one of the wild-type strains, but not the P13 escape mutant strain.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés permettant de distinguer des mutants d’échappement du virus de la maladie de Newcastle (NDV) (également connus sous le nom de souches de marqueurs) à partir de souches NDV de type sauvage. Les mutants d’échappement sont des souches qui contiennent des modifications génétiques qui amènent les mutants à échapper à la détection par des anticorps qui se lient normalement aux souches NDV du type sauvage. Les procédés de l’invention concernent en général une analyse de polymorphisme de restriction (RFLP) qui utilise des enzymes de restriction qui reconnaissent des sites de clivage d’enzymes de restriction uniques. De tels sites de clivage d’enzymes de restriction uniques sont présents dans les mutants d’échappement NDV, mais pas dans les souches NDV du type sauvage, ou vice-versa, et sont causés par une ou plusieurs mutations qui rendent les mutants d’échappement capables d’échapper à la détection par des anticorps anti-NDV. Les motifs des fragments de restriction produits par les procédés de la présente invention vont identifier de manière fiable la souche NDV sous forme d’un mutant d’échappement ou d’une souche de type sauvage, en fonction du motif de bande spécifique produit.
PCT/US2009/000907 2008-02-13 2009-02-12 Analyse génétique pour identifier des mutants d’échappement du virus de la maladie de newcastle WO2009102459A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6553308P 2008-02-13 2008-02-13
US61/065,533 2008-02-13

Publications (2)

Publication Number Publication Date
WO2009102459A2 true WO2009102459A2 (fr) 2009-08-20
WO2009102459A3 WO2009102459A3 (fr) 2009-11-12

Family

ID=40842682

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/000907 WO2009102459A2 (fr) 2008-02-13 2009-02-12 Analyse génétique pour identifier des mutants d’échappement du virus de la maladie de newcastle

Country Status (1)

Country Link
WO (1) WO2009102459A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104995315A (zh) * 2012-12-18 2015-10-21 梅里亚股份有限公司 Ibv、csfv和ndv的高分辨熔解基因分型

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040131640A1 (en) * 2002-12-06 2004-07-08 Wyeth Escape mutants of newcastle disease virus as marker vaccines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040131640A1 (en) * 2002-12-06 2004-07-08 Wyeth Escape mutants of newcastle disease virus as marker vaccines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KOU YIN-TAI ET AL: "Restriction fragment length polymorphism analysis of the F gene of Newcastle disease viruses isolated from chickens and an owl in Taiwan" JOURNAL OF VETERINARY MEDICAL SCIENCE, vol. 61, no. 11, November 1999 (1999-11), pages 1191-1195, XP002536893 ISSN: 0916-7250 *
UJVARI D ET AL: "Identification and subgrouping of pigeon type Newcastle disease virus strains by restriction enzyme cleavage site analysis" JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 131, no. 2, 1 February 2006 (2006-02-01), pages 115-121, XP025030342 ISSN: 0166-0934 [retrieved on 2006-02-01] *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104995315A (zh) * 2012-12-18 2015-10-21 梅里亚股份有限公司 Ibv、csfv和ndv的高分辨熔解基因分型
EP2935629A1 (fr) * 2012-12-18 2015-10-28 Merial, Inc. Génotypage par dénaturation à haute résolution d'od ibv, csfv et ndv

Also Published As

Publication number Publication date
WO2009102459A3 (fr) 2009-11-12

Similar Documents

Publication Publication Date Title
CN105132590B (zh) 一种牛传染性鼻气管炎病毒的lamp可视化检测方法
Saito et al. Application of RT‐PCR designed from the sequence of the local SRSV strain to the screening in viral gastroenteritis outbreaks
Magome et al. Single-strand conformation polymorphism analysis of apple stem grooving capillovirus sequence variants
WO2022257663A1 (fr) Procédé et kit pour la détection et le dépistage de la mutation n501y dans la covid-19
Liu et al. Detection of infectious bronchitis virus by multiplex polymerase chain reaction and sequence analysis
CN108165666B (zh) 检测CPV2a、CPV2b与CPV2c并鉴别野生型与疫苗型的方法
CN115466800A (zh) 一种检测猴痘病毒及其亚型的多重荧光定量pcr试剂盒
Dewey et al. Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products
Nuñez et al. Detection and typing of molluscum contagiosum virus in skin lesions by using a simple lysis method and polymerase chain reaction
Goswami et al. A Polymerase Chain Reaction–Based Method for the Detection of Hepatitis A Virus in Produce and Shellfish
WO2009102459A2 (fr) Analyse génétique pour identifier des mutants d’échappement du virus de la maladie de newcastle
CN112941240B (zh) 检测鹅星状病毒和鹅嵌杯病毒的引物对、试剂盒及方法
EP1185703B1 (fr) Dosage cort-pcr pour la discrimination entre l'activite de transcriptase inverse endogene dans des lignees de cellules eucaryotes et la contamination par retrovirus infectieux
JP5315519B2 (ja) コイヘルペスウイルス（ｋｈｖ）の検出方法
CN111621603A (zh) 一种鉴定非洲猪瘟病毒的特异性引物及其鉴定方法与检测试剂盒
CN115491431A (zh) 基于基因编辑技术检测日本乙型脑炎病毒核酸的检测试剂盒及应用
Katayama et al. Differentiation of mumps vaccine strains from wild viruses by single-strand conformation polymorphism of the P gene
WO2008008501A2 (fr) Dosage du polymorphisme de la longueur des fragments de restriction inverse et utilisations de celui-ci
KR100504763B1 (ko) 전염성 기관지염 바이러스의 뉴클레오캡시드 유전자
JP4676063B2 (ja) 弱毒水痘ウイルス岡株の遺伝子６２とこの遺伝子６２を用いる弱毒生水痘ワクチン用ウイルス株の同定方法
WO2006009260A1 (fr) Procédé de détection du virus de l'hépatite e
CN111254222B (zh) 流感病毒血凝素亚型分型引物、试剂盒和分型方法
CN117551818B (zh) 一种检测呼吸道合胞病毒的靶基因、引物探针组合以及试剂盒和应用
WO2022257664A1 (fr) Procédé et kit de détection et de criblage d'une mutation n439k d'un nouveau coronavirus
CN110438261B (zh) 用于检测f亚群禽白血病病毒的分子标记、引物、试剂盒及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09710840

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09710840

Country of ref document: EP

Kind code of ref document: A2