WO2009102459A2

WO2009102459A2 - A genetic assay to identify newcastle disease virus escape mutants

Info

Publication number: WO2009102459A2
Application number: PCT/US2009/000907
Authority: WO
Inventors: Harmen Jacob Geerligs; Cindy Aleida Maria Meinders
Original assignee: Wyeth
Priority date: 2008-02-13
Filing date: 2009-02-12
Publication date: 2009-08-20
Also published as: WO2009102459A3

Abstract

The present invention provides methods for distinguishing Newcastle Disease Virus (NDV) escape mutants (also known as marker strains) from wild-type NDV strains. Escape mutants are strains which contain genetic changes that cause the mutants to escape detection by antibodies that normally bind to wild-type NDV strains. The methods of the invention generally relate to a restriction fragment length polymorphism (RFLP) assay which utilizes restriction enzymes that recognize unique restriction enzyme cleavage sites. Such unique restriction enzyme cleavage sites are present in NDV escape mutants but not in NDV wild-type strains, or vice versa, and are caused by one or more mutations that render the escape mutants capable of escaping detection by anti-NDV antibodies. The restriction fragment patterns produced by the methods of the present invention will reliably identify the NDV strain as an escape mutant or a wild-type strain, depending on the specific band pattern produced.

Description

A GENETIC ASSAY TO IDENTIFY NEWCASTLE DISEASE VIRUS ESCAPE MUTANTS

BACKGROUND OF THE INVENTION

[0001] 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.

[0002] 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. As explained in U.S. 6,833,133, 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)).

[0003] 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. Although antibody-based assays can be used to distinguish NDV escape mutants from wild-type strains, there are circumstances in which alternative methods for distinguishing NDV escape mutants from wild-type strains would be useful. For example, in some situations, the use of antibodies and related reagents and equipment (e.g., secondary antibodies, detectable probes, fixing agents, microscopic detection equipment, etc.) is expensive and inconvenient for rapidly assaying for the presence an NDV escape mutant. In these circumstances, a rapid and easily analyzable method would be preferred. Until now, no such alternative methods have been available.

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention satisfies the aforementioned need in the art by providing alternative methods for distinguishing NDV escape mutants from wild-type NDV strains. In particular, 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.

[0005] Although 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. For example, 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. In fact, in the assays previously mentioned in the art, there is no direct correlation between the restriction fragment patterns observed and the functional or phenotypic characteristics of the NDV strains tested. In contrast, 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. From this initial detailed genomic analysis, specific mutations are identified which are responsible for NDV escape mutants to escape recognition by anti-NDV antibodies. The RFLP assays of the present invention are then carefully designed based on the identification of these functionally-significant mutations and the restriction enzyme cleavage sites that are created and/or destroyed therefrom.

[0006] Thus, 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. As a consequence, 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. Alternatively, or additionally, 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.

[0007] Thus, as described in more detail herein below, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] 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.

[0009] 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. The lanes, from left to right depict the following: (1 ) Nucleic acid marker with bands of 100, 200, 400, 800, 1200 and 2000 base pairs in length; (2) Strain NDW subjected to RT-PCR, uncut; (3) Strain NDW subjected to RT-PCR, treated with Tsp509l; (4) P13 escape mutant subjected to RT-PCR, uncut; (5) P13 escape mutant subjected to RT-PCR, treated with Tsp509l; (6) Negative control RT-PCR, uncut; (7) Negative control RT-PCR, treated with Tsp509l; and (8) Nucleic acid marker with bands of 125 and 500 base pairs in length.

[0010] 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 lanes, from left to right depict the following: (1 ) Nucleic acid marker with bands of 125, 250, 375 and 500 base pairs in length; (2) - (7) Hertz, La Sota, Ulster, P13, PMV-1 , and Ulster 2C NDV strains, respectively, subjected to RT-PCR and treated with Tsp509l; and (8) Nucleic acid marker with bands of 50, 125, 250, 375 and 500 base pairs in length.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention provides methods for distinguishing Newcastle Disease Virus (NDV) escape mutants from wild-type NDV strains.

NDV ESCAPE MUTANTS

[0012] As used herein, the term "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).

[0013] In general, 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 Docteur 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 STRAINS

[0014] The expression "wild-type NDV," as used herein means any NDV that is not an escape mutant. Thus, for purposes of the present disclosure, 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. Specific non-limiting examples of NDV strains that are considered "wild-type NDV" strains for purposes of this disclosure include, e.g., the Wiltenburg strain (also called "NDW," available under accession number 1-781 from the Collection Nationale de Cultures de Microorganismes, lnstitut Pasteur, Paris, France, see U.S. Patent No. 5,149,530), 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, Italien, Milano, Miyadera, Northants, Taiwan 95. Numerous other 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 (see, e.g., U.S. Patent No. 6,451 ,323) 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.

GENOMIC MUTATIONS AND UNIQUE RESTRICTION SITES

[0015] The methods of the present invention take advantage of the genetic changes that allow NDV escape mutants to escape recognition by mAb54. In particular, it was discovered that 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. As used herein and understood by persons of ordinary skill in the art, the "F gene" refers to a polynucleotide sequence that encodes the fusion protein (also referred to as the ¹¹F protein" or "F glycoprotein") of NDV.

[0016] Mutations which render NDV escape mutants incapable of being recognized by mAb54 were found to create and/or destroy restriction enzyme cleavage sites in the NDV genome. Such restriction sites are referred to herein as "unique restriction enzyme cleavage sites" or "URECS". Thus, by identifying one or more URECS in the genome of an NDV escape mutant, a restriction fragment length polymorphism (RFLP) assay can be designed and conducted which identifies the NDV escape mutant on the basis of the resulting unique restriction fragment pattern.

[0017] 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. For example, using standard molecular biological techniques (e.g., site directed mutagenesis), individual 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).

[0018] Once one or more nucleotide changes are identified that are responsible for the escape mutant phenotype, 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.

CLEAVAGE OF AN AMPLIFIED PORTION OF A CANDIDATE NDV ESCAPE MUTANT

[0019] The methods of the present invention comprise molecular assays involving restriction enzymes that recognize URECS. For example, according to certain embodiments of the present invention, 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. If the URECS is one that is present in the genome of an escape mutant but absent at the corresponding position in the genome of a wild-type strain, then 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). On the other hand, if 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, then 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.

[0020] 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. According to certain embodiments of the present invention, the polynucleotide molecule is obtained by amplifying a portion of the genome of a candidate NDV strain. For example, 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."

[0021] 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. For example, 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. The pattern of nucleic acid bands visualized within the gel is the restriction fragment pattern.

[0022] 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.

METHODS FOR SCREENING POULTRY

[0023] 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. First, a biological sample is removed from a poultry animal and viral RNA can be isolated therefrom. Next, 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). On the other hand, if the 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), then it is concluded that the animal was not vaccinated with an NDV escape mutant (vaccine strain), and/or that the animal was infected with a wild-type strain.

EXEMPLARY MUTATIONS WITHIN THE F-GENE OF NDV ESCAPE MUTANTS

[0024] The 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. (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. For example, 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. The methods of the present invention can be used to exploit such genomic changes in order to distinguish NDV escape mutants from wild-type NDV strains by analyzing the mutated sequences for URECS and using such URECS as the basis for RFLP analysis as described above. [0025] For example, in certain exemplary embodiments of the present invention, 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. Since escape mutants such as P12, P13, P14, P15 and P16 contain mutations in the codon encoding Ser157 of the F protein (i.e., nucleotides 469-471 of SEQ ID NO:1 ), the amplified portion, according to this exemplary embodiment, preferably comprises the region corresponding to the 462^nd through the 478^th nucleotide of the F gene (SEQ ID NO:1 ). For example, 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.

EXEMPLARY FORWARD AND REVERSE PRIMERS FOR AMPLIFICATION

[0026] 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. For example, if the URECS is one that is created or destroyed as a result of a mutation at the codon encoding Ser157 of the F protein, 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).

[0027] In certain exemplary embodiments, 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 ). In certain exemplary embodiments, 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. For example, the forward and reverse primers may be about 5, 10, 15, 20, 25 or 30 nucleotides in length. As used in this disclosure, 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 50⁰C to about 6O⁰C in a solution comprising about 1.2 mM MgSO₄. [0028] Forward and reverse primers can be designed using the sequence of a wild-type NDV F gene (e.g., SEQ ID NO:1 ). For example, 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 40⁰C to about 70⁰C). Alternatively, 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).

[0029] Specific examples of forward primers that can be used in the methods of the present invention include nucleic acid molecules comprising, consisting of, or consisting essentially of SEQ ID NOs: 11 , 13, 15, 17, 19. 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , or 43, or a sequence that is at least 90% identical to any one of SEQ ID NOs: 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29,

31 , 33, 35, 37, 39, 41 , or 43, and which hybridizes to a cDNA copy of the NDV genome at 57°C in the presence of 1.2 mM MgSO₄.

[0030] Specific examples of reverse primers that can be used in the methods of the present invention include nucleic acid molecules comprising, consisting of, or consisting essentially of SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44, or a sequence that is at least 90% identical to any one of SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,

32, 34, 36, 38, 40, 42, or 44, and which hybridizes to a cDNA copy of the NDV genome at 57°C in the presence of 1.2 mM MgSO₄.

EXEMPLARY UNIQUE RESTRICTION ENZYME CLEAVAGE SITES

[0031] According to the present invention, 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. According to the present invention, the URECS is preferably located within a region of the NDV genome that corresponds to the amplified portion of the candidate NDV strain.

[0032] In certain embodiments of the present invention, 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 ). For example, 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 --3¹ (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. In an escape mutant (e.g., P13) where the Serine at position 157 is changed to an Arginine, 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.

[0033] As explained in the examples presented herein, 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.

Table 1 : Additional URECS Created or Destro ed b Mutatin SeM 57 of the Wild-T e NDV F Protein

[0034] 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

[0035] The present invention also includes kits. The kits of the invention 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. In certain embodiments of the present invention, the 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.).

[0036] The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in molecular biology, microbiology, virology and chemistry which are obvious to those skilled in the art in view of the present disclosure are within the spirit and scope of the invention. EXAMPLES

EXAMPLE 1

GENERATION OF NDV ESCAPE MUTANTS

[0037] The purpose of this Example was to prepare mutant Newcastle Disease virus isolates that are easily distinguishable from wild-type NDV strains on the basis of their inability to interact with the monoclonal antibody designated mAb54. 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.

[0038] 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. The formation of plaques in the agar-overlaid cells, despite the presence of mAb54, indicated the presence of escape mutants. 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, P13, P14, P15, P16 and P17.

[0039] All eight escape mutants were confirmed to lack the epitope recognized by mAb54 by an indirect immunoperoxidase (IIP) assay. Briefly, tissue culture cells were infected with the various escape mutant virus stocks. The infected cells were then fixed and incubated with mAb54. The binding of mAb54 was visualized using a peroxidase conjugated anti-mouse antibody, followed by addition of substrate. As controls, IIP assays were also performed on cells infected with the escape mutants using a monoclonal antibody directed against an epitope on NDV hemagglutinin-neuraminidase protein and with an NDV polyclonal antiserum. A wild-type NDW control was also included in the assays. The results are shown in Table 2. Table 2: Indirect Immunoperoxidase Assays of Mother Virus Stocks of NDV escape mutants NDV Isolates

# Polyclonal NDV antiserum - = no binding of antibody + = binding of antibody

[0040] As indicated in Table 2, 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.

[0041] The P13 escape mutant was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), of the lnstitut Pasteur, Paris, France, 25 Rue de Docteur Roue, 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).

EXAMPLE 2 NUCLEOTIDE SEQUENCES OF NDV ESCAPE MUTANTS FUSION PROTEIN GENES

[0042] The purpose of this Example was to determine the genetic basis for the NDV escape mutants' inability to be recognized by mAb54. For this Example, only escape mutants P12, P13, P14, P15 and P16 were analyzed.

A. PRIMERS

[0043] Primers used for the PCR and Sequencing in this Example are set forth in Table 3.

F = Forward primer R = Reverse primer

B. POLYMERASE CHAIN REACTION

[0044] First, viral 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 μL) and 6.5 μL of dH₂O were added to 1.0 μL of forward primer (either Primer No. 7, 23 or 35 from Table 3). The mixture was heated at 95°C for 2 minutes, chilled on ice and then RNAsin (0.5 μL), 5X concentrated reverse transcriptase buffer (4.0 //L), dH₂O (4.0 μL), 40 mM dNTPs (1.0 μL) and M-MLV reverse transcriptase (0.5 μL) were added before incubation at 37°C for 1 hour.

[0045] Three overlapping PCR fragments, encompassing the entire fusion (F) protein reading frame of each virus were prepared using Primer Nos. 7 and 16, 23 and 17, 35 and 33 (Table 3). Each PCR reaction mix comprised 25 μL PCR Ready Mix x 2 (Applied Biosystems, Foster City, CA, USA), 18 μL dH2O and 1 μL (50 pmol) of both the forward and reverse primer. The components were well mixed, spun briefly and 5 μL of cDNA added before thermal cycling. Cycling parameters were 94⁰C 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 template¹ controls were included in each experiment to provide evidence, if any, of contamination.

[0046] After the PCR, 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. C. SEQUENCING

[0047] Purified PCR fragments of 968bp, 767bp and 797bp of NDW ("wild type¹), and escape mutants P13, P12, P14, PI5 and P16 were sequenced using primers listed in Table 3. Briefly, a reaction mix comprising 4 μL Terminator Ready reaction mix (Applied Biosystems, Foster City, CA, USA), 2-4 μL PCR template (volume depending on concentration of product), 1.6 μL sequencing primer and deionized water to bring the total volume to 10 μL was cycled on a heating block using the following cycling parameters: 96°C for 10 seconds/50°C for 5 seconds/60°C for 4 minutes (24 cycles).

[0048] The sequencing reaction products were precipitated by adding 1 μL of 25mM glycogen and 52 μL 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 μL 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 μl_ TSR, vortexed and then centrifuged (pulse) before heating again at 95⁰C 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 PRISM™ 310 genetic analyzer). Sequences were aligned and compared with one another.

D. RESULTS

[0049] 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.

[0050] In the case of escape mutants P12, P13 and P14, the C at position 471 of the NDW sequence (SEQ ID NO:1 ) is replaced with an A. This change causes the codon AGC in the wild-type sequence to change to AGA. Thus, at the amino acid level, the Serine at position 157 of the wild-type fusion protein (SEQ ID NO:1 ) is replaced with an Arginine in P12, P13 and P14.

[0051] In the case of escape mutants P15 and P16, the A at position 469 of the NDW sequence (SEQ ID NO:1 ) is replaced with a C. This change causes the codon AGC in the wild- type sequence to change to CGC. Thus, as with P12, P13 and P14, the Serine at position 157 of the wild-type fusion protein (SEQ ID NO:1) is also replaced with an Arginine in P15 and P16.

[0052] The sequence differences identified in this Example were used to establish a restriction fragment length polymorphism (RFLP) assay that can be used to identify NDW escape mutants as described in the following Example 3.

EXAMPLE 3

RT-PCR/RFLP ANALYSIS OF NDV ESCAPE MUTANTS

[0053] Escape mutants of NDV, by definition, 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. To this end, 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.

[0054] In this Example, the P13 escape mutant was analyzed along side several wild-type strains. It is emphasized, however, that this method can easily be adapted for the analysis of any NDV escape mutant.

A. IDENTIFICATION OF A UNIQUE RESTRICTION ENZYME CLEAVAGE SITE ("URECS")

[0055] As a preliminary step in establishing an RT-PCR/RFLP assay, the 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). In the NDW Ulster strain the sequence in this region is: [0056] 5'~ T AAA GAG AG^*C ATT GCT G -3¹ (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).

[0057] In P13, the sequence in this region is:

[0058] 5¹- T AAA GAG AG#A ATT GCT G -3^* (SEQ ID NO:46). Here, because of the single nucleotide change from C to A, the BsrDI site is lost and a Tsp509l cleavage site is created between the third G and sixth A (indicated with pound sign (#) in the foregoing sequence). Thus, in accordance with the terminology used herein, the Tsp509l site created by the mutation in P13 is considered a unique restriction enzyme cleavage site (URECS). (Since the BsrDI site is found in the wild-type sequence but not in the P13 escape mutant sequence, it too is appropriately considered a URECS).

B. RT-PCR AMPLIFICATION

[0059] In the next step, RNA was purified from the P13 escape mutant and from the NDW Ulster vaccine strain using the High Pure Viral RNA kit (Roche). RNA was isolated from virus in allantoic fluid and the RNA was stored at -70⁰C until use in the RT-PCR.

[0060] 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.

Table 4: Selected Primer Set for RT-PCR

Position refers to the nucleotide position of the primer within the F gene.

[0061] Alternative primer sets identified by the LaserGene program are shown in Table 5. Table 5: Alternative Primer Sets for RT-PCR

F = Forward primer R = Reverse primer # Position refers to the nucleotide position of the primer within the F gene

[0062] Using the selected primer set shown in Table 4, 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 MgSO₄ and 0.2 μM of each primer. The cycling conditions were as follows: 30 minutes 57⁰C (cDNA synthesis); 10 minutes 95⁰C (pre-denaturation); 30 cycles of 30 seconds 95⁰C (denaturation); 30 seconds 57⁰C (annealing); 45 seconds 72⁰C (extension); 10 minutes 72⁰C (final extension) and 5 minutes 4⁰C (reaction stop). The PCR products were stored at -2O⁰C until purification, gel-electrophoresis and RFLP.

C. RFLP ANALYSIS [0063] After RT-PCR, the products were purified from the primers using the QiaQuick PCR Purification Protocol (Qiagen, Valencia, CA, USA). The products were then incubated at 65⁰C in the presence of 5 units of Tsp509l restriction enzyme. The restriction digests were analyzed on a 3% agarose gel by electrophoresis. The results of the restriction digest, along with controls, are shown in Figure 2.

[0064] The RT-PCR/RFLP analysis described above was repeated using RNA isolated from the P13 escape mutant and from the following "wild-type" NDW strains: Hertz, LaSota, NDW Ulster, PMV-1 (pigeon isolate), and Ulster 2C. The results of this confirmatory experiment are shown in Figure 3.

D. RESULTS AND CONCLUSIONS

[0065] As shown in Figures 2 and 3, 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, on the other hand, when subjected to the same assay, produced a band of 439 bp, and the 80 bp band was absent.

[0066] These results confirm that the RT-PCR/RFLP assay of the present invention is a robust and reliable way to distinguish NDV escape mutants from wild-type vaccine and field strains of the virus.

EXAMPLE 4

USE OF RT-PCR/RFLP TO DISTINGUISH VACCINATED FROM INFECTED ANIMALS

[0067] In this Example, 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.

[0068] First, a virus-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. Second, 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. Third, 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.

[0069] If 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. On the other hand, if 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.

[0070] Variations on this assay will be immediately apparent to those of ordinary skill in the art. The assay described in this Example can easily be adapted to identify any NDV escape mutant.

[0071] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, this invention is not limited to the particular embodiments disclosed, but is intended to cover all changes and modifications that are within the spirit and scope of the invention as defined by the appended claims.

[0072] All publications and patents mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A method of distinguishing a Newcastle Disease Virus (NDV) escape mutant from a wild- type NDV strain, said method comprising:

(a) amplifying a portion of the genome of a candidate NDV strain, wherein said amplified portion includes the region corresponding to the 462^nd through the 478^th nucleotide of the NDV F glycoprotein coding sequence (SEQ ID NO:1);

(b) contacting said amplified portion with a restriction enzyme to produce a cleavage reaction mixture, wherein said restriction enzyme recognizes a unique restriction enzyme cleavage site (URECS), and wherein said URECS is either:

(i) present at a particular position between the 462^nd through the 478^th nucleotide of the genome of an NDV escape mutant but absent at the corresponding position in the genome of a wild-type NDV strain; or

(ii) absent at a particular position between the 462^nd through the 478^th nucleotide of the genome of an NDV escape mutant but present at the corresponding position in the genome of a wild-type NDV strain; and

(c) subjecting the cleavage reaction mixture to size separation to obtain a restriction fragment pattern for said candidate NDV strain;

wherein said candidate NDV strain is identified as an NDV escape mutant if the restriction fragment pattern obtained in (c) is different from the restriction fragment pattern obtained for a wild-type NDV strain subjected to steps (a) through (c).

2. The method of claim 1 , wherein said size separation is gel electrophoresis.

3. The method of claim 1 , wherein said amplified portion includes the region corresponding to the 400^th through the 500^th nucleotide of the NDV F glycoprotein coding sequence (SEQ ID NO:1 ).

4. The method of claim 3, wherein said amplified portion includes the region corresponding to the 300^th through the 600^th nucleotide of the NDV F glycoprotein coding sequence (SEQ ID NO:1).

5. The method of claim 4, wherein said amplified portion includes the region corresponding to the 200^th through the 700^th nucleotide of the NDV F glycoprotein coding sequence (SEQ ID NO:1 ).

6. The method of claim 1 , wherein said restriction enzyme is Tsp509l or a restriction enzyme that cleaves at the Tsp509l cleavage site.

7. The method of claim 1 , wherein said amplified portion is produced by a polymerase chain reaction (PCR) using a forward primer and a reverse primer, wherein said forward primer hybridizes to a segment of a cDNA copy of the NDV genome located between the 50^th and 470^th nucleotides of the F glycoprotein coding sequence, and wherein said reverse primer hybridizes to a segment of a cDNA copy of the NDV genome located between the 476^th and 800^th nucleotides of the F glycoprotein coding sequence.

8. The method of claim 7, wherein said PCR is a reverse transcription-PCR (RT-PCR)

9. The method of claim 7, wherein said forward primer comprises SEQ ID NOs: 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , or 43, or a sequence that is at least 90% identical thereto and that hybridizes to a cDNA copy of the NDV genome at 57°C in the presence of 1.2 mM MgSO₄.

10. The method of claim 7, wherein said reverse primer comprises SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44, or a sequence that is at least 90% identical thereto and that hybridizes to a cDNA copy of the NDV genome at 57°C in the presence of 1.2 mM MgSO₄.

11. The method of claim 1 , wherein said NDV escape mutant is NDV strain P13, deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on August 29, 2002 and having accession number I-2928.

12. A method of determining if a poultry animal has been vaccinated with a Newcastle Disease Virus (NDV) escape mutant vaccine strain, said method comprising:

(a) obtaining a tissue sample from a poultry animal;

(b) isolating viral RNA from said sample;

(c) amplifying a portion of said viral RNA by reverse transcription-polymerase chain reaction (RT-PCR), wherein said amplified portion includes the region corresponding to the 462^nd through the 478^th nucleotide of the NDV F glycoprotein coding sequence (SEQ ID NO:1 );

(d) digesting said amplified portion with Tsp509l or a restriction enzyme that cleaves at the Tsp509l cleavage site;

(e) subjecting the digested portion obtained in (d) to size separation to obtain a restriction fragment pattern;

wherein said poultry animal is identified as having been vaccinated with an NDV escape mutant if the restriction fragment pattern obtained in (e) includes a fragment of about 359 base pairs and a fragment of about 80 base pairs.

13. The method of claim 12, wherein said size separation is gel electrophoresis.

14. The method of claim 12, wherein said RT-PCR includes a forward primer comprising SEQ ID NO:11 , and a reverse primer comprising SEQ ID NO: 12.

15. The method of claim 12, wherein said NDV escape mutant is NDV strain P13, deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on August 29, 2002 and having accession number I-2928.

16. A kit comprising a forward primer and a reverse primer, wherein said forward primer hybridizes to a segment of a cDNA copy of the Newcastle Disease Virus (NDV) genome located between the 50^th and 470^th nucleotides of the F glycoprotein coding sequence, and wherein said reverse primer hybridizes to a segment of a cDNA copy of the NDV genome located between the 476^th and 800^th nucleotides of the F glycoprotein coding sequence.

17. The kit of claim 16, wherein said forward primer comprises SEQ ID NOs: 11 , 13, 15, 17, 19,

21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , or 43, or a sequence that is at least 90% identical thereto and that hybridizes to a cDNA copy of the NDV genome at 57°C in the presence of 1.2 mM MgSO₄.

18. The kit of claim 16, wherein said reverse primer comprises SEQ ID NOs: 12, 14, 16, 18, 20,

22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44, or a sequence that is at least 90% identical thereto and that hybridizes to a cDNA copy of the NDV genome at 57°C in the presence of 1.2 mM MgSO₄.

19. The kit of claim 16, further comprising NDV strain P13, deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on August 29, 2002 and having accession number I- 2928.