EP0481072A1 - Specific dna sequences related to an ibdv protein including vectors, hosts and vaccines - Google Patents

Abstract

A biologically pure polypeptide comprises an amino acid sequence of 30 to 1012 amino acids and has the antibody binding characteristics of at least one US variant of the VP2 protein of VMBF. Biologically pure RNA and DNA segments include sequences encoding at least one copy of said biologically pure polypeptide. A recombinant vector comprises at least one copy of a DNA segment coding for 1 to 20 polypeptides having the antibody binding characteristics of at least one United States variant of the VP2 protein of VMBF, and optionally the VP3 proteins and also VP4. A host is transformed using said recombinant vector. One method of protecting poultry against Fabricius bursa disease is to administer a dose of DNA or the recombinant vector effective to elicit an immunological response against VMBF.

Description

Specific DNA Sequences Related to a IBDV Protein including Vectors, Hosts and Vaccines

Technical Field

This invention relates to the infectious bursal disease virus (IBDV) that is associated with Gumboro disease of young chickens. More particularly, this invention relates to biologically pure DNA, RNA and polypeptide sequences associated with the VP2 protein of the virus, a broad spectrum IBDV vaccine and other related technologies. The present technology may be applied to a vaccine for the in vivo production of conformational epitopes which elicit an immunological response to the virus. In this manner, the administration of the vaccine affords protection against IBDV not only to a subject, e.g., poultry, that is being inoculated, but also to its progeny.

Description of the Background

Infectious bursal disease (IBD) or Gumboro disease is a highly contagious viral disease of young chickens which is characterized by the destruction of lymphoid follicles in the bursa of Fabricius. In a fully susceptible chicken flock of 3-6 weeks of age the clinical disease causes severe i munosuppression, and is responsible for losses due to impaired growth, decreased feed efficiency, and death. Susceptible chickens less than 3 weeks old do not exhibit outward clinical signs of the disease but have a marked infection characterized by gross lesions of the bursa.

The virus associated with the symptoms of the disease was called infectious bursal disease virus (IBDV) . IBDV is a pathogen of major economic importance to the nation and world's poultry industries. It causes severe immunodeficiency in young chickens by destruction of precursors of antibody-producing B cells in the bursa of Fabricius. Immunosuppression causes increased suscep- tibility to other diseases, and interferes with the effective vaccination against Newcastle disease, Marek's disease and infectious bronchitis disease viruses.

There are two known serotypes of IBDV. Serotype I viruses are pathogenic to chickens whereas serotype II viruses infect chickens and turkeys. The infection of turkeys is presently of unknown clinical significance.

Up until recently, the principal methods of controlling IBD in young chickens were by vaccination with an avirulent strain of IBDV or by transferring high levels of maternal antibody induced by the administration of live and killed IBD vaccines to breeder hens ( yeth, P.J. and Cullen, G.A., Vet. Rec. 104, 188-193, (1979)).

In recent years field outbreaks of IBD, particularly in the eastern United States, have shown infection of poultry with variant viruses which are not completely neutralized by antibodies against standard serotype I IBDV (Rosenberger, J.K. et al., Proc. of the 20th National Meeting on Poultry Health and Condemnations, 94-101 (1985); Snyder, D.B. et al., Proc. 23rd National Meeting on Poultry Health and Condemnations, Ocean City, Maryland (1988)).

IBDV belongs to a group of viruses called Birnaviridae which includes other bisegmented RNA viruses such as infectious pancreatic necrosis virus (fish), tellina virus and oyster virus (bivalve molluscs) and drosophila X virus (fruit fly). These viruses all contain high molecular weight (MW) double stranded RNA genomes.

The capsid of the IBDV virion consists of at least four structural proteins. As many as nine structural proteins have been reported but there is evidence that some of these may have a precursor-product relationship. The designation and molecular weights of the four viral proteins (VP) are as shown in Table 1 below. Table 1: Viral Proteins of IBDV

Viral Protein Molecular Weight

VP1 90 kDa

VP2 41 kDa

VP3 32 kDa

VP4 28 kDa

An additional protein, VPX, of 47 kDa was determined to be a precursor of the VP2 protein.

The nucleotide sequences of IBDV serotype I (ST-C, standard challenge virus and attenuated virus BB) and serotype II obtained from turkeys (OH, Ohio strain) have been compared and have provided preliminary information thereof (Jackwood, D.J. et al., 69th Annual Meeting of the Conference of Research Workers in Animal Disease, Abs. No. 346, Chicago, Illinois (1988)). Two segments of double stranded RNA were identified in the genome of IBDV. One contains 3400 base pairs and has a molecular weight of 2.06 x 10 , and the other base pairs and has a molecular weight of In vitro translation of the denatured f the virus has shown that the larger RNA segment encodes three structural proteins, i.e., VP2, VP3 and VP4, and the smaller RNA segment encodes only one protein, i.e., VP1.

Both genomic segments of an Australian strain of IBDV, that is different from the U.S. strains, were recently cloned and sequenced (Hudson, P.J. et al., Nucleic Acids Res. 14, 5001-5012, (1986); Morgan, M.M. et al., Virology 163, 240-242, (1988)). The complete nucleotide sequence of the larger segment has shown that these proteins are encoded in the order VP2, VP4 and VP3, and that they are contained in one open reading frame. In addition, further nucleotide sequence data confirmed that the smaller RNA segment encodes only the VPl protein (Morgan, M.M. et al., Virology 163, 240-243, (1988)). This protein is a minor component of the virion and it is presumed to be the viral RNA polymerase. In IBDV, the VPl protein binds tightly to both ends of the two genomic segments, and it effectively circularizes the molecule.

It has been recently demonstrated that the VP2 protein is the major host protective immunogen of IBDV, and that it contains the antigenic region responsible for the induction of neutralizing antibodies. The region containing the neutralization site has been shown to be highly conformation-dependent. The VP3 protein has been considered to be a group-specific antigen because it is recognized by monoclonal antibodies directed against it from strains of both serotype I and II viruses. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins. However, the precise manner in which the proteolytic break up takes place is not yet clear.

The occurrence of antigenic variations among IBDV isolates has been repeatedly reported. The use of monoclonal antibodies (MCA) B29, R63, B69, 179, BK9 and 57 raised against different strains of IBDV led to the recognition of the occurrence of three distinct antigenic types of IBDV in the field in the U.S. These data are shown in Table 2 below.

Table 2: AC-ELISA Characterization of Banked Field Isolates, Laboratory/Reference and Vaccine Strains of IBDV

Capture MCA

IBDV No . Virus Source B29 R63^a B69^a 179^a BK9 57^a Tested Type

Banked Isolates:

+

+ +

+ + + + + + + + + + + + + + + + + + + + + +

+ + + +

+ + + + + + + + + + + + + +

MCA neutralizes. Two of the MCAs discussed above, B69 and 57, made specifically against the Classic D78 and GLS strains of IBDV have been found by virus neutralization tests to neutralize only the parent virus. The third MCA, R63, also made against the IBDV Classic strain was shown to neutralize all serotype I IBDVs except the GLS variant virus. Two other MCAs, 179 and BK44, have been shown to be potent neutralizers of all serotype I IBDVs studied so far. All serotype I IBDVs bind to MCA B29 in an antigen- capture enzyme-linked immunosorbens assay (AC-ELISA) . However, the B29 MCA is not a neutralizing MCA. On the other hand, the B69 and R63 MCAs are both neutralizing MCAs. Predictions on new variants can be made on the basis of their reactivities with the B69 MCA. A virus that does not bind to this MCA in an AC-ELISA is very likely antigenically different from the standard type ("classic"), and would be termed as a variant virus. Neither the Delaware type E (E/DEL) nor the GLS variants of IBDV react with the B69 MCA. In addition, the E/DEL variant can be distinguished from the GLS variant virus on the basis of its reactivity with the R63 MCA. The GLS variant virus does not bind to the R63 MCA in AC-ELISA assay as is shown in Table 2 above. The new GLS variant was recently discovered on the basis of antigen-capture ELISA tests (Snyder, D.B. et al., Proc. 23rd Nat. Meeting Poultry Health and Condem., Ocean City, Maryland (1988)). This strain of IBDV is presently replacing the Delaware variant and has already become the most predominant IBDV type occurring in the Delmarva Peninsula. Data on IBDV types obtained with the monoclonal antibodies (MCAs) R63, B29, above are shown in Table 3 below. Table 3: Geographic Distribution of IBDV Types as Determined With an MCA R63, B69 and B29 Based AC-ELISA

There are currently 9 "live" attenuated avirulent vaccines available in the market. All the vaccine strains react with the B29, B69 and R63 in MCAs AC-ELISA tests. These viruses, therefore, are classified as the "Classic" type, as shown in Table 2 above. The brand name of these vaccines and their sources are given in Table 4 below. Table 4: Vaccines for IBDV

Vaccine Company

Clone-vac D78 Intervet America

Univax American Sci. Lab.

Bursine Salisbury

Bio-Burs KeeVet

Bio-Burs I KeeVet

IBD Blend Ceva

Bursa-vac Sterwin

VI-Bur-G Vineland

S706 Select

The above vaccine strains are not virulent like the variant viruses and they may be given "live." Thus, they do not have to be inactivated or "killed" in order to be used as vaccines. However, these vaccines are not fully effective in protecting against infection with variant viruses. A limited number of chickens immunized with the above vaccine strains are actually protected against challenge with Delaware (about 60%) and GLS (about 30%) variant viruses.

In addition, the immunization with the "Classic" strains of IBDV (see Table 4) that is routinely conducted nowadays renders the immunized birds partially protected only against the Delaware (DEL) and the GLS variant viruses.

A "killed" IBDV vaccine is also available from Intervet Co. in Millsboro, Delaware. This vaccine is called "Breeder-vac" and contains standard ("classic"), Delaware and GLS variant virus types. The use of the above "live" and "killed" vaccines has the following disadvantages, among others. The viruses have to be propagated in tissue culture, which is time-consuming and expensive. In "killed" vaccines, the viruses have to be inactivated prior to use, which requires an additional expensive step.

If the "killed" vaccines are not properly inactivated, a risk of an outbreak of the disease exists and does not provide broad protection to birds against the virus variants and the ensuing disease.

Thus, there is a palpable need for an improved vaccine which is effective in the treatment of IBD caused by various pathogenic IBDV strains.

Disclosure of the Invention

This invention relates to a biologically pure RNA segment that comprises at least one and up to 20 copies of an RNA sequence encoding at least one copy of a polypeptide of about 30 to 1012 amino acids, the polypeptide having the antibody binding characteristics of at least one US variant of the IBDV VP2 protein selected from the group consisting of E/DEL and GLS. This invention also relates to a biologically pure DNA segment that comprises a single stranded DNA sequence corresponding to the RNA sequence described above. This DNA segment is also provided as a double stranded DNA segment. Still part of this invention is a recombinant vector that comprises a vector capable of growing and expressing in a host structural DNA sequences attached thereto; and at least one and up to 20 copies of the DNA segment described above attached in reading frame to the vector. The tandem attachment of a plurality of copies of the DNA segment is also be provided as part of this invention.

Also provided herein is a host transformed with a recombinant vector comprising a vector capable of growing and expressing in a host structural DNA sequences attached thereto and at least one copy of the DNA segment of the invention attached in reading frame to the vector. This invention also relates to a broad spectrum IBD poultry vaccine that comprises a poultry protecting amount of the recombinant vector described above; and a physiologically acceptable carrier.

Encompassed by this invention is also a biologically pure polypeptide that comprises at least one and up to 20 copies of an amino acid sequence of about 30 to 1012 amino acids encoded by the RNA segment of the invention. A method of protecting poultry and its progeny from IBD is also part of this invention, the method comprising administering to the poultry an amount of the recombinant vector of the invention that is effective to attain an immunological response that will protect the poultry against the symptoms of IBD.

Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion.

Best Mode for Carrying Out the Invention

This invention arose from a desire to improve on prior art technology relating to the protection of poultry against the newly appearing variants of IBDV in the United States. This was attempted by studying the structural organization of the IBDV genome, and particularly that of the VP2, VP3 and VP4 proteins of the virus. This invention thus provides a DNA vaccine representative of more than one IBDV VP2 US variant. When this DNA is utilized for vaccinating poultry it conveys a broad protection against subsequent infection by known IBDV variants as well as, it is postulated, subsequently appearing variants. The breadth of protection afforded poultry by this DNA vaccine also extends to other strains of IBDV which are known to diverge to a greater extent from the U.S. strain(s) than the variants amongst themselves . Thus, it is provided herein a biologically pure RNA segment that comprises at least one and up to 20 copies of an RNA sequence encoding at least one copy of a polypeptide about 30 to 1012 amino acids long, the polypeptide having the antibody binding characteristics of at least one of the U.S. variants of the IBDV VP2 protein. Examples are the GLS and E/DEL variants. The layer segments encode more than sequences belonging to the VP2 protein. Each segment encoding at least about 1012 amino acid sequence comprises the binding capability of the VP2 protein, and sequences corresponding to the VP3 and VP4 IBDV proteins.

Such RNA sequence may encode only one copy of the polypeptide having the antibody binding characteristics of at least one of the U.S. IBDV variants or up to about 20 copies thereof, preferably about 1 to 5 copies thereof, an antibody binding functional fragment thereof, a functional precursor thereof, or combinations thereof. The RNA sequence may further encode at least one copy of a polypeptide having the antibody binding characteristics of the VP2 protein of another U.S. IBDV variant, e.g., the E/DEL, "classic" or GLS variant. The RNA sequence may encode either one of these polypeptides, functional fragments thereof, functional precursors thereof or func- tional analogs thereof as defined below. In addition, the RNA sequence may further encode the antibody binding activity of the VP2 protein of other IBDV strains, e.g., the Australian IBDV variant (W088/10298 published December 29, 1988; Hudson et al., Nucleic Acids Res. 14(12) .5001-5012 (1986)) or the European IBDV strain (Spies et al., Nucleic Acids Res. 17(19) 7982 (1989), the entire texts of which are incorporated herein by refer¬ ence insofar as they are necessary for the enablement of the German Cu-I (European) and Australian DNA, RNA, polypeptide and related sequences of the VP2 protein.

In one preferred embodiment, the polypeptide encoded by the RNA sequence comprises the antibody binding characteristics of amino acids 200 to 330 Of at least one US variant of the VP2 protein. In another preferred embodiment of the invention the RNA segment comprises about 90 to 9000 bases, more preferably about 150 to 5000 bases, and still more preferably about 300 to 750 bases. One particular clone obtained in the examples of this application is about 3.2 kilobases long.

The RNA sequence may preferably encode at least one copy of a polypeptide fragment of an amino acid sequence such as that of Tables 6 and 7, analogs thereof having at least one amino acid being different at a position such as positions 5, 74, 84, 213, 222, 239, 249, 253, 254, 258, 264, 269, 270, 272, 279, 280, 284, 286, 297, 299, 305, 318, 321, 323, 326, 328, 330, 332, 433 and combi¬ nations thereof, and up to 29 different amino acids, functional fragments thereof, functional precursors thereof and combinations thereof.

The functional precursors of the polypeptides having the antibody binding of the different IBDV VP2 US variants may be about 30 to 1012 amino acids long, and in some circumstances about 100 to 350 amino acids long. However, other polypeptide sizes are also considered to be within the definition of precursors as long as they contain a number greater than the final number of amino acids contained in the corresponding polypeptide having the antibody binding characteristics of at least one of the US variants of the VP2 protein.

The functional fragments of the polypeptide may be about 5 to 450 amino acids long, and more preferably about 10 to 30 amino acids long. These fragments comprise the binding characteristics and/or the amino acid sequence of an epitope that makes the polypeptide antigenic with respect to antibodies raised against IBDV as is known in the art.

The functional polypeptide analogs of the IBDV VP2 protein from the E/DEL and the GLS variants may have the size of the VP2 viral protein, or they may be larger or shorter as was described above for the precursors and fragments thereof. The analogs may have about 1 to 80 variations in the amino acid sequence, preferably about 1 to 30 variations, and more preferably at positions 5, 74, 84, 213, 222, 239, 249, 253, 254, 258, 264, 269, 270, 272, 279, 280, 284, 286, 297, 299, 305, 318, 321, 323, 326, 328, 330, 332, 433 or combinations thereof. However, other positions may be varied by themselves as long as the antigenic binding ability of the polypeptide is not destroyed.

In another embodiment of the invention, the RNA sequence encodes at least one copy of a VP2 protein selected from the group consisting of the GLS IBDV VP2 protein, the E/DEL IBDV VP2 protein, functional analogs thereof, functional fragments thereof, functional precursors thereof and combinations thereof. In a particularly preferred embodiment, the RNA sequence encodes at least one copy of the GLS and one copy of the E/DEL IBDV VP2 proteins, and up to 20 copies, and more preferably 5 to 10 copies thereof.

In still another embodiment, the RNA sequence encodes l to 20 copies of the entire sequence of the VP2, VP3 and VP4 proteins or the VP2 and VP4 proteins of IBDV E/DEL, GLS or both.

Also provided herein is a biologically pure DNA segment, comprising a single stranded DNA sequence corresponding to the RNA segment described above. In a particularly preferred embodiment of the invention the DNA segment is double stranded. This DNA sequence encodes the antibody binding characteristics of at least one of the US variants of the IBDV VP2 protein selected from GLS and E/DEL.

Because of the degeneracy of the genetic code it is possible to have numerous RNA and DNA sequences that encode a specified amino acid sequence. Thus, all RNA and DNA sequences which result in the expression of a polypeptide having the antibody binding characteristics described herein are encompassed by this invention.

In a particularly preferred embodiment of the invention, the DNA sequence comprises the DNA sequences shown in Tables 6 and 7, functional fragments thereof about 10 to 750 base pairs long, and more preferably about 20 to 350 base pairs long, functional precursors thereof about 100 to 1350 base pairs long, and more preferably about 200 to 1000 base pairs long, and analogs thereof about 30 to 1012 base pairs long, and more preferably about 15 to 450 base pairs long, corresponding to the amino acid variations described above for the polypeptide. A suitable proportion for variations: total number of the DNA, RNA and amino acid sequences is about 0.1 to 10%, and more preferably about 1 to 5%. However, other percentages are also contemplated as long as the func¬ tionality of the product as described above is preserved. Also provided herein is a recombinant vector that comprises a vector capable of growing and expressing in a host structural DNA sequences attached thereto; and at least one and up to about 20 copies of the DNA segment of the invention, the segment being operatively linked to the vector.

The recombinant vector may also comprise other necessary sequences such as expression control sequences, markers, amplifying genes, signal sequences, promoters, and the like, as is known in the art.

Useful vectors for this purpose are plasmids, and viruses such as baculoviruses, herpes viruses (HVT) and pox viruses, e.g., fowl pox virus, and the like. A particularly preferred vector comprises a known recombinant fowl pox virus system (Boyle and Coupar, Virus Research 10:343-356 (1988); Taylor, J. et al., J. Virology 64:1441-1450 (1990), the entire texts of which are incorporated herein by reference to the extent necessary to enable the preparation and use of the pox virus vector and its utilization in a poultry vaccine) .

In a particularly preferred embodiment of the invention, the recombinant vector comprises a further DNA sequence encoding at least one polypeptide affording protection against other diseases produced by agents such as bronchitis virus, avian reo virus, chicken anemia agent or Newcastle disease virus (NDV), among others. These DNA sequences are operatively attached to the recombinant vector in reading frame so they can be expressed in a host. The different structural DNA sequences carried by the vector may be separated by termination and start sequences so that the proteins will be expressed separately or they may be part of a single reading frame and therefore be produced as a fusion protein by methods known in the art (Taylor et al., supra) .

Also provided herein is a host transformed with the recombinant vector of the invention. The host may be a eukaryotic or a prokaryotic host. Suitable examples are E. coli, insect cell lines such as Sf-9, chicken embryo fibroblast (CEF) cells, chicken embryo kidney (CEK) cells, and the like. The latter two cell lines are useful in propagating the HVT and pox viruses. For combination vaccines, inactivated antigens can be added to the IBDV of the present invention in a dosage which fulfills the requirements or inactivated vaccines according to 99 C.F.R. 113-120, in particular, for combined vaccines containing New Castle Disease Virus (NDV), the requirements of 9 C.F.R. 113-125. However, other hosts and vectors may also be utilized as is known in the art.

Also part of this invention is a broad spectrum IBDV poultry vaccine comprising a poultry protecting amount of a recombinant vector comprising a vector that grows and expresses in a host structural DNA sequences attached thereto and at least one copy of a DNA segment in accordance with this invention attached in reading frame to the vector; and a physiologically acceptable carrier.

The vaccine according to the invention is administered in amounts sufficient to stimulate the immune system and confer resistance to IBD. The vaccine is preferably administered in a dosage ranging from about log 2 to about log 5 ^EI^Dc₀ (Embryo Infective Dose..,.), and more preferably about log 3 to about log 4 EID₅_. The amounts used when the vaccine is administered to poultry may thus be varied. Suitable amounts are about

10 2 to 106 plaque forming units (pfu) of the

₃ recombinant vector, and more preferably about 10 to

10 pfu units thereof. The animals, 6 weeks or older, may be administered about 0.01 to 2 ml of the vaccine, and more preferably about 0.1 to 1 ml of the vaccine with a needle by the, e.g., wing-web method. Suitably, the virus titre may be about 10 4 to 107 pfu/ml when reconstituted in a pharmaceutlcally-acceptable sterile carrier. The vaccine may be provided in powder form as a unit form, or in about 1-1000 doses of vaccine per sealed container, and more preferably about 10 to 100 doses.

Physiologically acceptable carriers for vaccination of poultry are known in the art and need not be further described herein. In addition to being physiologically acceptable to the poultry the carrier must not interfere with the immunological response elicited by the vaccine and/or with the expression of its polypeptide product.

Other additives, such as adjuvants and stabilizers, among others, may also be contained in the vaccine in amounts known in the art. Preferably, adjuvants such as aluminum hydroxide, aluminum phosphate, plant and animal oils, and the like, are administered with the vaccine in amounts sufficient to enhance the immune response to the IBDV. The amount of adjuvant added to the vaccine will vary depending on the nature of the adjuvant, generally ranging from about 0.1 to about 100 times the weight of the IBDV, preferably from about 1 to about 10 times the weight of the IBDV.

The vaccine of the present invention may also contain various stabilizers. Any suitable stabilizer can be used including carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, or glucose; proteins such as albumin or casein; and buffers such as alkaline metal phosphate and the like. A stabilizer is particularly advantageous when a dry vaccine preparation is prepared by lyophilization.

The attenuated vaccine can be administered by any suitable known method of inoculating poultry including nasally, ophthalmically, by injection, in drinking water, in the feed, by exposure, and the like. Preferably, the vaccine is administered by mass administration techniques such as by placing the vaccine in drinking water or by spraying the animals' environment. A vaccine according to the present invention can be administered by injection. When administered by injection, the vaccines are preferably administered parenterally. Parenteral adminstration as used herein means administration by intravenous, subcutaneous, intramuscular, or intra- peritoneal injection. Known techniques such as Beak-o-Vac administration are preferred.

The vaccine of the present invention is administered to poultry to prevent IBD anytime before or after hatching. Preferably, the vaccine is administered prior to the time of birth and after the animal is about 6 weeks of age. Poultry is defined to include chickens, roosters, hens, broilers, roasters, breeders, layers, turkeys and ducks . The vaccine may be provided in a sterile container in unit form or in other amounts. It is preferably stored frozen, below -20°C, and more preferably below -70°C. It is thawed prior to use, and may be refrozen immediately thereafter. For administration to poultry the recombi- nant DNA material or the vector may be suspended in a carrier m an amount of about 10 4 to 107 pfu/ml, and more preferably about 10 to 10 pfu/ml of a carrier such as a saline solution. Other carriers may also be utilized as is known in the art. Examples of pharma- ceutically acceptable carriers are diluents and inert pharmaceutical carriers known in the art. Preferably, the carrier or diluent is one compatible with the adminstration of the vaccine by mass administration techniques. However, the carrier or diluent may also be compatible with other administration methods such as injection, eye drops, nose drops, and the like.

Also provided herein is a biologically pure poly- peptide that comprises at least one copy of an amino acid sequence of about 30 to 1012 amino acids encoded by the DNA segment described above. The amino acid sequence of the polypeptide is also that encoded by the RNA segment of this invention. As in the case of the RNA and DNA segments described above, the amino acid sequence may comprise at least one and up to 20 copies of the about 30 to 1012 amino acids long polypeptide, the polypeptide having the antibody binding characteristics of at least one U.S. variant of the IBDV VP2 protein, functional precursors thereof, functional fragments thereof, functional analogs thereof and functional combinations thereof as described above.

Each amino acid sequence of the polypeptide may be about 30 to 1012 amino acids long, and more preferably about 100 to 800 amino acids long; each sequence of the functional precursors thereof may be about 40 to 2000 amino acids long, and more preferably about 50 to 1500 amino acids long; each sequence of the functional fragments thereof may be about 5 to 500 amino acids long, and more preferably about 10 to 350 amino acids long; and each sequence of the functional analogs may be about 30 to 1012 amino acids long, and more preferably about 100 to 800 amino acids long. The number and type of point variations the polypeptide has remains within that described above for the RNA and DNA segments.

In particularly preferred embodiments, the polypeptide comprises the amino acid sequence shown in Tables 6, 7 and/or 8. In another preferred embodiment it comprises the amino acid sequence shown in Tables 6, 7 and 8. In yet another preferred embodiment the poly¬ peptide comprises the binding characteristics of amino acids 200 to 330 of the VP2 protein. However, the polypeptide may also comprise other sequences such as those of the VP2 proteins of other IBDV variants or functional fragments thereof.

Also provided herein is a method of protecting poultry and its progeny from IBD comprising administering to the poultry an amount of the recombinant vector of this invention effective to attain the desired effect.

Although other amounts may also be administered, each animal may suitably be provided with about 10 2 to

10 pfu of the DNA, preferably in a carrier, and more preferably about 10³ to 10⁴ pfu of DNA per dose. The vaccine may be administered once to afford a certain degree of protection against IBD or it may be repeated at preset intervals. Or the vaccine may suitably be read inistered at anytime after hatching. A typical interval for revaccination is about 1 day to 6 months, and more preferably about 10 days to 4 months. However, the vaccine may be administered as a booster at other times as well.

The various products provided herein as part of this invention can be obtained by implementing standard technology available and known to the artisan and materials that are commercially available.

Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless so specified.

EXAMPLES

Example 1: IBDV Propagation in Chicken Bursae and its Purification

Two naturally occurring variants of serotype I IBDV were used that are prevalent in the Delmarva Peninsula, the Delaware strain (E/DEL or DEL) (Rosenberger, J.K. et al., Proc. 20th Nat. Meeting Poultry Health Condemn.

(1985)) and the GLS-5 strain (Snyder, D.B. et al., Proc.

23rd Nat. Meeting Poultry Health Condemn., Ocean City,

Maryland (1988)). The GLS and E/DEL strains of IBDV were propagated in the bursae of pathogen free white Leghorn chickens. Two to three week old chickens were orally inoculated with

GLS or E/DEL stock virus (Snyder, D.B. et al., Vet. Immunol. Immunopathol. 9:303-317 (1985)).

Four to five days after infection, the bursae was excised and homogenized in a buffer containing 10 mM

Tris-HCl (pH 7.5) and 150 mM NaCl (TNB buffer). An equal volume of TNB buffer was added to facilitate complete emulsification of the tissue.

The homogenate was freeze-thawed three times and sonicated with a large size probe with two 30 second bursts. Cellular debris from virus suspensions was pelleted by centrifugation at 15,000xg for 10 minutes. The supernate was then passed through a 0.8 μ filter and the filtrate separated. The virus present in the filtrate was then pelleted by centrifugation at 50,000xg for 1.5 hours at 4°C.

The pelleted virus was resuspended in 10 ml phosphate buffered saline (PBS) solution, pH 7.2, and then further purified by centrifugation at 90,000xg for 3 hours at 4°C on discontinuous sucrose gradients (30% to 55% sucrose)

(Snyder et al. (1985), supra).

The virus band was recovered, diluted with PBS, and repelleted by centrifugation at 50,000xg for 1.5 hours at 4°C.

Example 2: Isolation and Purification of Viral RNA

Total viral RNA was isolated from the virus by treating with proteinase K as follows. The pelleted virus was suspended in a reaction buffer containing 100 mM Tris-HCl, pH 7.5, 12 mM EDTA and 150 mM NaCl, and digested with proteinase K (200 μg/ml final concen¬ tration) for 1 hour at 37°C. The mixture was extracted twice with water-saturated phenol, and twice with a chloroform:isoamyl alcohol mixture (24:1). The RNA present in the aqueous phase was then precipitated by addition of 2.5 volumes of ethanol at -20°C, and recovered by centrifugation.

The extracted viral RNA was purified by fractionation on a low-melting temperature agarose gel (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1982)). The RNA sample was loaded onto a 1% agarose gel and the gel was electrophoresed using a buffer containing 89 mM Tris-borate, 1 mM EDTA and 0.05% ethidium bromide, pH 8.3. Lambda DNA standards digested with Bst EII were also applied to the gel and used as size markers.

The DNA and RNA fragments were stained with ethidium bromide under the above conditions and visualized under a UV light. Electrophoresis was carried out until a large and a small RNA segments of IBDV were well separated. The larger RNA segment of approximately 3400 base pairs was excised from the gel and recovered by phenol extraction as described above.

Example 3: Synthesis of First Strand cDNA and

Complementary Strand DNA of Large Genomic Segment of E/DEL and GLS Strains

Viral RNAs were denatured as follows prior to cDNA synthesis. About 5 μg of the larger segment of IBDV RNA were placed in 9 μl of 5 mM phosphate buffer, pH 6.8, heated at 100°C for 2 minutes and then snap-frozen. After thawing the RNA, 1 μl 100 mM methylmercury hydroxide was added thereto, and the mixture was left at room temperature for 10 minutes. Any methylmercury hydroxide excess was quenched by addition of 2 μl 700 mM 2-mercaptoethanol and further incubation for 5 minutes at room temperature.

Selected primers binding specifically the 3' end of the VP2 gene sequence and the 3' end of the large genomic segment sequence were synthesized and used to prime the cDNA synthesis on the basis of the published sequence of an Australian strain of IBDV (Hudson, P.J. et al., Nucleic Acids Res. 14, 5001-5012, (1986)), and the published sequence of the German Cu-I IBDV strain (Spies et al., Nucleic Acids Res. 17(19) 7982 (1989)), respectively.

VP2 primer: 5 *-CAATTGCATGGGCTAG-3 ' 3* end primer: 5 ^■-AACGATCCAATTTGGGAT-3 '

Random primers were also employed for use if, and when, the synthesized oligonucleotide failed to prime cDNA synthesis. Double stranded cDNA was synthesized according to the method of Gubler and Hoffman (Gubler, U. and Hoffman, B.J. Gene 25, 263-269 (1983)).

The synthesis of first-strand cDNA was carried out in a reaction volume of 50 μl containing 50 mM Tris-HCl, pH 8.3, 10 mM MgCl„, 10 mM dithiothreitol, 4 mM sodium pyrophosphate, 1.25 mM dGTP, 1.25 mM dATP, 0.5 mM dCTP, 20 μCi ³²P-dCTP, 5 μg primer, 5 μg RNA and 100 units reverse transcriptase for 1 hour at 42°C. The reaction was terminated by adding 2 μl of 0.5 M EDTA, pH 8.0. The reaction products were then extracted with phenol/chloroform (1:1) and precipitated with ethanol out of 2 M ammonium acetate.

The synthesis of the second strand of DNA and the formation of double stranded DNA fragments were carried out in a reaction volume of 100 μl containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 10 mM (NH₄)₂S0₄, 100 mM KC1, 0.15 mM β-NAD, 5 μg BSA, 40 μM dNTPs, 1 unit E. coli RNase H, 25 units DNA polymerase I and 1 unit E. coli DNA ligase. The reaction mixture was sequentially incubated at 12°C for 1 hour, and at 22°C for 1 hour, and terminated by addition of 10 μl of 0.5 M EDTA, pH 8.0. The reaction products were phenol- extracted and ethanol-precipitated as described above.

Example 4: Liga ion of DNA Fragments to a Vector

The double stranded cDNA was blunt-ended with T4 DNA polymerase and then fractionated on a low-melting agarose gel (Maniatis, T. et al.. Molecular Cloning: A

Laboratory Manual. Cold Spring Harbor Laboratory, New

York (1982)) . Large size fragments, e.g., greater than 500 base pairs were recovered from the gel by phenol extraction as described above. EcoRI adapters (Promega Biotech) were ligated to the blunt-ended cDNAs by incubating the mixture at 14°C overnight in the presence of T4 DNA ligase (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1982)) .

EcoRI-ended cDNAs were then phosphorylated in the presence of T4 polynucleotide kinase, ligated with dephosphorylated EcoRI cut pGEM-7Z vector (Promega Biotech), and then used for transformation.

Example 5: Transformation of Bacterial Host With the Recombinant Vector

E. coli JM 109 cells were made competent as follows.

An overnight culture of E. coli JM 109 in 5 ml of Luria-

Bertani (LB) broth were used to inoculate 40 ml LB broth

(1:100) in a 250 ml Erlenmeyer flask which was gently shaken at 37°C. When the OΩ 5_5_ _n0nm reached about 0.5 the flask was chilled in ice and the cells pelleted by centrifugation at 4000xg for 5 minutes at 4°C. The supernate was discarded, and the cells suspended in 20 ml of transformation buffer containing 50 mM CaCl_, 250 mM KC1, 5 mM Tris-HCl, pH 7.5 and 5 mM gCl₂. The cells were then incubated on ice for 20 minutes, pelleted once again by centrifugation, resuspended in 2 ml transfor¬ mation buffer and left overnight at 4°C.

0.2 ml competent cells were added to the ligated cDNAs and the mixture was first incubated on ice for 1 hour and then at 42°C for 2 minutes. One ml LB broth was then added and the mixture was incubated at 37°C for 1 hour.

Example 6: Selection of Hosts Carrying Viral DNA Inserts

The pGEM-72 plasmid contains a beta-galactosidase gene marker. The transformed mixture was thus plated on culture plates containing ampicillin, isopropylthio-P-D- galactopyranoside (IPTG) and a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) for selection of the recombinants . Ampicillin resistant white colonies with inserts were selected, propagated and stored in 15% glycerol at -70°C.

Example 7: Screening of Recombinant Clones

The ampicillin resistant white colonies obtained in Example 8 above were screened for the presence of viral-specific sequences by Southern hybridization (Southern, E.M., J. Mol. Biol. 98, 503-517 (1975)).

Briefly, recombinant bacteria harboring the plasmid DNA were propagated in 2 ml of LB broth and plasmid DNA was isolated by an established method (Birnboim, H.C. and Doly, J., Nucleic Acids Res. 7, 1513-1520 (1979)). The purified plasmid DNA was then digested with EcoRI enzyme and separated on 1% agarose gel to determine the size of the inserts. Fragments of Lamda DNA digested with Hind III and Eco RI were used as size markers. The identity of the released inserts was determined by transferring the DNA to a Gene screen plus membrane (DuPont, Inc.) and hybridizing with a 32P labeled probe.

The probe was prepared by 5 '-end labeling the base-hydrolyzed larger segment of the viral RNA with

32 γ- P-ATP and polynucleotide kinase (Richardson,

C.C., Proc. Nucleic Acid Res. 2, 815-819 (1971)) and denatured by heating at 100°C for 2 minutes, and then fast cooling to 0°C. The membrane was pre-hybridized for at least 15 minutes at 42°C with a pre-hybridization mixture containing 50% formamide, 1 M NaCl, 1% SDS and 20% dextran sulfate. 100 μg/ml of denatured salmon sperm DNA and a denatured radioactive probe were then added to the pre-hybridization mixture. The hybridization was carried out for 16 hours at 42°C with constant agitation.

After hybridization, the membrane was washed twice successively with a buffer containing 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0 (2xSSC) , and 1% SDS at 65°C for 20 minutes and with 0.1 SSC buffer at room temperature for 20 minutes. Hybridization was detected by autoradiography and positive cDNA clones were then selected with the largest inserts.

The overlapping clones were identified both by hybridization and restriction enzyme mapping.

The different clones mapped as shown in Table 5 below.

Example 8: Napping of GLS and E/DEL cDNA clones

The GLS-1, GLS-2, GLS-3, and GLS-4 and E.DEL-2 cDNA clones were completely sequenced and their sequences were compared with the DNA sequence of the Australian strain of IBDV using the "Microgenie" computer program. On the basis of the sequence homology, the above clones were mapped on the IBDV genomes as shown in Table 5 below.

Table 5: IBDV-Large Segment cDNA Clones (GLS-1, GLS-2, GLS-3, GLS-4 and T./OEL-2)

Ikb 2kb 3k

3'

VP2 VP4 VP3

_________

GLS-2

GL5-3

E.Del-2 Example 9: Sequencing of VP2 Gene Fragments of E/Del and GLS IBDV Strains

Recombinant bacteria, each harboring a cDNA segment of the E/DEL and GLS strains of IBDV, were propagated in LB broth containing 100 μg/ml/ampicillin. The large- scale isolation of plasmid DNA was carried out by the alkali lysis method (Birnboim, H.C. and Doly, J. , Nucleic Acids Res. 7, 1513-1520, (1979)). The plasmid DNA was then purified by cesium chloride gradient centrifugation (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1982)).

The nucleotide sequence of these cDNA clones was determined by a modification of the dideoxy chain termi¬ nation method (Sanger, F. et al., Proc. Natl. Acad. Sci. 74, 5463-5467 (1977)) using a Sequenase^R System kit (U.S. Biochemical Corp.) with SP6 and T7 promoter primers (Promega Biotech) .

A set of selected oligonucleotides, corresponding to the VP2 region of the large genomic segment of IBDV, was synthesized and used as primers in the sequencing reactions. Examples are as follows (5* to 3' end).

(1) TATTCTGTAACCAGGTT

(2) CACTATCTCCAGTTTGAT

(3) TACGAGGACTGACGGGTCTT The labeled fragments were fractionated on 45 or 60 cm 8% polyacrylamide-urea gels and detected by autoradiography.

Example 10: Sequencing of VP3 and VP4 Gene Fragments of GLS IBDV Strain

The nucleotide sequence of the cDNA clones, GLS-3 and

GLS-4, was determined by a modification of the dideoxy chain termination method (for reference, see Example 9) using a "Sequenase" System kit (U.S. Biochemical Corp.) with SP6 and T7 promoter primers (Promega Biotech) .

A set of selected oligonucleotides corresponding to the VP3 and VP4 region of the large genomic segment of IBDV, was synthesized and used as primers in the sequencing reactions. Examples are as follows (5' to 3 ' end) . (1) TTCAAAGACATAATCCGG

(2) GGGTGAAGCAAGAATCCC

(3) GTGCGAGAGGACCTCCAA

(4) GTATGGAAGGTTGAGGTA (5) GGGATTCTTGCTTCACCC

(6) ACGTTCATCAAACGTTTCCC

(7) TTGCAAACGCACCACAAGCA

(8) CGGATCCAATTTGGGAT

( ) GTGTCGGGAGACTCCCA Sequences were obtained for several fragments and the information put together in accordance with the information obtained from overlapping segments. The DNA sequences obtained for the E/DEL viral fragment is shown in Table 6 below and for the GLS fragments in Table 7 below.

Example 11: Comparison of DNA Sequences with Computerized Program

Nucleotide sequence data were entered into an IBM computer with the aid of a gel reader and analyzed with a

"Microgenie" software program (Beckman) . This program provides information of the following characteristics of the strains of IBDV.

(1) The presence of an open reading frame from a major initiation site within the consensus eukaryotic initiation site sequences (Kozak, M. , Microbiol. Rev. 47, 1-49, (1983)).

(2) The complete predicted (deduced) amino acid sequences. (3) Comparisons and homology alignments of obtained sequences with the published sequence data of an Australian strain of IBDV (Hudson, P.J. et al., Nucleic Acids Res. 14, 5001-5012, (1986)) and the German Cu-I IBDV strain (Spies et al. (1989), supra).

Example 12: DNA and Deduced Amino Acid Sequences of E/DEL-2 Clone

The following Table 6 shows the DNA sequence obtained for an E/DEL-2 clone containing 1471 nucleotides. Table 6 also shows the corresponding amino acid sequence deduced from the DNA sequence as discussed above.

Table 6: DNA and Deduced Amino Acid Sequences of E/DEL-2 Clone

30 60

CTA CAA TGC TAT CAT TGA TGG TTA GTA GAG ATC AGA CAA ACG ATC GCA GCG ATG ACA AAC Leu Gin Cys Tyr His End Trp Leu Val Glu lie Arg Gin Thr He Ala Ala Met Thr Asn

90 120

CTG CAA GAT CAA ACC CAA CAG ATT GTT CCG TTC ATA CGG AGC CTT CTG ATG CCA ACA ACC Leu Gin Asp Gin Thr Gin Gin He Val Pro Phe He Arg Ser Leu Leu Met Pro Thr Thr

150 180

GGA CCG GCG TCC ATT CCG GAC GAC ACC CTG GAG AAG CAC ACT CTC AGG TCA GAG ACC TCG Gly Pro Ala Ser He Pro Asp Asp Thr Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser

210 240

ACC TAC AAT TTG ACT GTG GGG GAC ACA GGG TCA GGG CTA ATT GTC TTT TTC CCT GGA TTC Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu He Val Phe Phe Pro Gly Phe

270 300

CCT GGC TCA ATT GTG GGT GCT CAC TAC ACA CTG CAG AGC AAT GGG AAC TAC AAG TTC GAT Pro Gly Ser He Val Gly Ala His Tyr Thr Leu Gin Ser Asn Gly ASn Tyr Lys Phe Asp

330 360

CAG ATG CTC CTG ACT GCC CAG AAC CTA CCG GCC AGC TAC AAC TAC TGC AGG CTA GTG AGT Gin Met Leu Leu Thr Ala Gin Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu Val Ser

390 420

CGG AGT CTC ACA GTA AGG TCA AGC ACA CTC CCT GGT GGC GTT TAT GCA CTA AAC GGC ACC Arg Ser Leu Thr Val Arg Ser Ser Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr

450 480

ATA AAC GCC GTG ACC TTC CAA GGA AGC CTG AGT GAA CTG ACA GAT GTT AGC TAC AAC GGG He Asn Ala Val Thr Phe Gin Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly

510 540 TTG ATG TCT GCA ACA GCC AAC ATC AAC GAC AAA ATT GGG AAC GTC CTA GTA GGG GAA GGG

Leu Met Ser Ala Thr Ala Asn He Asn Asp Lys He Gly Asn Val Leu Val Gly Glu Gly

570 600

GTA ACC GTC CTC AGC TTA CCC ACA TCA TAT GAT CTT GGG TAT GTG AGG CTT GGT GAC CCC Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro

630 660

ATA CCC GCT ATA GGG CTT GAC CCA AAA ATG GTA GCA ACA TGT GAC AGC AGT GAC AGG CCC He Pro Ala He Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro

690 720

AGA GTC TAC ACC ATA ACT GCA GCC GAT AAT TAC CAA TTC TCA TCA CAG TAC CAA ACA GGT Arg Val Tyr Thr He Thr Ala Ala Asp Asn Tyr Gin Phe Ser Ser Gin Tyr Gin Thr Gly Table 6: DNA and Deduced Amino Acid Sequences of E/DEL-2 Clone (Concluded)

750 780

GGG GTA ACA ATC ACA CTG TTC TCA GCC AAC ATT GAT GCC ATC ACA AGT CTC AGC GTT GGG Gly Val Thr He Thr Leu Phe Ser Ala Asn He Asp Ala He Thr Ser Leu Ser Val Gly

810 840

GGA GAG CTC GTG TTC AAA ACA AGC GTC CAA AGC CTT GTA CTG GGC GCC ACC ATC TAC CTT Gly Glu Leu Val Phe Lys Thr Ser Val Gin Ser Leu Val Leu Gly Ala Thr He Tyr Leu

870 900

ATA GGC TTT GAT GGG ACT GCG GTA ATC ACC AGA GCT GTG GCC GCA AAC AAT GGG CTG ACG He Gly Phe Asp Gly Thr Ala Val He Thr Arg Ala Val Ala Ala Asn Asn Gly Leu Thr

930 960

GCC GGC ATC GAC AAT CTT ATG CCA TTC AAT CTT GTG ATT CCA ACC AAT GAG ATA ACC CAG Ala Gly He Asp Asn Leu Met Pro Phe Asn Leu Val He Pro Thr Asn Glu He Thr Gin

990 1020

CCA ATC ACA TCC ATC AAA CTG GAG ATA GTG ACC TCC AAA AGT GAT GGT CAG GCA GGG GAA Pro He Thr Ser He Lys Leu Glu He Val Thr Ser Lys Ser Asp Gly Gin Ala Gly Glu 1050 1080

CAG ATG TCA TGG TCG GCA AGT GGG AGC CTA GCA GTG ACG ATC CAT GGT GGC AAC TAT CCA Gin Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr He His Gly Gly Asn Tyr Pro

1110 1140 GGA GCC CTC CGT CCC GTC ACA CTA GTG GCC TAC GAA AGA GTG GCA ACA GGA TCT GTC GTT

Gly Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val

1170 1200

ACG GTC GCT GGG GTG AGC AAC TTC GAG CTG ATC CCA AAT CCT GAA CTA GCA AAG AAC CTG Thr Val Ala Gly Val Ser Asn Phe Glu Leu He Pro Asn Pro Glu Leu Ala Lys Asn Leu

1230 1260

GTT ACA GAA TAT GGC CGA TTT GAC CCA GGA GCC ATG AAC TAC ACG AAA TTG ATA CTG AGT Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu He Leu Ser

1290 1320

GAG AGG GAC CGC GTT GGC ATC AAG ACC GTC TGG CCA ACA AGG GAG TAC ACT GAC TTT CGT Glu Arg Asp Arg Leu Gly He Lys Thr Val Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg 1350 1380

GAG TAC TTC ATG GAG GTG GCC GAC CTC AAC TCT CCC CTG AAG ATT GCA GGA GCA TTT GGC Glu Tyr Phe Met Glu Val Ala Asp Leu Asn Ser Pro Leu Lys He Ala Gly Ala Phe Gly

1410 1440 TTC AAA GAC ATA ATC CGG GCC ATA AGG AGG ATA GCT GTA CCG GTG GTC TCT ACA TTG TTC

Phe Lys Asp He He Arg Ala He Arg Arg He Ala Val Pro Val Val Ser Thr Leu Phe

1470 CCA CCT GCC GCT CCT GTA GCC CAT GCA ATT G Pro Pro Ala Ala Pro Val Ala His Ala He Example 13 : DNA and Deduced Amino Acid Sequences for GLS-1, GLS-2, GLS-3 and GLS-4 Clones

The following Table 7 provides the DNA sequence of the GLS-1, GLS-2, GLS-3 and GLS-4 clone obtained above and the amino acid sequence deduced therefrom obtained from the DNA sequences with the aid of a computerized program.

Table 7: DNA Sequence and Deduced Amino Acid Sequence for GLS-1, GLS-2, GLS-3 and GLS-4 Clones

cc 32 62

CCG GGG GAG TCA CCC GGG GAC AGG CCG TCA AGG CCT TGT TCC AGG ATG GAA CTC CCC CTT Pro Gly Glu Ser Pro Gly Asp Arg Pro Ser Arg Pro Cys Ser Arg Met Glu Leu Pro Leu

92 122 CTA CAA TGC TAT CAT TGA TGG TTA GTA GAG ATC GGA CAA ACG ATC GCA GCG ATG ACA AAC

Leu Gin Cys Tyr His End Trp Leu Val Glu He Gly Gin Thr He Ala Ala Met Thr Asn

152 182

CTG CAA GAT CAA ACC CAA CAG ATT GTT CCG TTC ATA CGG AGC CTT CTG ATG CAA ACA ACC Leu Gin Asp Gin Thr Gin Gin He Val Pro Phe He Arg Ser Leu Leu Met Pro Thr Thr

212 242

GGA CCG GCG TCC ATT CCG GAC GAC ACC CTG GAG AAG CAC ACT CTC AGG TCA GAG ACC TCG Gly Pro Ala Ser He Pro Asp Asp Thr Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser

272 302

ACC TAC AAT TTG ACT GTG GGG GAC ACA GGG TCA GGG CTA ATT GTC TTT TTC CCT GGA TTC Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu He Val Phe Phe Pro Gly Phe

332 362

CCT GGC TCA ATT GTG GGT GCT CAC TAC ACA CTG CAG AGC AAT GGG AAC TAC AAG TTC GAT Pro Gly Ser He Val Gly Ala His Tyr Thr Leu Gin Ser Asn Gly Asn Tyr Lys Phe Asp

392 422 CAG ATG CTC CTG ACT GCC CAG AAC CTA CCG GCC AGC TAC AAC TAC TGC AGG CTA GTG AGT

Gin Met Leu Leu Thr Ala Gin Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu Val Ser

452 482

CGG AGT CTC ACA GTA AGG TCA AGC ACA CTC CCT GGT GGC GTT TAT GCA CTA AAC GGC ACC Arg Ser Leu Thr Val Arg Ser Ser Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr

512 542

ATA AAC GCC GTG ACC TTC CAA GGA AGC CTG AGT GAA CTG ACA GAT GTT AGC TAC AAT GGG He Asn Ala Val Thr Phe Gin Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly

572 602

TTG ATG TCT GCA ACA GCC AAC ATC AAC GAC AAA ATT GGG AAC GTC CTA GTA GGG GAA GGG Leu Met Ser Ala Thr Ala Asn He Asn Asp Lys He Gly Asn Val Leu Val Gly Glu Gly Table 7: DNA Sequence and Deduced Amino Acid Sequence for GLS-1, GLS-2, GLS-3 and GLS-4 Clones (Continued)

632 662

GTT ACT GTC CTC AGC TTA CCC ACA TCA TAT GAT CTT GGG TAT GTG AGG CTT GGT GAC CCC Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro

692 722

ATA CCC GCT ATA GGG CTT GAC CCA AAA ATG GTA GCA ACA TGT GAC AGC AGT GAC AGG CCC He Pro Ala He Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro

752 782 AGA GTC TAC ACC ATA ACT GCA GCT GAT GAT TAC CAA TTC TCA TCA CAG TAC CAA ACA GGT

Arg Val Tyr Thr He Thr Ala Ala Asp Asp Tyr Gin Phe Ser Ser Gin Tyr Gin Thr Gly

812 842

GGG GTA ACA ATC ACC CTG TTC TCA GCC AAC ATT GAT GCC ATC ACA AGC CTC AGC GTT GGG Gly Val Thr He Thr Leu Phe Ser Ala Asn He Asp Ala He Thr Ser Leu Ser Val Gly

872 902

GGA GAG CTC GTG TTT AAA ACA AGC GTC CAC AGC CTT GTA CTG GGC GCC ACC ATC TAC CTT Gly Glu Leu Val Phe Lys Thr Ser Val His Ser Leu Val Leu Gly Ala Thr He Tyr Leu

932 962

ATA GGC TTT GAT GGG TCT GCG GTA ATC ACT AGA GCT GTG GCC GCA AAC AAT GGG CTG ACG He Gly Phe Asp Gly Ser Ala Val He Thr Arg Ala Val Ala Ala Asn Asn Gly Leu Thr

992 1022

ACC GGC ACC GAC AAT CTT ATG CCA TTC AAT CTT GTG ATT CCA ACC AAC GAG ATA ACC CAG Thr Gly Thr Asp Asn Leu Met Pro Phe Asn Leu Val He Pro Thr Asn Glu He Thr Gin

1052 1082 CCA ATC ACA TCC ATC AAA CTG GAG ATA GTG ACC TCC AAA AGT GGT GGT CAG GAA GGG GAC

Pro He Thr Ser He Lys Leu Glu He Val Thr Ser Lys Ser Gly Gly Gin Glu Gly Asp

1112 1142

CAG ATG TCA TGG TCG GCA AGT GGG AGC CTA GCA GTG ACG ATT CAT GGT GGC AAC TAT CCA Gin Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr He His Gly Gly Asn Tyr Pro

1172 1202

GGG GCC CTC CGT CCC GTC ACA CTA GTA GCC TAC GAA AGA GTG GCA ACA GGA TCT GTC GTT Gly Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val

1232 1262

ACG GTC GCT GGG GTG AGC AAC TTC GAG CTG ATC CCA AAT CCT GAA CTA GCA AAG AAC CTG Thr Val Ala Gly Val Ser Asn Phe Glu Leu He Pro Asn Pro Glu Leu Ala Lys Asn Leu

1292 1322

GTT ACA GAA TAC GGC CGA TTT GAC CCA GGA GCC ATG AAC TAC ACA AAA TTG ATA CTG AGT Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu He Leu Ser

1352 1382 GAG AGG GAC CGC CTT GGC ATC AAG ACA GTC TGG CCG ACA AGG GAG TAC ACC GAC ITT CGT

Glu Arg Asp Arg Leu Gly He Lys Thr Val Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg Table 7: DNA Sequence and Deduced Amino Acid Sequence for GLS-1, GLS-2, GLS-3 and GLS-4 Clones (Continued)

1412 1442

GAG TAC TTC ATG GAG GTG GCC GAC CTC AGC TCT CCC CTG AAG ATT GCA GGA GCA TTT GGC Glu Tyr Phe Met Glu Val Ala Asp Leu Ser Ser Pro Leu Lys He Ala Gly Ala Phe Gly

1472 1502

TTC AAA GAC ATA ATC CGG GCC ATA AGG AGG ATA GCT GTG CCG GTG GTC TCC ACA TTG TTC Phe Lys Asp He He Arg Ala He Arg Arg He Ala Val Pro Val Val Ser Thr Leu Phe

1532 1562

CCA CCT GCC GCT CCC CTG GCC CAT GCA ATT GGG GAA GGT GTA GAC TAC CTG CTG GGT GAT Pro Pro Ala Ala Pro Leu Ala His Ala He Gly Glu Gly Val Asp Tyr Leu Leu Gly Asp

1592 1622

GAG GCA CAG GCT GCT TCA GGA ACT GCT CGA GCC GCG TCA GGA AAA GCA AGG GCT GCC TCA Glu Ala Gin Ala Ala Ser Gly Thr Ala Arg Ala Ala Ser Gly Lys Ala Arg Ala Ala Ser

1652 1682

GGC CGC ATA AGG CAG CTG ACT CTC GCC GCC GAC AAG GGG TAC GAG GTA GTC GCG AAT CTA Gly Arg He Arg Gin Leu Thr Leu Ala Ala Asp Lys Gly Tyr Glu Val Val Ala Asn Leu

1712 1742

TTC CAG GTG CCC CAG AAT CCC GTA GTC GAC GGG ATT CTT GCT TCA CCC GGG ATA CTC CGC Phe Gin Val Pro Gin Asn Pro Val Val Asp Gly He Leu Ala Ser Pro Gly He Leu Arg

1772 1802

GGT GCA CAC AAC CTC GAC TGC GTG TTA AGA GAG GGC GCC ACG CTA TTC CCT GTG GTC ATC Gly Ala His Asn Leu Asp Cys Val Leu Arg Glu Gly Ala Thr Leu Phe Pro Val Val He

1832 1862 ACG ACA GTG GAA GAC GCC ATG ACA CCC AAA GCA CTA AAC AGC AAA ATG TTT GCT GTC ATT

Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu Asn Ser Lys Met Phe Ala Val He

1892 1922

GAA GGC GTG CGA GAG GAC CTC CAA CCT CCA TCT CAA AGA GGA TCC TTC ATA CGA ACT CTC Glu Gly Val Arg Glu Asp Leu Gin Pro Pro Ser Gin Arg Gly Ser Phe He Arg Thr Leu

1952 1982

TCC GGA CAC AGA GTC TAT GGA TAT GCT CCA GAT GGG GTA CTT CCA CTG GAG ACT GGG AGA Ser Gly His Arg Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg

2012 2042

GAC TAC ACC GTT GTC CCA ATA GAT GAT GTC TGG GAC GAC AGC ATT ATG CTG TCC AAA GAC Asp Tyr Thr Val Val Pro He Asp Asp Val Trp Asp Asp Ser He Met Leu Ser Lys Asp

2072 2102

CCC ATA CCT CCT ATT GTG GGA AAC AGT GGA AAC CTA GCC ATA GCT TAC ATG GAT GTG TTT Pro He Pro Pro He Val Gly Asn Ser Gly Asn Leu Ala He Ala Tyr Met Asp Val Phe

2132 2162 CGA CCC AAA GTC CCC ATC CAT GTG GCC ATG ACG GGA GCC CTC AAC GCT TGT GGC GAG ATT

Arg Pro Lys Val Pro He His Val Ala Met Thr Gly Ala Leu Asn Ala Cys Gly Glu He Table 7: DNA Sequence and Deduced Amino Acid Sequence for GLS-1, GLS-2, GLS-3 and GLS-4 Clones (Continued)

2192 2222

GAG AAA ATA AGC TTT AGA AGC ACC AAG CTC GCC ACC GCA CAC CGG CTT GGC CTC AAG TTG Glu Lys He Ser Phe Arg Ser Thr Lys Leu Ala Thr Ala His Arg Leu Gly Leu Lys Leu

2252 2282

GCT GGT CCC GGA GCA TTT GAT GTA AAC ACC GGG CCC AAC TGG GCA ACG TTC ATC AAA CGT Ala Gly Pro Gly Ala Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe lie Lys Arg

2312 2342 TTC CCT CAC AAT CCA CGC GAC TGG GAC AGG CTC CCC TAC CTC AAC CTT CCA TAC CTT CCA

Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr Leu Asn Leu Pro Tyr Leu Pro

2372 2402

CCC AAT GCA GGA CGC CAG TAC CAC CTC GCC ATG GCC GCA TCA GAG TTC AAG GAG ACC CCT Pro Asn Ala Gly Arg Gin Tyr His Leu Ala Met Ala Ala Ser Glu Phe Lys Glu Thr Pro

2432 2462

GAA CTC GAG AGC GCC GTC AGG GCC ATG GAA GCA GCA GCC AGT GTA GAC CCA CTG TTC CAA Glu Leu Glu Ser Ala Val Arg Ala Met Glu Ala Ala Ala Ser Val Asp Pro Leu Phe Gin

2492 2522

TCT GCA CTC AGT GTG TTC ATG TGG CTG GAA GAG AAT GGG ATT GTG ACT GAC ATG GCC AAC Ser Ala Leu Ser Val Phe Met Trp Leu Glu Glu Asn Gly He Val Thr Asp Met Ala Asn

2552 2582

TTC GCA CTC AGC GAC CCG AAC GCC CAT CGG ATG CGA AAC TTT CTT GCA AAC GCA CCA CAA Phe Ala Leu Ser Asp Pro Asn Ala His Arg Met Arg Asn Phe Leu Ala Asn Ala Pro Gin

2612 2642

GCA GGT AGC AAG TCT CAA AGG GCC AAA TAC GGG ACA GCA GGC TAC GGA GTG GAG GCC CGG Ala Gly Ser Lys Ser Gin Arg Ala Lys Tyr Gly Thr Ala Gly Tyr Gly Val Glu Ala Arg

2672 2702

GGC CCC ACA CCA GAA GAA GCA CAG AGG GAA AAA GAC ACA CGG ATC TCA AAG AAG ATG GAG Gly Pro Thr Pro Glu Glu Ala Gin Arg Glu Lys Asp Thr Arg He Ser Lys Lys Met Glu

2732 2762

ACC ATG GGC ATC TAC TTT GCA ACA CCA GAA TGG GTA GCA CTC AAT GGG CAC CGA GGG CCA Thr Met Gly He Tyr Phe Ala Thr Pro Glu Trp Val Ala Leu Asn Gly His Arg Gly Pro

2792 2822

AGC CCC GGC CAG CTA AAG TAC TGG CAG AAC ACA CGA GAA ATA CCG GAC CCA AAC GAG GAC Ser Pro Gly Gin Leu Lys Tyr Trp Gin Asn Thr Arg Glu He Pro Asp Pro Asn Glu Asp

2852 2882

TAT CTA GAC TAC GTG CAT GCA GAG AAG AGC CGG TTG GCA TCA GAA GAA CAA ATC CTA AGG Tyr Leu Asp Tyr Val His Ala Glu Lys Ser Arg Leu Ala Ser Glu Glu Gin He Leu Arg

2912 2942

GCA GCT ACG TCG ATC TAC GGG GCT CCA GGA CAG GCA GAG CCA CCC CAA GCT TTC ATA GAC Ala Ala Thr Ser lie Tyr Gly Ala Pro Gly Gin Ala Glu Pro Pro Gin Ala Phe He Asp Table 7: DNA Sequence and Deduced Amino Acid Sequence for GLS-1, GLS-2, GLS-3 and GLS-4 Clones (Concluded)

2972 3002

GAA GTT GCC AAA GTC TAT GAA ATC AAC CAT GGA CGT GGC CCA AAC CAA GAA CAG ATG AAA

Glu Val Ala Lys Val Tyr Glu He Asn His Gly Arg Gly Pro Asn Gin Glu Gin Met Lys

3032 3062

GAT CTG CTC TTG ACT GCG ATG GAG ATG AAG CAT CGC AAT CCC AGG CGG GCT CCA CCA AAG

Asp Leu Leu Leu Thr Ala Met Glu Met Lys His Arg Asn Pro Arg Arg Ala Pro Pro Lys

3092 3122

CCC AAG CCA AGA CCC AAC GCT CCA ACG CAG AGA CCC CCT GGT CGG CTG GGC CGC TGG ATC

Pro Lys Pro Arg Pro Asn Ala Pro Thr Gin Arg Pro Pro Gly Arg Leu Gly Arg Trp He

3152 3182

AGG ACT GTC TCT GAT GAG GAC CTT GAG TGA GGC TCC TGG GAG TCT CCC GAC ACC ACC CGC

Arg Thr Val Ser Asp Glu Asp Leu Glu End Gly Ser Trp Glu Ser Pro Asp Thr Thr Arg

3212 GCA GGC GTG GAC ACC AAT TCG GCC TTA CAA CAT CCC AAA TTG GAT CCG Ala Gly Val Asp Thr Asn Ser Ala Leu Gin His Pro Lys Leu Asp Pro

The DNA sequence of the GLS-1 clone starts at nucleotide 1 and ends at nucleotide 348, and is therefore 348 base pairs long. The sequence of the GLS-2 clone starts at nucleotide 283 and ends at nucleotide 1252, and is 970 base pairs long. The sequence of the GLS-4 clone starts at nucleotide 999 and ends at nucleotide 2620, and is 1622 base pairs long. The sequence of the GLS-3 clone starts at nucleotide 1722 and ends at nucleotide 3230, and is 1509 base pairs long.

Example 14 Localization of Virus Neutralizing Epitopes Of IBDV

A panel of three monoclonal antibodies (MCAs) generated against IBDV is used to localize antigenic determinant(s) responsible for the induction of neutralizing antibodies. Two of the MCAs, B69 and 57, were raised specifically against the Classic D78 and GLS IBDV strains respectively, and both of them neutralize only the parent IBDV strain. The second MCA, R63, was raised against the D78 IBDV strain and neutralizes all serotype I IBDVs, except for the GLS variant of the virus. All of these neutralizing antibodies bind to the VP2 (41 kDa) structural protein of IBDV in the radioimmunoprecipitation assay (unpublished data). The MCAs thus recognize a region of epitopes located on the VP2 protein. Some sites have been found to be of importance for binding and are therefore considered associated with the epitopes. Examples are the sites corresponding to amino acids 74, 84, 213, 222, 249, 253, 254, 258, 264, 269, 270, 272, 279, 280, 284, 286, 297, 299, 305, 318, 321, 323, 326, 328, 330, 332 and 433, among others, of the VP2 protein. Information on these amino acid sites is provided in Table 12 below.

These sites are, individually or in groups, responsible for or associated with the binding of specific MCAs. Variations of the complementary DNA sequences (or viral RNAs) at the sites encoding these amino acids may provide a basis for genetic drift leading to failure of specific vaccines raised against known viral strains.

Example 15: VP2 DNA and Amino Acid Homologies and Specific Amino Acid Variations of GLS-5 and E/DEL IBDV The DNA sequences and the amino acid sequences deduced therefrom by the computerized method described above were examined, and a comparison of the GLS-5 clone and the E/DEL clone. Table 9 below shows the homology found for these US variants of the virus both at the DNA and the amino acid level.

Table 9 : Comparisons of VP2 Gene and Protein Sequences of GLS-5 and E/DEL Variant Viruses

Percent Homology

At Nucleotide Level 98.1%

Of Deduced Amino Acids 98.0% Tables 9 and 10 below show variations of amino acids found between the VP2 sequences of GLS-5 and E/DEL clones.

Table 10 : Com aris n of VP2 Protein Se uences of GLS-5

GLS-5 Gin Asp Thr Lys His Ser Ser Ala Asn Thr Thr Gly Glu Asp Ser Ser Ser E.DEL Gin Asn Thr Lys Gin Ser Thr Ala Asn Ala He Asp Ala Glu Ser Ser Asn

Example 16: IBDV VP2 DNA and Amino Acid Homologies Found Between the Australian Variant and GLS and The Australian Variant and E/DEL

The homologies found for the VP2 DNA of the

Australian strain (002-783) and the GLS-5 viral DNA segment as well as for the Australian and E/DEL segment are shown in Table 12 below. Also shown are amino acid homologies found between the Australian and U.S. GLS-5 variants as well as between the Australian and E/DEL variants . Table 12: Comparison of VP2 Gene and Protein Sequences of Australian and American Isolates of IBDV

Percent Homology at Nucleotide Level

GLS-5 91 . 9%

E . DEL 92 . 1%

Percent Homology of Deduced Amino Acids

GLS-5 96 . 2%

E . DEL 95 . 6%

Example 17: Changes in the Amino Acid Residues for VP2

Protein Among U.S. GLS-5 and E/DEL Variants, and Australian and German Cu-I IBDV Strains

Comparisons of the deduced amino acid sequences between two U.S. variants, GLS-5 and E/DEL, and the Australian and the German Cul strains of IBDV showed various differences in the amino acids occupying specific positions of the VP2 protein. Changes in the amino acid residues in specific region of the VP2 protein for two U.S. variants, GLS-5 and E/DEL, the Australian strain and the German Cul strain of IBDV are shown in Table 13 below.

Table 13: Amino Acid Changes in VP2 of IBD US Variants and Australian and German Viruses

Amino Acid Residue Number in VP2

Variant

Viruses 5 74 84213 222239249253 254258 264269270 272 279280 284286297 299305 318 321 323 326 328330 332433

Australian Ser Met Gin Asp Pro Asn Gin Gin Gly Asn Val Thr Thr Thr Gly Asn Ala Thr Pro Ser Val Gly Ala Asp Ser Leu Ser Asn Asn

GLS-5 Gin Leu Gin Asp Thr Ser Lys His Ser Gly He Ser Ala He Asn Asn Thr Thr Pro Asn He Gly Glu Asp Ser Ser Ser Ser Ser

E. Delaware Gin Leu Gin Asn Thr Ser Lys Gin Ser Gly He Thr Ala He Asn Asn Ala He Pro Asn He Asp Ala Glu Ser Ser Ser Ser Asn

German Cu-I Gin Leu Gin Asp Pro Ser Gin His Gly Gly He Thr Thr He Asn Asn Thr Thr Ser Asn He Gly Ala Asp Ser Ser Lys Ser Asn

Example 18: Preparation of Synthetic Peptides

The nucleotide sequences of the genes encoding the structural protein VP2 for three IBDV strains GLS-5, E/Delaware, and German Cu-I are compared. On the basis of nucleotide sequence-predicted amino acid change(s), selected polypeptides are synthesized on an automated peptide synthesizer according to the manufacturer's instructions (Biosearch) . The peptides are purified by reverse phase (C18) high performance liquid chroma- tography using acetonitrile gradients in 0.1% trifluoroacetic acid, and are analyzed for amino acid content in an Amino Quant analyzer (Hewlett Packard) .

Synthetic peptides are dissolved in a 0.05 M Tris/0.25 M NaCl, pH 7.5 buffer if freely water soluble, or otherwise in a 8 M urea, 1% 2-mercaptoethanol/0.05 M Tris, pH 8.3, buffer, and stored at -70°C until used.

Example 19: Competitive Antigen Binding Assays

Radiolabeling of the IBDV proteins is carried out as described (Muller, H. and Becht, H., J. Virol. 44, 384- 392 (1982)). Monolayers of CEF cells are infected with IBDV at a multiplicity of infection of 10 pfu/cell and incubated at 37°C. After 1 hour, the cells are washed twice and incubated for 1 hour with Eagle's minimum essential medium (MEM) without methionine. Two hours after infection the above media are removed and replaced with MEM containing 100 μCi of 35S-methionine. After a pulse with 35S-methionme for 12 hours, labeled virus particles are sedimented from the culture medium and purified further by sucrose gradient centrifugation as described above.

Competitive binding assays are performed as described by Robertson et al. (Robertson, B.H. et al., Virus

Res. 1, 489-500, (1984)) except that purified

35 S-labeled virus particle antigen is used as the assay antigen.

Briefly, MCAs are pretitrated against labeled virus to bind 70-80% of input virus in the absence of inhibitor. Synthetic peptides are added into dilution sets immediately before the assays are performed. Titration endpoints are determined at the 50% inhibition of the maximum binding (I₅₀ dose) by logit-log transformed linear regression analysis (Trautman, R. and Harris, W.F., Scand. J. Immunol. 6, 831-841 (1977)). The results are plotted as percent inhibition v. log 10 molar quantity of inhibitor added.

Example 20: Some Predicted IBDV Epitopes for IBDV MCA

Binding

A number of neutralizing MCAs against various strains of IBDV were used to test their reactivity with different

IBDV antigenic variants. Table 14 below shows the reactivity pattern of some MCAs with different antigenic variants of IBDV in an AC-ELISA system. (Snyder, D.B., et al., Proc. 23rd Nat. Meeting Poultry Health Condemn.,

Ocean City, Maryland (1988)).

Table 14: Antigenic variants of IBDV reacting with Neutralizing MCAs in AC-ELISA systems

IBDV Variant B69 R63 BK44 179 8 42 57

"Classic" + + +

Delaware + + +

GLS + +

The MCAs are all neutralizing MCAs

On the basis of the available nucleotide sequences and the corresponding deduced amino acid sequences a prediction of regions of amino acids that may be involved in the binding with these MCAs can be made. In other words, these amino acids may be part of the neutralizing epitopes of IBDV and the base pairs encoding them may be part of a special sequence (conformational epitope) minimizing the outer binding area of the protein. Since the BK44, BK179 and BK8 MCAs react with all the IBDVs, they must recognize a region(s) of amino acids that are almost identical in all viruses. Therefore, the binding region(s) for these MCAs cannot be predicted. However, on the basis of radioimmunoprecipitation assays it is known that all these MCAs bind to the VP2 protein of IBDV. These MCAs, thus, may recognize either a linear continuous epitope(s) or a conformational eρitope(s) . Binding of the above four MCAs to VP2 amino acid residues can be predicted on the basis of the available nucleotide sequences as shown in Table 15 below.

Table 15: Some Predicted Epitopic Sites in The Amino Acid Sequence of IBDV

MCA B69

Residue No. 314 315 316 317 318 319 320 321 322 323 324 Thr Ser Lys Ser Gly Gly Gin Ala Gly Asp Gin

MCA 42 Residue No, 279 280 281 282 283 284 285 286 287 288 289 Asn Asn Gly Leu Thr Thr Gly Thr Asp Asn Leu

MCA R63 Residue No, 264 265 266 267 268 269 270 271 272 273 274 lie Gly Phe Asp Gly Thr Thr Val lie Thr Arg

MCA 57 Residue No, 315 319 320 321 322 323 324 325 326 327 328 Gly Gly Gin Glu Gly Asp Gin Met Ser Trp Ser

329 330 Ala Ser

These sequences are, therefore, considered to be present in all types of IBDVs and DNA segments encoding them may be utilized for the vaccination of birds. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.

Claims

1. A biologically pure RNA segment, comprising an RNA sequence encoding at least one copy of a polypeptide having the antibody binding characteristics of the IBDV VP2 protein derived from at least one US variant selected from the group consisting of E/DEL and

GLS strains.

2. The RNA segment of claim 1, wherein the polypeptide encoded comprises the antibody binding characteristics of amino acids 200 to 330 of the VP2 protein.

3. The RNA segment of claim 1, comprising about 3.2 K bases.

4. The RNA segment of claim 1, wherein the RNA sequence encodes a polypeptide fragment having an amino acid sequence selected from the group consisting of the amino acid sequences of Tables 6 and 7, analogues thereof having a different amino acid in at least one position selected from the group consisting of positions 5, 74, 84, 213, 222, 239, 249, 253, 254, 258, 264, 269, 270, 272, 279, 280, 284, 286, 297, 299, 305, 318, 321, 323, 326, 328, 330, 332 and 433, functional fragments thereof, functional precursors thereof and combinations thereof.

5. The RNA sequence of claim 4, comprising the RNA sequence encoding the VP2, VP3 and VP4 proteins of GLS and E/DEL IBDV.

6. A biologically pure DNA segment, comprising a single stranded DNA sequence corresponding to the RNA segment of claim 1.

7. The DNA segment of claim 6 in double stranded form.

8. The DNA segment of claim 6, wherein the DNA sequence comprises the DNA sequences of Tables 6, 7 and 8.

9. A recombinant vector, comprising a vector capable of growing and expressing in a host structural DNA sequences attached thereto; and at least one copy of the DNA segment of claim 7 attached in reading frame to the vector.

10. The recombinant vector of claim 9, wherein the vector comprises a recombinant fowl pox virus or a herpes virus from turkeys (HVT) .

11. The recombinant vector of claim 9, further comprising a further DNA sequence encoding at least one polypeptide affording protection against the "Classic" US IBDV variant, or the Australian, German, or European IBDV strains, wherein the further DNA sequence is attached in reading frame to the vector.

12. A broad spectrum IBDV poultry vaccine, comprising a poultry protecting amount of the recombinant vector of claim 9; and a physiologically acceptable carrier.

13. The broad spectrum vaccine of claim 12, comprising

4 7 about 10 to 10 pfu units of the recombinant vector per ml of carrier.

14. A broad spectrum IBDV poultry vaccine, comprising a poultry protecting amount of the biologically pure

DNA segment of claim 7; and a physiologically acceptable carrier.

15. The broad spectrum IBDV poultry vaccine of claim 14, comprising ₄ Λ *7 aabboouutt 1100 ttoo 10 pfu units of the recombinant vector per ml of carrier

16. A host carrying the recombinant vector of claim 9.

17. The host of claim 16 being selected from the group consisting of

E. coli: insect cell-line Sf-9; chicken embryo fibroblast (CEF) cells; chicken embryo kidney (CEK) cells; and vero cells.

18. A biologically pure polypeptide, comprising at least one copy of an amino acid sequence encoded by the RNA segment of claim 1.

19. The polypeptide of claim 18, wherein the amino acid sequence comprises a sequence selected from the group consisting of the amino acid sequences of Tables 6, 7 and 8, analogues thereof having a different amino acid in at least one position selected from the group consisting of positions 5, 74, 84, 213, 222, 239, 249, 253, 254, 258, 264, 269, 270, 272, 279, 280, 284, 286, 297, 299, 305, 318, 321, 323, 326, 328, 330, 332 and 433, functional fragments thereof, functional precursors thereof and combinations thereof.

20. A method of protecting poultry and its progeny from IBD comprising administering to the poultry an amount of the recombinant vector of claim 9 effective to attain the desired effect.

21. The method of claim 20, wherein the recombinant vector is administered to the subject ophthalmically, by injection, nasally or orally.

22. The method of claim 20, wherein the recombinant vector is administered to the subject

2 6 in an amount of about 10 to 10 pfu.

23. A method of protecting poultry and its progeny from IBD comprising administering to the poultry an amount of the biologically pure DNA segment of claim 7 effective to attain the desired effect.

24. The method of claim 23, wherein the DNA is administered to the subject ophthal¬ mically, by injection, nasally or orally.

25. The method of claim 23, wherein the DNA is administered to the subject in an amount of about 10² to 10⁶ pfu.