AU667782B2

AU667782B2 - Specific DNA sequences related to an IBDV protein including vectors, hosts and vaccines

Info

Publication number: AU667782B2
Application number: AU68841/94A
Authority: AU
Inventors: Vikram Vakharia
Original assignee: University of Maryland at Baltimore; University of Maryland at College Park
Current assignee: University of Maryland at Baltimore
Priority date: 1990-05-04
Filing date: 1994-07-28
Publication date: 1996-04-04
Anticipated expiration: 2011-04-30
Also published as: DK792D0; AU6884194A; KR920703100A; DK792A

Description

~Lm~C_~_Jii 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 Sresponse to the virus. In this manner, the administration of the vaccine affords protection against IBDV not only to a subject, poultry, that is being inoculated, but also to its progeny.

pescription of the Background Infectious bursal disease (IBD) or Gumboro disease is 1 °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 Schicken flock of 3-6 weeks of age the clinical disease causes severe immunosuppression, 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 susceptibility to other diseases, and interferes with the i li ii ii- 2 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 (Wyeth, P.J. and Cullen, 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 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)).

o IBDV belongs to a group of viruses called Birnaviridae which includes other bisegmented RNA viruses S° such as infectious pancreatic necrosis virus (fish), 25 tellina virus and oyster virus (bivalve molluscs) and drosophila X virus (fruit fly). These viruses all o 06 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.

3 Table 1: Viral Proteins of IBDV o o o o uu a a o O oy

U

Dr a «f 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 6 has a molecular weight of 2.06 x 10 and the other contains 2900 base pairs and has a molecular weight of 6 1.76 x 106 In vitro translation of the denatured genomic RNA of the virus has shown that the larger RNA segment encodes three structural proteins, VP2, VP3 and VP4, and the smaller RNA segment encodes only one protein, 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 i- i 4 that the smaller RNA segment encodes only the VP1 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 VP1 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 o'o recognized by monoclonal antibodies directed against it 15 from strains of both serotype I and II viruses. The VP4 Sprotein appears to be a virus-coded protease that is a C° 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 o 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.

IL. awe Tabe 2 AC-ELISA Characterization of Isolates, Laboratory/Reference Strains of EBDV Banked Field and vaccine I BDV No. Virus Source 1329 R63a B69a 1 79 a BK9 57a Tested Type Banked Isolates: Pure Classic Pure DEL Pure GLS Classic Delaware Purt OLS Laboratory Virus:

IM

STO

Edgar 2512

LUK

F52/70

MD

A/DEL

D/DEL

E/DEL

G/DEL

1 Classic Classic Classic Classic Classic classic Classic Delaware Delaware Delaware Delaware a a 0 Vaccines: D78 Univax Bursine Bio-Burs Bio-Burs I IBD Glend Burs a-vac VI-Bur-G S706 Classic Classic Classic Classic Classic Classic Classic Classic Classic MCA neutralizes.

6 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 antigencapture 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 O that does not bind to this MCA in an AC-ELISA is very likely antigenically different from the standard type So ("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 0:0 on the basis of its reactivity with the R63 MCA. The GLS variant virus does not bind to the R63 MCA in AC-ELISA o«0 assay as is shown in Table 2 above.

0*00 25 The new GLS variant was recently discovered on the basis of antigen-capture ELISA tests (Snyder, D.B.

0 et al., Proc. 23rd Nat. Meeting Poultry Health and 00 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.

I

r 7 Table 3: Geographic Distribution of IBDV Types as Determined With an MCA R63, B69 and B29 Based

AC-ELISA

Total No.

IBDy T0.e, of State Classic Delaware GLS Isolates Delmarva 8 42 50 319 Alabama 15 67 18 52 North Carolina 19 52 29 67 Mississippi 20 70 10 Georgia 22 52 26 27 Arkansas 36 45 19 53 Tennessee 50 50 0 2 Missouri 50 50 0 2 Indiana 57 43 0 7 Florida 73 20 7 California 84 8 8 Oklahoma 87 0 13 8 Texas 100 0 0 Oregon 100 0 0 7 Minnesota 100 0 0 7 Washington 100 0 0 3 25 Virginia 0 100 0 8 Pennsylvania 0 100 0 Total No.: 627 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.

04 4 o oc 0040 oo o oo 0 4 00 0 0 4 B 0 6 8 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 o° 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 0 0 challenge with Delaware (about 60%) and GLS (about variant viruses.

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

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.

I

00 0 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 According to a broad format of this invention there is provided a biologically pure RNA lo segment, comprising at least one copy of an RNA sequence encoding at least one copy of a polypeptide of at least about 30 amino acids, and having all the antibody binding characteristics of the IBDV VP2 protein derived from at least one US variant selected from E/DEL or GLS strains.

It is preferred that the segment comprises up to 20 copies of the RNA sequence and that the polypeptide has up to 1012 anmino acids.

This invention also relates to a biologically purc 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 copy 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 copy of an amino acid sequence of at least about 30 amino acids encoded by the RNA segment of the invention.

36 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.

[N:\L1BC]G0309:MHiT

L

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 organisation 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 utilised 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.

o0 0 0o IN:\LIBC]00309:JOC 11 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 le jt one of the U.S. IBDV variants or up to about 20 copies '-hereof, preferably about 1 to 5 copies 0o0060 thereof, an antibody binding functional fragment -hereof, Sa functional precursor thereof, or combinations thereof.

The RNA sequence may further encode at least one copy of o 20 a polypeptide having the antibody binding characteristics of the VP2 protein of another U.S. IBDV varin) e.g., the E/DEL, "classic" or GLS variant. The RNA sequence may encode either one of these polypeptides, functional o:-:0o fragments thereof, functional precursors thereof or functional analogs thereof as defined below. In addition, :0 the RNA sequence may further encode the antibody binding ,a activity of the VP2 protein of other IBDV strains, e.g., o 0 the Australian IBDV variant (W088/10298 published December 29, 1988; Hudson et al., Nucleic Acids Res.

30 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 reference 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 i 12 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 combinations 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 Uo variants may be about 30 to 1012 amino acids long, and in S 20 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 S° 25 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 30 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 8C u- P-n^^i-^iMMilTilfm.al~lt~~ l>aMlWjl^ 13 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 1 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 25 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 r o nc, onoa a o ua a 000 900ac 14 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 and more preferably about 1 to However, other percentages are also contemplated as long as the functionality of the product as described above is preserved.

Also provided herein is a recombinant vector that comprises o" 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, o' 25 and the like, as is known in the art.

Useful vectors for this purpose are plasmids, and Q'a °viruses such as baculoviruses, herpes viruses (HVT) and pox viruses, 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 15 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 0" o0 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 °0 20 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 0 25 (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.

o i Also part of this invention is a broad spectrum IBDV poultry vaccine comprising 30 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 cr.

16 is preferably administered in a dosage ranging from about log 2 to about log 5 EID 50 (Embryo Infective Dose 5 0 and more preferably about log 3 to about log 4 EID 5 0.

The amounts used when the vaccine is administered to poultry may thus be varied. Suitable amounts are about to 10 plaque forming units (pfu) of the recombinant vector, and more preferably about 103 to 104 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, wing-web method. Suitably, the virus titre may be about 104 to 10 7 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

O

l described herein. In addition to being physiologically o 20 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 o. 4 25 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 o 64 30 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 u- I ii 17 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 intraperitoneal 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 0oooo frozen, below -20 0 C, and more preferably below -70 0 C. It is thawed prior to use, and may be refrozen immediately thereafter. For administration to poultry the recombinant DNA material or the vector may be suspended in a carrier in an amount of about 104 to 107 pfu/ml, and more preferably about 105 to 106 pfu/ml of a carrier such as a saline solution. Other carriers may also be utilized as is known in the art. Examples of pharmaceutically 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 18 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 polypeptide 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 o "to 1012 amino acids long, and more preferably about 100 .4 4 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 4ow\ lor- In another preferred embodiment it comprises the amino acid sequence shown in Tables 6 and 7. In yet another preferred embodiment the polypeptide comprises the binding characteristics of amino acids 200 to 330 of the VP2 protein. However, the j A polypeptide may also comprise other sequences such as 1.9 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 102 to 106 pfu of the DNA, preferably in a carrier, and more preferably about 13to 104I 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 readministered at anytime after hatching. A typical interval for revaccination is about 1 day to 6 months, 0and more preferably about 10 days to 4 months. However, 0 o 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 o :20:technology available and known to the artisan and materials that are commercially available.

000$Having now generally described this invention, the 0000 same will be better understood by reference to certain specific examples, which are included herein for purposes 0. of illustration only and are not intended to be limiting 0 of the invention or any embodiment thereof, unless so specified.

XPE

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)).

~II~LI; i/ L_ 20 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 NaC1 (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 S°pelleted by centrifugation at 15,000xg for 10 minutes.

The supernate was then passed through a 0.8 p 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 0

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) oo, (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 0

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 NaC1, and digested with proteinase K (200 pg/ml final concentration) for 1 hour at 37 0

C.

The mixture was extracted twice with water-saturated phenol, and twice with a chloroform:isoamyl alcohol mixture The RNA present in the aqueous phase was 21 then precipitated by addition of 2.5 volumes of ethanol at -20 0 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 0 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 ooo o Segment of E/DEL and GLS Strains Viral RNAs were denatured as follows prior to cDNA synthesis. About 5 pg of the larger segment of IBDV °o00o0 25 RNA were placed in 9 p1 of 5 mM phosphate buffer, pH 6.8, heated' at 100 0 C for 2 minutes and then 0 00 0snap-frozen. After thawing the RNA, 1 P1 100 mM 0 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 pI 700 mM 2-mercaptoethanol and further incubation for 5 minutes at room temperature.

Selected primers binding specifically the 31 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 22 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' (SEQ. ID No:l) 3' end primer: 5'-AACGATCCAATTTGGGAT-3' (SEQ. ID No:2) 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 pl containing 50 mM Tris-HCl, pH 8.3, 10 mM MgCl 2 10 mM dithiothreitol, 4 mM sodium pyrophosphate, 1.25 mM dGTP, 1.25 mM dATP, 0.5 mM dCTP, 32 15 20 pCi P-dCTP, 5 pg primer, 5 pg RNA and 100 S0 units reverse transcriptase for 1 hour at 42 0 C. The reaction was terminated by adding 2 pl of 0.5 M EDTA, pH 8.0. The reaction products were then extracted with phenol/chloroform and precipitated with ethanol out of 2 M ammonium acetate.

oThe synthesis of the second strand of DNA and the formation of double stranded DNA fragments were carried .o out in a reaction volume of 100 pl containing 20 mM .O Tris-HC1 (pH 5 mM MgCl 2 10 mM (NH 4 2

SO

4 100 mM KC1, 0.15 mM B-NAD, 5 pg BSA, 40 pM dNTPs, 1 0. unit E. li RNase H, 25 units DNA polyme-ase I and 1 unit E. coli DNA ligase. The reaction mixture was sequentially incubated at 12 0 C for 1 hour, and at 22°C for 1 hour, and terminated by addition of 10 pl of 0.5 M EDTA, pH 8.0. The reaction products were phenolextracted and ethanol-precipitated as described above.

Example 4: Ligation 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)).

-LIIC'i-- 23 Large size fragments, 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 0 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.

S° Example 5: Transformation of Bacterial Host With the 15 Recombinant Vector E. coli JM 109 cells were made competent as follows.

o° An overnight culture of E. coli JM 109 in 5 ml of Luria- Bertani (LB) broth were used to Anoculate 40 ml LB broth (1:100) in a 250 ml Erlenmeyer flask which was gently shaken at 37 0 C. When the OD 5 5 0 n m reached about 0.5 the flask was chilled in ice and the cells pelleted by °centrifugation at 4000xg for 5 minutes at 4 0 C. The supernate was discarded, and the cells suspended in 20 ml of transformation buffer containing 50 mM CaC1 2 250 mM KC1, 5 mM Tris-HCl, pH 7.5 and 5 mM MgCl 2 The cells were then incubated on ice for 20 minutes, pelleted once again by centrifugation, resuspended in 2 ml transfor- Smation buffer and left overnight at 4 0

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 0 C for 2 minutes. One ml LB broth was then added and the mixture was incubated at 37 0 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 24 culture plates containing ampicillin, isopropylthio-P-Dgalactopyranoside (IPTG) and a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-B3-D-galactoside (X-Gal) for selection of the recombinants.

Ampicillin resistant white colonies with inserts were selected, propagated and stored in 15% glycerol at 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, J. Mol. Biol. 98, 503-517 k1975)).

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, 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 y- 32P-ATP and polynucleotide kinase (Richardson, 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 minutes at 42°C with a pre-hybridization mixture containing 50% formamide, 1 M NaCI, 1% SDS and dextran sulfate. 100 pg/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 agitati-n.

After hybridization, the membrane was washed twice successively with a buffer containing 0.3 M NaCI, 0.03 M 00

'*Y

25 sodium citrate, pH 7.0 (2xSSC), and 1% SDS at 65 0 C for 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: Mapping 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.

15 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 Z/OEL-2) od Ikb 2kb 3kb o. 5 a 3' 0oo0 VP2 I VP4 V P3 CLS-1 GLS-2 GLS-3 E.Del-2 i; -i 26 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 pg/ml/ampicillin. The largescale isolation of plasmid DNA was carried out by the alkali lysis method (Birnboim, H.C. and Doly, 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 termination method (Sanger, F. et al., Proc. Natl. Acad. Sci.

74, 5463-5467 (1977)) using a Sequenase R System kit Biochemical Corp.) with SP6 and T7 promoter primers (Promega Biotech).

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

TATTCTGTAACCAGGTT (SEQ. ID No:3) CACTATCTCCAGTTTGAT (SEQ. ID No:4) TACGAGGACTGACGGGTCTT (SEQ. ID 000 25 The labeled fragments were fractionated on 45 or 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 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 to 3' end).

I

27 TTCAAAGACATAATCCGG (SEQ ID No:6) GGGTGAAGCAAGAATCCC (SEQ ID No:7) GTGCGAGAGGACCTCCAA (SEQ ID No:8) GTATGGAAGGTTGAGGTA (SEQ ID No:9) GGGATTCTTGCTTCACCC (SEQ ID ACGTTCATCAAACGTTTCCC (SEQ ID No:11) TTGCAAACGCACCACAAGCA (SEQ ID No:12) CGGATCCAATTTGGGAT (SEQ ID No:13) GTGTCGGGAGACTCCCA (SEQ ID No:14) 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 0:0. 15 below.

SExample 11: Comparison of DNA Sequences with oo Computerized Program Nuclebtide sequence data were entered into an IBM computer with the aid of a gel reader and analyzed with a o "Microgenie" software program (Beckman). This program provides information of the following characteristics of the strains of IBDV.

S(1) The presence of an open reading frame from 9000 25 a major initiation site within the consensus 0 eukaryotic initiation site sequences (Kozak, M., Microbiol. Rev. 47, 1-49, (1983)).

The complete predicted (deduced) amino acid sequences.

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.

28 Table 6 also shows the corresponding amino acid sequence deduced from the DNA sequence as discussed above.

TablQ 6: DNA and Deduced Amino Acid Sequences of E/DEL-2 Clone (SEQ. ID No:15) (SEQ. ID No:16) CTA CAA TGC TAT CAT TGA TGG TTA GTA GAG ATC AGA Leu Gin Cys Tyr His End Trp Leu Val Giu Ile Arg CMA ACG ATC GCA GCG ATG ACA Gin Thr Ile Ala Ala Met Thr 0 00 00 0 00 0 00,0 0000 o 0 o o Old 4 4 CTG CMA GAT CMA ACC CMA CAG AT7 GTT CCG TTC ATA CGG AGC CTT CTG ATG CCA ACA Leu Gin Asp Gin Thr Gin Gin Ile Val Pro Phe Ile Arg Ser Leu Leu Met Pro Thr 150 GGA CCG GCG TCC ATT CCG GAC GAC ACC CTG GAG MAG CAC ACT CTC AGG TCA GAG ACC Gly Pro Aia Ser Ile Pro Asp Asp Thr Leu Giu Lys His Thr Leu Arg Ser Giu Thr 21i0 ACC TAC MAT TTG ACT GTG GGG GAC ACA GGG TCA GGG CTA Arr GTC TTT TTC CCT GGA Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Giy 270 240

TTC

Phe 2 5 CCT GGC TCA ATT GTG GGT GCT CAC TAC ACA CTG CAG AGC MAT GGG MAC TAG MAG Pro Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gin Ser Asn Cly ASn Tyr Lys 330 CAG ATG CTC CTG ACT GCC CAC MAC CTA CCG GCC AGC TAC MAC TAG TGC AGG CTA Gin Met Leu Leu Thr Aia Gin Asn Leu Pro Aia Ser Tyr Asn Tyr Cys Arg Leu 390 TTC CAT Phe Asp 360 GTG AGT Val Ser 420 CGG ACT CTC ACA GTA AGG TCA AGC ACA CTC CCT CCT GGC GT17 TAT GCA CTA MAC CGC Arg Ser Leu Thr Vai Arg Ser Ser Thr Leu Pro Giy Giy Val Tyr Ala Leu Asn Gly ATA MAC GCC GTG ACC TTC CMA Ile Asn Aia Val Thr Phe Gin 450 GGA AGC CTG ACT GMA CTG Giy Ser Leu Ser Cli, Leu AGA GAT GTT AGC TAG MAC

ACC

Thr 480

GGC

Thr Asp Val Ser Tyr Asn Gly 4 0 TTG ATG TCT GCA ACA CCC MAC Leu Met Ser Ala Thr Ala Asn 510 ATC MAC GAG Ile Asn Asp 570 ACA TCA TAT AAA ATT CCC MAC CTC CTA CTA CCC GMA Lys Ile Gly Asn Val Leu Val Cly Ciu CTA ACC Val Thr CTC CTC AGC TTA CCC Val Leu Ser Leu Pro Thr Ser Tyr Asp 630 ATA CCC GCT ATA CCC CTT GAG CCA AMA ATG CIA Ilie Pro Ala Ile Gly Leu Asp Pro Lys Met Val 690 AGA CTC TAC ACC ATA ACT CCA CCC CAT MAT TAC Arg Val Tyr Thr Ile Thr Ala Ala Asp Asn Tyr CTT CCC TAT C TG AGG CTT CCT GAG Leu Gly Tyr Val Arg Leu Cly Asp GCA ACA TGT GAC ACC ACT GAG AG Aia Thr Cys Asp Ser Ser Asp Arg CMA TTC TCA TCA CAG TAC CMA ACA Gin Phe Ser Ser Gin Tyr Gin Thr 29- T~ eE DNA and Deduced Amino Acid Sequences of E/DEL-2 Clone (SEQ. ID No:15) (Continued) -750 780 GGG GTA ACA ATC ACA CTG rrC TCA GCC MAC ATT GAT GCC ATC ACA AGT CTC AGC GTT GGG Gly Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala le Thr Ser Leu Ser Val Gly 810 840 GGA GAG CTC GTG TTC AMA ACA AGC GTC CAA AGC CTT GTA CTG GGC GCC ACC ATC TAC CTT Gly Glu Leu Va& Phe Lys Thr Ser Val Gin Ser Leu Val Leu Gly Ala Thr Ile Tyr Leu 870 900 ATA GGC TTT GAT GGG ACT GCG GTA ATC ACC AGA GCT GTG GCC GCA MAC MAT GGG CTG ACG Ile Gly Phe Asp Gly Thr Ala Val Ile Thr Arg Ala Val Ala Ala Asn Asn Gly Leu Thr 930 960 GCC GGC ATC GAC MAT CTT ATG CCA TTC MAT CTT GTG ATT CCA ACC MAT GAG ATA ACC CAG Ala Giy Ile Asp Asn Leu Met Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gin 990 1020 CCA ATC ACA TCC ATC AMA CTG GAG ATA GTG ACC TCC AMA AGT GAT GGT CAG GCA GGG GMA oPro Ile Thr Ser Ile Lys Leu Glu Ile Vai Thr Ser Lys Ser Asp Gly Gin Aia Gly Glu CA T C G C C G G G 1050 1080 0A T C G C C G G G CTA GCA GTG ACG ATC CAT GGT GGC MAC TAT CCA Gin Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr Ile His Gly Gly Asn Tyr Pro 1110 1140 0 ,30 GGA GCC CTC CGT CCC GTC ACA CTA GTG GCC TAC GMA AGA GTG GCA ACA GGA TCT GTC GTT Gly Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Giu Arg Val Ala Thr Gly Ser Val Vai 1170 1200 ACG GTC GCT GGG GTG AGC MAC TTC GAG CTG ATC CCA MAT CCT GMA CTA GCA MAG MAC CTG Thr Val Ala Gly Val Ser Aso Phe Giu Leu Ile Pro Asn Pro Giu Leu Ala Lys Asn Leu 1230 1260 GTT ACA GMA TAT GGC CGA TTr GAC CCA GGA GCC ATG MAC TAC ACG AMA TTG ATA CTG AGT o 1Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu Ile Leu Ser o *1290 1320 GAG AGG GAC CGC GTT GGC ATC MAG ACC GTC TGG CCA ACA AGC GAG TAC ACT GAC TrT CCT Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr Arg Giu Tyr Thr Asp Phe Arg 1350 1380 GAG TAC T7C ATG GAG GTG GCC GAC CTC MAC TCT CCC CTC MAG ATT GCA GCA GCA TTT GGC Ciu Tyr Phe Met Glu Val Ala Asp Leu Aso Ser Pro Leu Lys Ile Ala Gly Ala Phe Giy 1410 1440 TTC AMA GAC ATA ATC CGG GCC ATA AGG AGG ATA GCT GTA CCG GTG GTC TCT ACA TTG TTC Phe Lys Asp Ile Ile Arg Ala Ile Arg Arg Ile Ala Val Pro Val Val Ser Thr Leu Phe 1470 CCA CCT GCC GCT CCT GTA CCC CAT GCA ATT G Pro Pro Ala Ala Pro Val Ala His Ala Ile 30 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 (SEQ. ID No:16) coono 0 0 o" o o a 0. .00 0 0 0 0010 Su .i I 00 0 0 6 4 o CCG GGG GAG TCA CCC GGG Pro Gly Glu Ser Pro Gly CTA CAA TGC TAT CAT TGA Leu Gin Cys Tyr His End CTG CAA GAT CAA ACC CAA Leu Gin Asp Gin Thr Gin GGA CCG GCG TCC ATT CCG Gly Pro Ala Ser Ile Pro ACC TAC AAT TTG ACT GTG Thr Tyr Asn Leu Thr Val CCT GGC TCA ATT GTG GGT Pro Gly Ser Ile Val Gly CAG ATG CTC CTG ACT GCC Gin Met Leu Leu Thr Ala CGG AGT CTC ACA GTA AGG Arg Ser Leu Thr Val Arg ATA AAC GCC GTG ACC TTC lie Asn Ala Val Thr Phe TTG ATG TCT GCA ACA GCC Leu Met Ser Ala Thr Ala GAC AGG Asp Arg TGG TTA Trp Leu CAG ATT Gin Ile GAC GAC Asp Asp GGG GAC Gly Asp GCT CAC Ala His CAG AAC Gin Asn TCA AGC Ser Ser CAA GGA Gin Gly AAC ATC Asn Ile TCA AGG CCT Ser Arg Pro 92 GAG ATC GGA Glu Ile Gly 152 CCG TTC ATA Pro Phe lle 212 CTG GAG AAG Leu Glu Lys 272 GGG TCA GGG Gly Ser Gly 332 ACA CTG CAG Thr Leu Gin 392 CCG GCC AGC Pro Ala Ser 452 CTC CCT GGT Leu Pro Gly 512 CTG AGT GAA Leu Ser Glu 572 GAC AAA ATT Asp Lys Ile TGT TCC Cys Ser CAA ACG Gin Thr CGG AGC Arg Ser CAC ACT His Thr CTA ATT Leu ile AGC AAT Ser Asn TAC AAC AGG ATG Arg Met ATC GCA Ile Ala CTT CTG Leu Leu CTC AGG Leu Arg GTC TTT Val Phe GGG AAC Gly Asn TAC TGC GAA CTC Glu Leu GCG ATG Ala Met ATG CAA Met Pro TCA GAG Ser Glu TTC CCT Phe Pro TAC AAG Tyr Lys AGG CTA Arg Leu Tyr Asn Tyr Cys GTT TAT GCA CTA Val Tyr Ala Leu ACA GAT GTT AGC Thr Asp Val Ser AAC GTC CTA GTA Asn Val Leu Val AAC GGC Asn Gly TAC AAT Tyr Asn GGG GAA Gly Glu OMMUMONOWEEd 31 DNA Sequence and Deduced Amino for GLS-1, GLS-2, GLS-3 and (SEQ. ID No:16)(Gontinued) Acid Sequence GLS-4 Clones 632 GTT ACT GTC CTC AGC TTA CCC ACA TCA TAT GAT CTT GGG TAT Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr 662 GTG AGG CTT GGT GAC CCC Val Arg Leu Gly Asp Pro 692 ATA CCC GCT ATA GGG CTT GAC CCA MAA ATG GTA GCA ACA TGT GAC AGC Ile Pro Ala Ile Giy Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser 752 AGA GTC TAC ACC ATA ACT GCA GCT GAT GAT TAG GMA TTC TCA TCA GAG Arg Val Tyr Thr Ile Thr Ala Ala Asp Asp Tyr Gin Phe Ser Ser Gin GGG GTA ACA ATG AGC CTG TTC TCA GCC 2 0 Gly Vai Thr Ile Thr Leu Phe Ser Ala 812 MGC AT7 Asn Ile CAT CCC ATC ACA AGG Asp Ala Ile Thr Ser 872 GGA GAG CTC GTG TTT AAA ACA AGC GC GAG AGC CT]' GTA CTG CCC CC Cly Giu Leu Vai Phe Lys Thr Ser Val His Ser Leu Val Leu Cly Ala 932 ATA GGG TTT CAT CCC TCT CC CTA ATG ACT AGA GCT GTG GGG GCA MGC Ilie Cly Phe Asp Gly Ser Aia Vai Ile Thr Arg Ala Val Ala Ala Asn 992 ACC GGG ACC GAG MAT CTI' ATG GGA T7C MAT GTT GTG AT-r GGA ACC MGC Thr Gly Thr Asp Asn Leu Met Pro Phe Asn Leu Val Ile Pro Thr Asn 0000 722 ACT GAG AGG CCC Ser Asp Arg Pro 782 TAG GMA ACA GGT Tyr Gin Thr Giy 842 CTG AGG GTT GGG Leu Ser Vai Giy 9G02 ACC ATC TAG Crr Thr Ile Tyr Leu 962 MAT CCC GTG AG Asn Gly Leu Thr 1022 GAG ATA ACC GAG Giu Ile Thr Gin 1082 GAG GMA CCC GAG Gin Giu Gly Asp i1142 GGG MGC TAT GGA Giy Asn Tyr Pro 1202 GGA TCT GTCGCT]' Gly Ser Vai Val 1262 GGA MAG MG GTG Ala Lys Asn Leu 1322 TTG ATA GTG AGT Leu Ile Leu Ser 1382 ACC GAG ITT' GGT Thr Asp Phe Arg GCA ATC ACA TGC ATG MAA CTG GAG Pro Ilie Thr Ser Ile Lys Leu Giu GAG ATG TCA TGG TGG GGA ACT GCC 4 0 Gin Met Ser Trp Ser Ala Ser Gly GGG CCC CTG CGT GGC GTG ACA CTA Gly Ala Leu Arg Pro Val Thr Leu ACG GTG GGT CCC GTG AGG MAC 'FTC Thr Val Ala Cly Vai Ser Asn Phe GTT ACA GMA TAG CCC GGA TTT GAG Val Thr Giu Tyr Gly Arg Phe Asp 1052 ATA CTG ACC Ile Val Thr 1112 AGC CTA GCA Ser Leu Ala 1172 GTA GGC TAG Val Ala Tyr 1232 GAG CTG ATG Ciu Leu Ile 1292 CCA GCA CC Pro Cly Ala TGG MAA ACT GCT CCT Ser Lys Ser Gly Gly CTG AGG ATT CAT CCT Val Thr Ile His Gly GMA AGA GTC GCA ACA Giu Arg Val Ala Thr CCA MAT GCT GMA GTA Pro Asn Pro Clu Leu ATG MAC TAG ACA A Met Asn Tyr Thr Lys GAG AGG GAG CGG CT]' GGG ATG MAG ACA GTC TGGCGCG ACA AGG GAG TAG Giu Arg Asp Arg Leu Cly Ile Lys Thr Val Trp Pro Thr Arg Giu Tyr -9-rr~lllla~f~ri 32 DNA Sequence and Deduced Amino for GLS-1, GLS-2, GLS-3 and Acid Sequence GLS-4 Clones (SEQ. ID No:16)(Continued) onov 14 i) Inoi O""0 Ui) OCI D 1)0 (14 (tn C1 WO Il OU

O

UUOU

GAG TAC TTC ATG GAG GTG GCC GACG Glu Tyr Phe Met Glu Val Ala Asp TTC AAA GAC ATA ATC CGG GCC ATA Phe Lys Asp Ile Ile Arg Ala Ile 1 5 CCA CCT GCC GCT CCC CTG GCC CAT Pro Pro Ala Ala Pro Leu Ala His GAG GCA CAG GCT GCT TCA GGA ACT Glu Ala Gln Ala Ala Ser Gly Thr GGC CGC ATA AGG CAG CTG ACT CTC Gly Arg Ile Arg Gin Leu Thr Leu TTC CAG GTG CCC CAG AAT CCC GTA Phe Gin Val Pro Gin Asn Pro Val GGT GCA CAC AAC CTC GAG TGC GTG Gly Ala His Asn Leu Asp Cys Val 35 ACG ACA GTG GAA GAC GCC ATG ACA Thr Thr Val Glu Asp Ala Met Thr GAA GGC GTG CGA GAG GAC CTC CAA 40 Glu Gly Val Arg Glu Asp Leu Gin 1412 CTC AGC Leu Ser 1472 AGG AGG Arg Arg 1532 GCA ATT Ala Ile 1592 GCT CGA Ala Arg 1652 GCC GCC Ala Ala 1712 GTC GACG Val Asp 1772 TTA AGA Leu Arg 1832 CCC AAA Pro Lys 1892 CCT CCA Pro Pro 1952 GCT CCA Ala Pro 2012 GAT GTC Asp Val 2072 AGT GGA Ser Gly 2132 GCC ATG Ala Met TCT CCC CTG AAG ATT GCA GGA GCA Ser Pro Leu Lys Ile Ala Gly Ala ATA GCT GTG CCG GTG GTC TCC ACA Ile Ala Val Pro Val Val Ser Thr GGG GAA GGT GTA GAC TAC CTG CTG Gly Glu Gly Val Asp Tyr Leu Leu GCC GCG TCA GGA AAA GCA AGG GCT Ala Ala Ser Gly Lys Ala Arg Ala 1682 GAC AAG GGG TAC GAG GTA GTC GCG AAT CTA Asp Lys Gly Tyr Glu Val Val Ala Asn Leu 1742 GGG ATT CTT GCT TCA CCC GGG ATA CTC CGC Gly Ile Leu Ala Ser Pro Gly Ile Leu Arg 1802 GAG GGC GCC ACG CTA TTC CCT GTG GTC ATC Glu Gly Ala Thr Leu Phe Pro Val Val Ile 1862 GCA CTA AAC AGC AAA ATG TTT GCT GTC ATT Ala Leu Asn Ser Lys Met Phe Ala Val Ile 1922 TCT CAA AGA GGA TCC TTC ATA CGA ACT CTC Ser Gin Arg Gly Ser Phe le Arg Thr Leu 1442 TTT GGC Phe Gly 1502 TTG TTC Leu Phe 1562 GGT GAT Gly Asp 1622 GCC TCA Ala Ser 00 0000 S o ro TCC GGA CAC AGA GTC TAT GGA TAT Ser Gly His Arg Val Tyr Gly Tyr GAC TAC ACC GTT GTC CCA ATA GAT Asp Tyr Thr Val Val Pro Ile Asp CCC ATA CCT CCT ATT GTG GGA AAC Pro Ile Pro Pro Ile Val Gly Asn GAT GGG GTA CTT CCA CTG GAG AGT Asp Gly Val Leu Pro Leu Glu Thr TGG GAC GAC AGC ATT ATG CTG TCC Trp Asp Asp Ser Ile Met Leu Ser AAC CTA GCC ATA GCT TAC ATG GAT Asn Leu Ala Ile Ala Tyr Met Asp ACG GGA GCC CTG AAC GCT TGT GGC Thr Gly Ala Leu Asn Ala Cys Gly 1982 GGG AGA Gly Arg 2042 AAA GAC Lys Asp 2102 GTG TTT Val Phe 2162 GAG ATT Glu Ile CGA CCC AAA GTC CCC ATC CAT GTG Arg Pro Lys Val Pro Ile His Val 1 33 T2b1.eJ: DNA Sequence and Deduced Amino Acid Sequence for GLS-1, GLS-2, GLS-3 and GLS-4 Clones (SEQ. ID No:16)(Continued) 2192 ACC MAG CTC GAG AMA ATA AGC M1~ AGA AGC GCC ACC GCA CAC CGG 2222 CTT GGC CTC MAG 170 Lou Gly Lou Lys Leu Glu Lys Ile Sor !'he Arg Ser Thr Lys Lou Ala Thr Ala His Arg GCT GGT CCC GGA GCA TT GAT Ala Gly Pro Gly Ala Phe Asp 2252 GTA MAC ACC Val Asn Thr GGG CCC MAC TGG GCA ACG TTC ATC Gly Pro Asn Trp Ala Thr Phe Ilie TTC CCT CAC MAT CCA CGC Phe Pro His Asn Pro Arg 2312 GAC TGG GAC AGG Asp Trp Asp Arg CTC CCC TAC CTC MAC CTT CCA TAC Lou Pro Tyr Lou Asn Lou Pro Tyr CCC MAT GCA GGA CGC CAG TAC CAC CTC GCC ATG GCC GCA TCA GAG TTC MAG GAG Pro Asn Ala Gly Arg GIn Tyr His Lou Ala Met Ala Ala Ser Giu Phe Lys Glu 2282 MAA CGT Lys Arg 2342 CTT CCA Leu Pro 2402 ACC OCT Thr Pro 2462 TTC CAA Pho Gin 2522 GCC MAC Ala Asn 2582 CCA CMA Pro Gin GMA CTO GAG AGC GCC GTC AGG GCO Glu Lou Glu Ser Ala Val Arg Ala TOT GCA CTO AGT GTG TTC ATG TGG Sor Ala Lou Ser Val Plie Het Trp 77C GOA CTC AGC GAC CCG MAC GC Phe Ala Lou Sor Asp Pro Asn Ala 2432 ATG GMA GCA Met Glu Ala 2492 CTG GMA GAG Lou Glu Glu 2552 GCA GCC AGT GTA GAO CCA CTG Ala Ala Sor Val Asp Pro Leu MAT GG AT' GTG ACT GAC ATG AMn Gly Ile Val Thr Asp Hot

GOA

Ala CAT CGG His Arg 2612 3 5 GCA GGT AGC MAG TCT CMA AGG GCC AMA TAC Ala Gly Scr Lys Ser Gin Arg Ala Lys Tyr 2672 GGC CCC ACA CCA GMA GMA GCA CAG AGG GMA Gly Pro Thr Pro Glu Giu Ala Gin Arg Giu 2732 ACC ATG GGC ATC TAC TT GCA ACA CCA GMA Thr Het Gly Ile Tyr Pho Ala Thr Pro Giu 2792 AGC CCC GGC CAG OTA MAG TAC TGG CAG MAC Ser Pro Gly Gin Leu Lys Tyr Trp Gln Asn ATG OGA MAC TTT CTT- GCA MAC Met Arg Asn Phe Leu Ala Asn GGG ACA GCA GGC TAC GGA GTG Gly Thr Ala Gly Tyr Gly Val AMA GAO ACA CGG ATC TCA MAG Lys Asp Thr Arg Ile Ser Lys TGG GTA GCA OTC MAT GGG CAC Trp Val Ala Lou Azn Gly His 2642 GAG GCC CGG Giu Ala Arg 2702 MAG ATG GAG Lys Met Giu 2762 OGA GGG CCA Arg Giy Pro ACA CGA GMA ATA CCG GAC CCA MAC Thr Arg Giu Ile Pro Asp Pro Msn 2822 GAG GAO Giu Asp 2852 2882 TAT CTA GAC TAC GTG CAT GCA GAG MAG AGC CGG TTG GCA TCA GMA GM CMA ATC CTA AGG Tyr Lou Asp Tyr Vai His Ala Giu Lys Ser Arg Lou Ala Ser Giu Giu Gin Ile Lou Arg 2912 2942 GCA OCT ACG TCG ATC TAC GGG GOT COA GGA CAG GCA GAG CCA CCC CMA GOT TTO ATA GAC Ala Ala Thr Ser Ile Tyr Gly Ala Pro Gly Gin Ala Giu Pro Pro Gin Ala Phe Ile Asp i I-I 34 Table_7: DNA Sequence and Deduced Amino for GLS-1, GLS-2, GLS-3 and (SEQ. ID No:16)(Concluded) Acid Sequence GLS-4 Clones GAA GTT GCC AAA GTC Glu Val Ala Lys Val GAT CTG CTC TTG ACT Asp Leu Leu Leu Thr CCC AAG CCA AGA CCC Pro Lys Pro Arg Pro AGG ACT GTC TCT GAT Arg Thr Val Ser Asp GCA GGC GTG GAC ACC Ala Gly Val Asp Thr 2972 3002 TAT GAA ATC AAC CAT GGA CGT GGC CCA AAC CAA GAA CAG ATG AAA Tyr Glu Ile Asn His Gly Arg Gly Pro Asn Gin Glu Gin Met Lys 3032 3062 GCG ATG GAG ATG AAG CAT CGC AAT CCC AGG CGG GCT CCA CCA AAG Ala Met Glu Met Lys His Arg Asn Pro Arg Arg Ala Pro Pro Lys 3092 3122 AAC GCT CCA ACG CAG AGA CCC CCT GGT CGG CTG GGC CGC TGG ATC Asn Ala Pro Thr Gin Arg Pro Pro Gly Arg Leu Gly Arg Trp Ile 3152 3182 GAG GAC CTT GAG TGA GGC TCC TGG GAG TCT CCC GAC ACC ACC CGC Glu Asp Leu Glu End Gly Ser Trp Glu Ser Pro Asp Thr Thr Arg 3212 AAT TCG GCC TTA CAA CAT CCC AAA TTG GAT CCG Asn Ser Ala Leu Gin His Pro Lys Leu Asp Pro o uj.?o a n a se a or o crros

O

r)r(La U 1 0~10 O I

II

09(11

D

a a 11 The DNA sequence of the GLS-1 clone starts at nucleotide I and ends at nucleotide 348, and is therefore 348 base pairs long. The sequence of the GLS-2 clone 30 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 I.BDV strain. The second MCA, R63, was raised against the D78 IBDV strain and neutralizes all 35 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, o 15 responsible for or associated with the binding of oo° specific MCAs. Variations of the complementary DNA o 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 0°0 Specific Amino Acid Variations of GLS-5 and E/DEL IBDV 0 25 The DNA sequences and the amino acid sequences 'o.o 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 Sfound for these US variants of the virus both at the DNA and the amino acid level.

Table 8: Comparisons of VP2 Gene and Protein Sequences of GLS-5 and E/DEL Variant Viruses Percent Homology At Nucleotide Level 98.1% S/ Of Deduced Amino Acids 98.0% 1. 0_ :i 36 Tables 9 and 10 below show variations of amino acids found between the VP2 sequences of GLS-5 and E/DEL clones.

Table Comparison of VP2 Protein Sequences of and E/DEL Variant Viruses Differences in Amino Acids 1 9 Asp His Ser Thr Thr Gly Glu Asp Ser E.DEL Asn Gln Thr Ala Ile Asp Ala Glu Ash Table 0 Amino Acid Changes for IBDV VP2 Variants Amino Acid Residue Numbers in VP2 84 213 222 249 253 254 269 270 280 284 286 318 321 323 326 330 443 Gin Asp Thr Lys His Ser Ser Ala Asn Thr Thr Gly Glu Asp Ser Ser Ser Gin Asn Thr Lys Gin Ser Thr Ala Asn Ala 11e Asp Ala Glu Ser Ser Asn E.DEL o o o fta a a a (r 0 ai

I

Example 16: IBDV VP2 DNA and Amino Acid Homologies Found Between the Australian Variant and GLS and 25 The Australian Variant and E/DEL The homologies found for the VP2 DNA of the Australian strain (002-783) and the GLS-5 *-iral DNA segment as well as for the Australian and E/DEL segment are shown in Table 11 below. Also shown are amino acid 30 homologies found between the Australian and U.S. variants as well as between the Australian and E/DEL variants.

37 Table I Comparison of VP2 Gene and Protein Sequences of Australian and American Isolates of IBDV Percent Homology at Nucleotide Level 91.9% E.DEL 92.1% Percent Homology.of Deduced Amino Acids 96.2% E.DEL 95.6% ue a ooo o oa~ re d(l L

L

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 (9 below.

06- Table Ja Amino Acid Changes in VP2 of IBD US Variants and Australian and German Viruses Amino Acid Residue Number in VP2 Variant Viruses 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 Australian Ser Met Gln Asp Pro Asn Gin Gin Gly Asn Val Thr Thr Thr Gly Asn Ala Tbr Pro Ser Val Gly Ala AsjF Ser Leu Ser Asn Asn Gin Leu Gin Asp Thr Ser Lys His Ser Gly Ile Ser Ala Ile Asn Asn Thr Thr Pro Asn Ile Gly Glu Asp Ser Ser Ser Ser Ser E. Delaware Gin Leu Gin Asn Thx- Ser Lys Gin Ser Gly Ile Thr Ala Ile Asn Asn Ala Ile Pro Asn Ile Asp Ala Glu Ser Ser Sex- Ser Asn German Cu-I Gin Leu Gin Asp Pro Ser Gin His Gly Gly Ile Thr Th- Ile Mn Aso Thx- Thx- Ser Asn Ile Gly Ala Asp Ser Ser Lys Sex- Asn 39 Example 18: Preparation of Synthetic Peptides The nucleotide sequences of the genes encoding, the rtirc'.ural protein VP2 for three IBDV strains GLS-b, E/Dclaware, 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 chromatography 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 NaCI, pH 7.5 buffer if freely water soluble, ~15 or otherwise in a 8 M urea, 1% 2-mercaptuethanol/0.05 M oTris, pH 8.3, buffer, and stored at -70°C until used.

°Example 19: Competitive Antigen Binding Assays 0° Radiolabeling of the IBDV proteins is carried out as described (Muller, H. and Becht, J. Virol. 44, 384- 392 (1982)). Monolayers of CEF cells are infected with IBDV at a multiplicity of infection of 10 pfu/cell and 0. :incubated at 37°C. After 1 hour, the cells are washed twice and incubated for 1 hour with Eagle's minimum S0 25 essential medium (MEM) without methionine. Two hours 0 after infection the above media are removed and replaced with MEM containing 100 pCi of 35 S-methionine. After 935 a pulse with 3S-methionine for 12 hours, labeled virus 0. 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 3 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 40 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 (150 dose) by logit-log transformed linear regression analysis (Trautman, R. and Harris, Scand. J. Immunol. 6, 831-841 (1977)). The results are plotted as percent inhibition v. logl 0 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 13 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 13: Antigenic variants of IBDV reacting with Neutralizing MCAs in AC-ELISA systems o oa 0o oa o o io o r

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.

1 41 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 epitope(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 Is- below.

Table 1 Some Predicted Epitopic Sites in The Amino 15 Acid Sequence of IBDV MCA B69 Residue No. 314 315 316 317 318 319 320 321 322 323 324 (SEQ.ID No:17) Thr Ser Lys Ser Gly Gly Gln Ala Gly Asp Gln MCA 42 Residue No. 279 280 281 282 283 284 285 286 287 288 289 (SEQ.ID No:18) Asn Asn Gly Leu Thr Thr Gly Thr Asp Asn Leu 25 MCA R63 Residue No. 264 265 266 267 268 269 270 271 272 273 274 (SEQ.ID No:19) Ile Gly Phe Asp Gly Thr Thr Val Ile Thr Arg SMCA 57 30 Residue No. 315 319 320 321 322 323 324 325 326 327 328 (SEQ.ID No:20) Gly Gly Gln Glu Gly Asp Gln 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 e set forth herein.

42 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: Nucleic Acid s STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic PUBLICATION INFORMATION: AUTHORS: Hudson, P.J. et al.

JOURNAL: Nucleic Acids Research VOLUME: 14 PAGES: 5001-5012 DATE: 1986 SEQUENCE DESCRIPTION: SEQ ID No: 1: 5'-CAATTGCATGGGCTAG-3' o INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 18 S 20 TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear o MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic PUBLICATION INFORMATION: oo.. AUTHORS: Spies et al.

o JOURNAL: Nucleic Acids Research VOLUME: 17(19) PAGES: 7982 S 3so DATE: 1989 SEQUENCE DESCRIPTION: SEQ ID No: 2: '-AACGATCCAATTTGGGAT-3' INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 17 TYPE: Nucleic Acid .STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic ILUMIN0601 1 00C 42 of 9 SEQUENCE DESCRIPTION: SEQ ID No: 3 5'-TATTCTGTAACCAGGTT-3' INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 18 TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 4: 5'-CACTATCTCCAGTTTGAT-3' INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear S MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic oO SEQUENCE DESCRIPTION: SEQ ID No: 5'-TACGAGGACTGACGGGTCTT-3' INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: 25 LENGTH: 18 TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: 30 DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 6: 5'-TTCAAAGACATAATCCGG-3' INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 18 TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic UbMIW6011: JOC 4 3 of 9 SEQUENCE DESCRIPTION: SEQ ID No: 7: 5'-GGGTGAAGCAAGAATCCC-3' INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 18 TYPE:. Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: to DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 8: 5'-GTGCGAGAGGACCTCCAA-3' INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 18 TYPE: Nucleic Acid S STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 9: 5'-GTATGGAAGGTTGAGGTA-3' INFORMATION FOR SEQ ID NO: SSEQUENCE CHARACTERISTICS: LENGTH: 18 0 0o. TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: S 30 DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 5'-GGGATTCTTGCTTCACCC-3' INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic I I 1 1. l_ -m C* ~LI SEQUENCE DESCRIPTION: SEQ ID No: 11: 5'-ACGTTCATCAAACGTTTCCC-3' INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: s LENGTH: TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 12: 5'-TTGCAAACGCACCACAAGCA-3' INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 17 TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 13: 5'-CGGATCCAATTTGGGAT-3' INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 17 S TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: 30 DESCRIPTION: Synthetic SEQUENCE DESCRIPTION: SEQ ID No: 14: 5'-GTGTCGCCAGACTCCCA-3' INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1453 TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Viral 46 ORIGINAL SOURCE: ORGANISM: Infectious bursal disease virus (IBDV) STRAIN: E/DEL IMMEDIATE SOURCE: CLONE: E/DEL-2 PUBLICATION INFORMATION: JOURNAL: European Molecular Biology Laboratory (EMBL) Data Base DATE: 18 OCT 1990 DOCUMENT NUMBER: X54858 RELEVANT RESIDUES IN SEQ ID No: 15: 52 to 1471 900000 0 g o a ,a ,tug4 A ll ILibMl\06011 :JOC dR nf 46 f SEQUENCE DESCRIPTION: SEQ ID No: CTA CMA TGC TAT CAT TGA TGG TTA GTA GAG ATC AGA CAA ACG ATC GCA GCG ATG Leu Gin Cys Tyr His End Trp Leu Val Giu Ile Arg Gin Thr Ile Ala Ala Met CTG CMA GAT CAA ACC CMA CAG ATT GTT CCG TTC ATA CGG AGC CTT CTG ATG CCA Leu Gin Asp Gin Thr Gin Gin Ile Val Pro Phe Ile Arg Ser Leu Leu Met Pro 150 GGA CCG GCG TCC ArT CCG GAC GAC ACC CTG GAG MAG CAC ACT CTC AGG TCA GAG Gly Pro Ala Ser Ile Pro Asp Asp Thr Leu Giu Lys His Thr Leu Arg Ser Giu 210 ACC TAC MAT TTG ACT GTG GGG GAC ACA GGG TCA GGG CTA ArT GTC TTT TTC CCT Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Giy Leu Ile Val Phe Phe Pro 270 CCT GGC TCA At GTG GGT GCT CAC TAC ACA CTG CAG AGC MAT GGG MAC TAC MAG Pro Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gin Ser Asn Gly ASn Tyr Lys 330 CAG ATG CTC CTG ACT GCC CAG MAC CTA CCG GCC AGC TAC MAC TAC TGC AGG CTA Gin Met Leu Leu Thr Ala Gin Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu 390 CGG ACT CTC ACA GTA AGG TCA AGC ACA CTC CCT GGT GGC GTT TAT GCA CTA MAC Arg Ser Leu Thr Vai Arg Ser Ser Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn ACA MAC Thr Asn 120 ACA ACC Thr Thr 180 ACC TCG Thr Ser 240 GGA rTC Gly Phe 300 rtC CAT Phe Asp 360 GTG AGT Val Ser 420 GGC ACC Gly Thr 480 MAC GGG Asn Gly 540 GMA GGG ATA MAC GCC G TG ACC TTC CMA GGA AGC Ile Asn Ala Val Thr Phe Gin Gly Ser 450 CTG AGT Leu Ser GMA CTG ACA GAT GTT AGC TAG Giu Leu Thr Asp Val Ser Tyr o 4 0 004 TTG ATG TCT GCA ACA GCC MAC ATC MAC GAC AAA At Leu Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile GGG MAC GTC CTA GTA GGG Gly Aso Val Leu Vai Gly Giu Gly 570 GTA ACC GTC CTC AGC TTA CCC ACA TCA TAT GAT CT]' GGG TAT GTG AGO CTT GGT Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly 630 ATA CCC GCT ATA GGG CTT GAC CCA AMA ATG GTA GCA ACA TGT GAC AGC AGT GAC Ile Pro Ala Ile Giy Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp 690 AGA GTC TAC ACC ATA ACT GCA GCC GAT MAT TAC CMA rTC TCA TCA CAG TAG CMA Arg Val Tyr Thr Ile Thr Ala Ala Asp Asn Tyr Gin Phe Ser Ser Gin Tyr Gin 600 GAG CCC Asp Pro 660 AGG CCC Arg Pro 720 ACA GGT Thr Giy ILibMIN0601 1:JOC GGG GTA ACA ATC ACA CTG TTC TCA GCC MAC ATT GAT GCC ATC ACA AGT CTC AGC GTT GGG Gly Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu Ser Val Gly o 0 on 0 00 0 GGA GAG CTC GTG TTC Giy Glu Leu Val Phe ATA GGC IT GAT GGG Ile Cly Phe Asp Gly GCC GGC ATC GAC MAT Ala Cly Ile Asp Asn CCA ATC ACA TCC ATC Pro Ile Thr Ser Ile CAG ATG TCA TGG TCG Gin Met Ser Trp Ser GGA GCC CTC COT CCC Oly Aia Leu Arg Pro ACG GTC GCT GGG GTG Thr Val Ala Giy.Vai 017 ACA GMA TAT GGC Val Thr Giu Tyr Gly GAG AGO CAC COC 017 Ciu Arg Asp Arg Leu GAG TAC TTC ATG GAG Giu Tyr Phe Met Oiu TTC AMA GAC ATA ATC Phe Lys Asp Ile Ile CCA CCT CCC OCT CCT Pro Pro Ala Ala Pro AAA ACA AGC Lys Thr Ser ACT GCG GTA Thr Ala Val CTT ATG CCA Leu Met Pro MAA CTG GAG Lys Leu Giu GCA ACT GG Ala Ser Cly CTC ACA CTA Val Thr Leu AGC AAC TTC Ser Asn Phe CGA TIT CAC Arg Phe Asp GC ATC MAG Oly Ile Lys GTG CCC GAC Val Ala Asp COO GCC ATA Arg Ala Ile OTA GCC CAT Val Ala His 810 GTC CAA AGC Val Gin Ser 870 ATC ACC AGA Ile Thr Arg 930 TTC MAT CTT Phe Asn Leu 990 ATA CTG ACC Ile Val Thr 1050 ACC CTA OCA Ser Leu Ala 1110 GTG CCC TAC Val Ala Tyr 1170 GAG CTG ATC Clu Leu Ile 1230 CCA OGA GCC Pro Gly Ala 1290 ACC GTC TGG Thr Val Trp 1350 CTC MAC TCT Leu Asn Ser 1410 ACG AGO ATA Arg Arg Ile 1470 OCA ATT G Ala Ile CTT GTA CTG GC CCC ACC Leu Val Leu Giy Ala Thr Ile Tyr Leu 900 OCT OTO GCC OCA MAC MT COO CTG ACO Ala Val Ala Ala Asn Asn Giy Leu Thr 960 GTG ATT CCA ACC MAT GAG ATA ACC CAG Val Ile Pro Thr Asn Clu Ile Thr Gin 1020 TCC AAA ACT CAT GOT CAG OCA CCC GMA Ser Lys Ser Asp Giy Gin Ala Gly Giu 1080 CTG ACO ATC CAT OCT CCC MAC TAT CCA Val Thr Ile His Cly Cly Asn Tyr Pro 1140 CMA AGA GTG OCA ACA GCA TCT CTC GTT Giu Arg Vai Ala Thr Oly Ser Val Val 1200 CCA MAT CCT GMA CTA OCA MOG MC CTG Pro Asn Pro Oiu Leu Ala Lys Asn Leu 1260 ATO MAC TAC ACO AMA TTC ATA CTG ACT Met Asn Tyr Thr Lys Leu Ile Leu Ser 1320 CCA ACA AGO GAG TAC ACT GAC TTT COT Pro Thr Arg Oiu Tyr Thr Asp Phe Arg 1380 CCC CTC MOG NIT OCA OGA OCA TTT GC Pro Leu Lys Ile Ala Oly Ala Phe Oly 1440 OCT CTA CCG OTO CTC TCT ACA TO TTC Ala Val Pro Val Val Ser Thr Leu Phe ATC TAC I _I~X~ INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 3230 TYPE: Nucleic Acid STRANDEDNESS: Both TOPOLOGY: Linear MOLECULE TYPE: OTHER NUCLEIC ACID: DESCRIPTION: Viral ORIGINAL SOURCE: ORGANISM: Infectious bursal disease virus (IBDV) STRAIN: GLS oao ~o a o- ~aoo ao a u; o I o oaoc n oJ o ao ,soo o oa~c o o o o ao o~ r o~~ D o a o urr IMMEDIATE SOURCE: CLONE: GLS-I; GLS-2; GLS-3; GLS-4.

SEQUENCE DESCRIPTION: SEQ ID No: 16: CCG GGG GAG TCA CCC GGG GAC AGG CCG Pro Gly Glu Ser Pro Gly Asp Arg Pro 32 TCA AGG CCT Ser Arg Pro TGT TCC AGG ATG GAA CTC CCC Cys Ser Arg Met Glu Leu Pro CTA CAA TGC TAT CAT TGA TGG TTA GTA GAG ATC GGA CAA ACG ATC GCA GCG ATG ACA Leu Gin Cys Tyr His End Trp Leu Val Glu lie Gly Gin Thr Ile Ala Ala Met Thr CTG CAA GAT CAA ACC CAA CAG ATT GTT Leu Gin Asp Gin Thr Gin Gln Ile Val GGA CCG GCG TCC ATT CCG GAC GAC ACC Gly Pro Ala Ser Ile Pro Asp Asp Thr ACC TAC AAT TTG ACT GTG GGG GAC ACA Thr Tyr Asn Leu Thr Val Gly Asp Thr CCT GGC TCA ATT GTG GGT GCT CAC TAC Pro Gly Ser lie Val Gly Ala His Tyr CAG ATG CTC CTG ACT GCC CAG AAC CTA Gin Met Leu Leu Thr Ala Gin Asn Leu CGG AGT CTC ACA GTA AGG TCA AGC ACA Arg Ser Leu Thr Val Arg Ser Ser Thr ATA AAC GCC GTG ACC TTC CAA GGA AGC Ile Asn Ala Val Thr Phe Gin Gly Ser 152 CCG TTC Pro Phe 212 CTG GAG Leu Glu 272 GGG TCA Gly Ser 332 ACA CTG Thr Leu 392 CCG GCC Pro Ala 452 CTC CCT Leu Pro 512 CTG AGT Leu Ser ATA CGG AGC CTT CTG ATG CAA ACA Ile Arg Ser Leu Leu Met Pro Thr AAG CAC ACT CTC AGG TCA GAG ACC Lys His Thr Leu Arg Ser Glu Thr GGG CTA ATT GTC TTT TTC CCT GGA Gly Leu Ile Val Phe Phe Pro Gly CAG AGC AAT GGG AAC TAC AAG TTC Gin Ser Asn Gly Asn Tyr Lys Phe AGC TAC AAC TAC TGC AGG CTA GTG Ser Tyr Asn Tyr Cys Arg Leu Val GGT GGC GTT TAT GCA CTA AAC GGC Gly Gly Val Tyr Ala Leu Asn Gly GAA CTG ACA GAT GTT AGC TAC AAT Glu Leu Thr Asp Val Ser Tyr Asn TTG ATG TCT GCA ACA GCC Leu Met Ser Ala Thr Ala 572 AAC ATC AAC GAC AAA Asn Ile Asn Asp Lys ATT GGG AAC GTC Ile Gly Asn Val 602 CTA GTA GGG GAA GGG Leu Val Gly Glu Gly 632 662 GT ACT GTC CTC AGC TTA CCC ACA TCA TAT GAT Cr1' GGG TAT GTG AGG CTT GGT GAC CCC Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Cly Tyr Val Arg Leu Gly Asp Pro 692 722 ATA CCC GCT ATA GGG CTT GAG CCA AAA ATG GTA GCA ACA TGT GAG AGG AGT GAC AGG CCC Ile Pro Ala Ile Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro 752 782 AGA GTG TAC ACC ATA ACT GCA GGT GAT GAT TAC CMA TTC TCA TCA CAG TAGCAGM ACA GGT Arg Val Tyr Thr Ile Thr Ala Ala Asp Asp Tyr Gin Phe Ser Ser Gin Tyr Gin Thr Gly 812 842 GGG GTA ACA ATC ACC GTG TTC, TCA CCC MAC ATT CAT GCG ATG AGA ACC CTC AGC GTT GGG Giy Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu Ser Val Cly GGA GAG GTC CTC TTT AMA ACA AGC GTC Cly Ciu Leu Val Phe Lys Thr Ser Val AGC GTT GTA GTG GGC CCC ACC Ser Leu Val Leu Giy Ala Thr AGA CCT GTC CCC GCA MGC AAT Arg Ala Val Ala Ala Asn Asn 902 ATC TAG GTT Ile Tyr Leu 962 CCC GTG AG Gly Leu Thr ATA CCC TTT CAT CCC TCT CC GTA Ile Gly Phe Asp Gly Ser Ala Val 932 ATC ACT Ile Thr 00 0 ACCCGC ACC GAG MAT CT1' ATG CCA TTG MAT Cr1' CTC Ar1' GCA ACC MGC GAG ATA Thr Gly Thr Asp Asn Leu Met Pro Phe Asn Leu Val Ile Pro Thr Asn Giu Ile CGA ATG ACA TGC ATG AMA CTG GAG Pro Ile Thr Ser Ile Lys Leu Glu 1052 ATA GTG ACC Ile Val Thr TCC AMA ACT GCT GGT GAG GMA Ser Lys Ser Gly Cly Gin Ciu GTG ACG ATT CAT GGT CCC MAC Val Thr Ile His Gly Gly Asn 1022 ACC GAG Thr Gin 1082 CCC GAG Cly Asp 1142 TAT GCA Tyr Pro 1202 GTC CTT Val Val GAG ATC TGA TGG TCG GGA ACT Gin Met Ser Trp Ser Ala Ser 1112 CCC AGC CTA GGA Ciy Ser Leu Ala 1172 CCC CCC CTC CCT CCC CTC AGA CTA GTA CCC TAG CMA AGA CTC GGA AGA CGA TCT Gly Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Giu Arg Val Ala Thr Cly Ser 1232 AG GTG GCT CCC GTC ACC MAC TTC GAG CTG ATC CCA MAT CCT Thr Val Ala Cly Val Ser Asn Phe Giu Leu Ile Pro Asn Pro 1292 CT1' AGA GMA TAG CCC GGA TTT GAG CCA CGA CCC ATG MAC TAG Val Thr Ciu Tyr Cly Arg Phe Asp Pro Cly Ala Met Asn Tyr 1352 GAG AGG GAG CCC CTI2 CCC ATC MCG AGA CTC TGC CCC AGA AGC Glu Arg Asp Arg Leu Cly Ile Lys Thr Val Trp Pro Thr Arg 1262 GMA CTA GCA MAG MAC GTG Giu Leu Ala Lys Asn Leu 1322 ACA AMA TTG ATA CTC ACT Thr Lys Leu Ile Leu Ser 1382 GAG TAG ACC GAG ITT CGT Glu Tyr Thr Asp Phe Arg i_ 1412 GAG TAC TTC ATG GAG GTG GCC GAC CTC AGC TCT CCC CTG AAG ATT GCA GGA GCA Glu Tyr Phe Met Glu Val Ala Asp Leu Ser Ser Pro Leu Lys Ile Ala Gly Ala 1472 TTC AAA GAC ATA ATC CGG GCC ATA AGG AGG ATA GCT GTG CCG GTG GTC TCC ACA Phe Lys Asp Ile Ile Arg Ala Ile Arg Arg Ile Ala Val Pro Val Val Ser Thr 1532 CCA CCT GCC GCT CCC CTG GCC CAT GCA ATT GGG GAA GGT GTA GAC TAC CTG CTG Pro Pro Ala Ala Pro Leu Ala His Ala Ile Gly Glu Gly Val Asp Tyr Leu Leu 000200 o o 0000 00 0 60 o e ,go 04 sees GAG GCA CAG GCT GCT TCA GGA ACT Glu Ala Gin Ala Ala Ser Gly Thr GGC CGC ATA AGG CAG CTG ACT CTGC Gly Arg Ile Arg Gin Leu Thr Leu TTC CAG GTG CCC CAG AAT CCC GTA Phe Gin Val Pro GIn Asn Pro Val GGT GCA CAC AAC CTC GAC TGC GTG Giy Ala His Asn Leu Asp Cys Val ACG ACA GTG GAA GAC GCC ATG ACA Thr Thr Val Glu Asp Ala Met Thr GAA GGC GTG CGA GAG GAC CTC CAA Glu Gly Val Arg Glu Asp Leu Gin 1592 GCT CGA GCC GCG Ala Arg Ala Ala 1652 GCC GCC GAC AAG Ala Ala Asp Lys TCA GGA AAA GCA AGG GCT Ser Gly Lys Ala Arg Ala GGG TAC GAG GTA GTC GCG Gly Tyr Glu Val Val Ala 1712 GTC GAC GGG Val Asp Gly ATT CTT GCT TCA CCC GGG Ile Leu Ala Ser Pro Gly 1442 TTT GGC Phe Gly 1502 TTG TTC Leu Phe 1562 GGT GAT Gly Asp 1622 GCC TCA Ala Ser 1682 AAT CTA Asn Leu 1742 CTC CGC Leu Arg 1802 GTC ATC Val Ile 1862 GTC ATT Val Ile 1922 ACT CTC Thr Leu 1982 GGG AGA Gly Arg

ATA

Ile 1772 TTA AGA Leu Arg 1832 CCC AAA Pro Lys 1892 CCT CCA Pro Pro 1952 GCT CCA Ala Pro GAG GGC GCC ACG CTA TTC CCT GTG Glu Gly Ala Thr Leu Phe Pro Val GCA CTA AAC AGG AAA ATG TTT GCT Ala Leu Asn Ser Lys Met Phe Ala TCT CAA AGA GGA TCC TTC ATA CGA Ser Gin Arg Gly Ser Phe Ile Arg GAT GGG GTA CTT CCA CTG GAG ACT Asp Gly Val Leu Pro Leu Glu Thr

TAT

Tyr TCC GGA CAC AGA GTC TAT GGA Ser Gly His Arg Val Tyr Gly GAC TAC ACC GTT GTC CCA ATA Asp Tyr Thr Val Val Pro Ile 2012 GAT GAT GTC Asp Asp Val TGG GAC GAC AGC ATT ATG Trp Asp Asp Ser Ile Met 2042 CTG TCC AAA GAC Leu Ser Lys Asp 2072 CCC ATA CCT CCT ATT GTG GGA AAC ACT GGA AAC CTA GCC Pro Ile Pro Pro Ile Val Gly Asn Ser Gly Asn Leu Ala 2132 CGA CCC AAA GTC CCC ATC CAT GTG GCC ATG ACG GGA GCC Arg Pro Lys Val Pro lie His Val Ala Met Thr Gly Ala 2102 ATA GCT TAC ATG GAT GTG TTT Ile Ala Tyr Met Asp Val Phe 2162 CTC AAC GCT TGT GGC GAG ATT Leu Asn Ala Cys Gly Glu Ile 0000 00 0 GAG AAA ATA AGC TTT AGA AGC ACC Glu Lys Ile Ser Phe Arg Ser Thr GCT GGT CCC GGA OCA TT' GAT GTA Ala Gly Pro Gly Ala Phe Asp Val TTC CCT CAC MAT CCA CGC GAC TGG Phe Pro His Asn Pro Arg Asp Trp CCC MAT GCA GGA CGC CAG TAC CAC Pro Asn Ala Cly Arg Gin Tyr His GMA CTC GAG AGC GCC GTC AGG GCC Giu Leu Giu Ser Ala Val Arg Ala TCT GCA CTC AGT GTG TTC ATG TGG Ser Ala Leu Ser Val Phe Met Trp, TTC GCA CTC AGC GAC CCG MAC CC Phe Ala Leu Ser Asp Pro Asn Ala GCA GGT AGC MAG TCT CAA AGG GCC Ala Gly Ser Lys Ser Gin Arg Aia GGC CCC ACA CCA GMA GMA GCA GAG Gly Pro Thr Pro Giu Glu Ala Gin ACC ATG GGC ATG TAC TTT GCA AGA Thr Met Gly Ile Tyr Phe Ala Thr AGC CCC GGC GAG CTA MAG TAC TGG Ser Pro Gly Gin Leu Lys Tyr Trp TAT CTA GAC TAC GTC CAT GCA GAG Tyr Leu Asp Tyr Val His Ala Clu GGA GCT AGG TGG ATG TAC GGG GCT Ala Ala Thr Ser Ile Tyr Gly Ala 2252 MAC ACCGGCG CCC MAC TGG GA ACG TTC ATG Asn Thr Giy Pro Asn Trp Ala Thr Phe Ile 2312 GAG AGG GTC CCC TAG CTC Asp Arg Leu Pro Tyr Leu 2372 GTC CCC ATG GCCGCA TGA Leu Ala Met Ala Ala Ser MAC C1I7 CGA TAG Asn Leu Pro Tyr GAG T7C MAG GAG Giu Phe Lys Giu 2282 MAA GGT Lys Arg 2342 Cr1' GCA Leu Pro 2402 ACC CGT Thr Pro 2462 TTC CMA Phe Gin 2522 GCC MAC Ala Asn 2582 GCA CMA Pro Gin 2192 MAG GTC GGC ACC GGA GAG GGG CT7 Lys Leu Ala Thr Ala His Arg Leu 2432 ATG GMA GGA GGA Met Giu Ala Ala 2492 CTG GMA GAG MAT Leu Glu Clu Asn 2552 CAT GGG ATG GGA His Arg Met Arg 2612 AMA TAG CCC ACA Lys Tyr Gly Thr 2672 AGG GMA AMA GAG Arg Glu Lys Asp 2732 CGA GMA TGG GTA Pro Giu Trp Val 2792 GAG MAC ACA CGA Gin Asn Thr Arg 2852 MAG AGC GGG TTG Lys Ser Arg Leu 2912 CGA GGA GAG GCA Pro Gly Gin Ala GCC AGT GTA GAG CCA CTG Ala Ser Val Asp Pro Leu COG ATT GTG ACT GAG ATG Gly Ile Val Thr Asp Met MGC TTT GTT GGA MAC GCA Asn. Phe Leu Ala Asn Ala 2222 GGG CTC MAG TTG Gly Leu Lys Leu GCA CCC TAG CCA Ala Gly Tyr Gly 2642 GTG GAG CCC CCC Val Giu Ala Arg 2702 ACA CCC ATC TGA MAG MG ATG GAG Thr Arg Ile Ser Lys Lys Met Giu 2762 OCA CTC MAT CCC GAG GGA CCC GGA Ala Leu Asn Gly His Arg Oly Pro 2822 GMA ATA CCC GAG GGA MAC GAG GAG Glu Ile Pro Asp Pro Asn Giu Asp 2882 OCA TGAA M C CAA ATC CTA AGO Ala Ser Clii Giu Gin Ile Leu Arg 2942 GAG GCA CCC CMA GGT TTG ATA GAG Olu Pro Pro Gin Ala Phe Ile Asp 2972 GAA GTT GCC AAA GTC TAT GAA ATC AAC CAT GGA CGT GGC CCA AAC CAA Glu Val Ala Lys Val Tyr Glu lie Asn His Gly Arg Gly Pro Asn Gln 3002 GAA CAG ATG AAA Glu Gin Met Lys GAT CTG CTC TTG ACT GCG ATG Asp Leu Leu Leu Thr Ala Met CCC AAG CCA AGA CCC AAC GCT Pro Lys Pro Arg Pro Asn Ala AGG ACT GTC TCT GAT GAG GAC Arg Thr Val Ser Asp Glu Asp 3032 GAG ATG AAG CAT CGC AAT Glu Met Lys His Arg Asn 3092 CCA ACG CAG AGA CCC CCT Pro Thr Gln Arg Pro Pro 3152 CTT GAG TGA GGC TCC TGG Leu Glu End Gly Ser Trp 3062 CCC AGG CGG GCT CCA CCA AAG Pro Arg Arg Ala Pro Pro Lys 3122 GGT CGG CTG GGC CGC TGG ATC Gly Arg Leu Gly Arg Trp Ile 3182 GAG TCT CCC GAC ACC ACC CGC Glu Ser Pro Asp Thr Thr Arg t 0"i GCA GGC GTG Ala Gly Val 3212 GAC ACC AAT TCG GCC TTA CAA CAT CCC AAA TTG GAT CCG Asp Thr Asn Ser Ala Leu Gin His Pro Lys Leu Asp Pro 0 eoo 0 o o a a INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 11 TYPE: Amino Acid MOLECULE TYPE: Peptide FRAGMENT TYPE: Internal SEQUENCE DESCRIPTION: SEQ ID No: 17: Thr Ser Lys Ser Gly Gly Gin Ala Gly Asp Gin INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 11 TYPE: Amino Acid MOLECULE TYPE: Peptide SEQUENCE DESCRIPTION: SEQ ID No: 18: is Asn Asn Gly Leu Thr Thr Gly Thr Asp Asn Leu INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 9 TYPE: Amino Acid MOLECULE TYPE: Peptide FRAGMENT TYPE: Internal IMMEDIATE SOURCE: CLONE: MCA R63 SEQUENCE DESCRIPTION: SEQ ID No: 19: Ile Gly Phe Asp Gly Thr Val Ile Thr

A

54 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 13 TYPE: Amino Acid s MOLECULE TYPE: Peptide FRAGMENT TYPE: Internal IMMEDIATE SOURCE: CLONE: MCA 57 SEQUENCE DESCRIPTION: SEQ ID No: o1 Gly Gly Gin Glu Gly Asp Gin Met Ser Trp Ser Ala Ser o o oo

D

a~si ~7 at i) 1ibM \OB5t1: JOC SO 1 50 of 9 1

Claims

1. A biologically pure RNA segment, comprising at least one copy of an RNA sequence encoding at least one copy of a polypeptide of at least about 30 amino acids, and having all the antibody binding characteristics of the IBDV VP2 protein derived from at least one US variant selected from E/DEL or GLS strains.

2. The RNA segment of claim 1, which includes up to 20 copies of the RNA sequence.

3. The RNA segment of claim 1 or claim 2 wherein the polypeptide has up to 1012 amino acids.

4. The RNA segment of any one of claims 1 to 3, wherein the polypeptide encoded comprises the antibody binding characteristics of amino acids 200 to 330 of the VP2 protein. The RNA segment of any one of claims 1 to 4, comprising about 3.2K bases.

6. The RNA segment of any one of claims 1 to 5, wherein the RNA sequence encodes a polypeptide fragment having an amino acid sequence encoded by a nucleic acid sequence of Table 6 or 7, an analogue thereof having a different amino acid in at least one position 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 or 433, a o functional fragment thereof, a functional precursor thereof or combinations thereof.

7. The RNA segment of claim 6, having a different amino acid in at least one and less than 29 of said positions.

8. The RNA sequence of claim 7, comprising the RNA sequence encoding a VP2, VP3 or VP4 protein of GLS or E/DEL IBDV.

9. A biologically pure RNA segment encoding a polypeptide having all the antibody binding characteristics of the IBDV VP2 protein, substantially as hereinbefore described with reference to Example 2. A biologically pure DNA segment, comprising a single stranded DNA sequence corresponding to the RNA segment of any one of claims 1 to 9.

11. The DNA segment of claim 10 in double stranded form.

12. The DNA segment of claim 10, wherein the DNA sequence comprises a DNA sequence of Table 6 or 7.

13. A biologically pure DNA segment which codes for a polypeptide having all the antibody binding characteristics of the IBDV VP2 protein, substantially as hereinbefore described with reference to Example 3.

14. 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 11 attached in reading frame to the vector. The recombinant vector of claim 14, wherein the vector comprises a X) 0 recombinant fowl pox virus or a herpes virus from turkeys (IHVT). [N:\LIBC]00309:MHT ~mnua;F~~ FP~- -ICCsDL~B rab I ;i 56

16. The recombinant vector of claim 14 or claim 15, 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.

17. A recombinant vector comprising a DNA sequence which codes for a polypeptide having all the antibody binding characteristics of the IBDV VP2 protein derived from at least one US variant, substantially as hereinbefore described with reference to Example 4.

18. A broad spectrum IBDV poultry vaccine, comprising a poultry protecting amount of the recombinant vector of any one of claims 14 to 17; and a physiologically acceptable carrier.

19. The broad spectrum vaccine of claim 18, comprising about 104 to 107 pfu units of the recombinant vector per mL of carrier. A broad spectrum IBDV poultry vaccine, comprising a poultry protecting amount of the biologically pure DNA segment of claim 11; and a physiologically acceptable carrier.

21. The broad spectrum IBDV poultry vaccine of claim 20, comprising about 104 to 107 pfu units of the recombinant vector per mL of carrier.

22. A host carrying the recombinant vector of any one of claims 13 to 16.

23. A host of claim 22, being E. coli; insect cell-line Sf-9; chicken embryo fibroblast (CEF) cells; chicken embryo kidney (CEK) cells; or vero cells.

24. A biologically pure polypeptide, comprising at least one copy of an amino acid sequence encoded by the RNA segment of any one of claims 1 to 9. 0 25. The polypeptide of claim 24, wherein the amino acid sequence comprises an °a 25 amino acid sequence encoded by a nucleic acid sequence of Table 6 or 7, an analogue thereof having a different aminc acid in at least one 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 or 433, a functional fragment thereof, a functional precursor thereof or combinations thereof.

26. The polypeptide of claim a5, having a different amino acid in at least one and less than 29 of said positions.

27. A biologically pure polypeptide, substantially as hereinbefore described with reference to Example 18.

28. A method of protecting poultry and its progeny from IBD comprising administering to the poultry an amount of the recombinant vector of any one of claims 14 to 17 effective to attain the desired effect.

29. The method of claim 28, wherein the recombinant vector is administered to P the subject ophthalmically, by injection, nasally or orally. [N:\LIBC]00309:MHT 57 The method of claim 28 or claim 29, wherein the recombinant vector is administered to the subject in an amount of about 102 to 106 pfu.

31. 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 11, effective to attain the desired effect.

32. The method of claim 31, wherein the DNA is administered to the subject ophthalmically, by injection, nasally or orally.

33. The method of claim 31 or claim 32, wherein the DNA is administered to the subject in an amount of about 102 to 106 pfu.

34. A method of protecting poultry and its progeny from IBD comprising administering to the poultry an amount of a vaccine of any one of claims 18 to 21 effective to attain the desired effect. f 4 Dated 25 November, 1994 0 University of Maryland At College Park o 15 Patent Attorneys for the Applicant/Nominated Person °0 SPRUSON FERGUSON a 0 8 0 4 s o% [N:\LIBC]00309:MHT i- i .F