AU1349100A - Recombinant herpesvirus of turkeys and uses thereof II - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

0 'ese 0 00 0 0 Name of Applicant: of: Actual Inventors: Address for Service: Syntro Corporation 9669 Lackman Road, Lenexa, KS 66219, United States of America Mark D. COCHRAN; David E. JUNKER; Martha A. WILD; and Philip A. SINGER DAVIES COLLISON CAVE, Patent Attorneys, of 1 Little Collins Street, Melbourne, Victoria 3000, Australia Invention Title: Recombinant herpesvirus of turkeys and uses thereof H The following statement is a full description of this invention, including the best method of performing it known to us: -1- P:OPER\MR0254914SPE -21/1100 -1A- RECOMBINANT HERPESVIRUS OF TURKEYS AND USES THEREOF

II

Throughout this application various publications are referenced by Arabic numerals in parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are in their entirety hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

Throughout this specification and the claims which follow, unless the context requires otherwise, the work "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

9 BACKGROUND OF THE INVENTION The ability to isolate DNA and clone such isolated DNA into bacterial plasmids has.greatly expanded the approaches available to make viral vaccines. The methods used to make the present invention involve modifying cloned DNA sequences from various viral pathogens of animals, by insertions, deletions, single or multiple base changes, and subsequent insertions of these modified sequences into the genome of the virus. One utility of the addition of a foreign sequence is achieved when the foreign sequence encodes a foreign protein that is expressed during viral infection of the animal. The resulting live virus may then be used in a vaccine to elicit an immune response in a host animal and provide protection to the animal against disease.

A virus with these characteristics is referred to as a viral vector, because it becomes a living vector that will carry and express the foreign protein in the host animal. In effect it becomes an elaborate delivery system for the foreign protein(s).

More specifically, the present invention relates to the use of herpesvirus of turkeys (HVT) as a viral vector for vaccination of birds against disease. The group of herpesviruses comprise various pathogenic agents that infect and cause disease in a number of target species: 15 swine, cattle, chickens, horses, dogs, cats, etc. Each herpesvirus is specific for its host species, but they are all related in the structure of their genomes, their mode of replication, and to some extent in the pathology they cause in the host animal and in the mechanism of the host immune response to the virus infection.

The application of recombinant DNA techniques to animal viruses has a relatively recent history. The first viruses to be engineered have been those with the smallest genomes. In the case of the papovaviruses, because these viruses are so small and cannot accommodate much extra DNA, their use in genetic engineering has been as defective replicons. Foreign gene expression from these viruses requires a wild-type helper virus and is limited to cell culture systems.

For adenoviruses, there is a small amount of nonessential DNA that can be replaced by foreign sequences. The only foreign DNA that seems to have been expressed in adenoviruses are the T-antigen genes from papovaviruses (Mansour, et al., Proc. Natl. Acad.

Sci. US, 1985; Thummel, et al., Cell, 1983; Scolnick, et al., Cell, 1981; Thummel, et al., Cell, 1981), and the herpes simplex virus (HSV) thymidine kinase gene (Haj-Ahmed and Graham, J. of Virology, 1986). These publications do not identify the nonessential regions in HVT wherein foreign DNA may be inserted, nor do they teach how to achieve the expression of foreign genes in HVT, which promoter sequence and termination sequence to use.

o 15 Another group of viruses that have been engineered are the poxviruses. One member of this group, vaccinia, has been the subject of much research on foreign gene expression. Poxviruses are large DNA-containing viruses that replicate in the cytoplasm of the infected 20 cell. They have a structure that is unique in that they do not contain any capsid that is based upon icosahedral symmetry or helical symmetry. The poxviruses are most likely to have evolved from bacterial-like microorganisms through the loss of function and degeneration. In part due to this uniqueness, the advances made in the genetic engineering of poxviruses cannot be directly extrapolated to other viral systems, including herpesviruses and HVT. Vaccinia recombinant virus constructs have been made in a number of laboratories that express the following inserted foreign genes: HSV thymidine kinase gene (Mackett, et al., Proc. Natl.

Acad. Sci. USA, 1982; Panicali and Paoletti, Proc.

Natl. Acad. Sci. USA, 1982, hepatitis B surface antigen (Paoletti, et al., Proc. Natl. Acad. Sci. USA, 1984; Smith et al., Nature, 1983), HSV glycoprotein D gene, influenzae hemagglutinin gene (Panicali, et al., Proc.

Natl. Acad. Sci. USA, 1983; Smith, et al., Proc. Natl.

Acad. Sci. USA, 1983), malaria antigen gene (Smith, et al., Science, 1984, and vesicular stomatitis glycoprotein G gent (Mackett, et al., Science, 1986).

The general overall features of vaccinia recombinant DNA work are similar to the techniques used for all the viruses, especially as they relate to the techniques in reference (Maniatis, et al., Molecular Cloning, 1982).

However in detail, the vaccinia techniques are not Sapplicable to herpesviruses and HVT. The utility of vaccinia as a vaccine vector is in question because of its close relationship to human smallpox and its known pathogenicity to humans. Thus, the use of the hostspecific herpesvirus HVT is a better solution to vaccination of poultry.

Among the primate herpesviruses, only HSV of humans and, to a limited extent, herpes saimiri of monkeys have been engineered to contain foreign DNA sequences.

The first use of recombinant DNA to manipulate HSV involved cloning a piece of DNA from the L-S junction region into the unique long region of HSV DNA, specifically into the thymidine kinase gene (Moccarski, et al., Cell, 1980). This insert was not a foreign piece of DNA, rather it was a naturally occurring piece of herpesvirus DNA that was duplicated at another place in the genome. This piece of DNA was not engineered to specifically express a protein, and thus this work does not involve expression of protein in herpesviruses.

The next manipulation of HSV involved the creation of deletions in the virus genome by a combifration of recombinant DNA techniques and thymidine kinase selection. Using this approach, the HSV alpha-22 gene has been deleted (Post, et al., Cell, 1981), and a 15,000 basepair sequence of DNA has been deleted from the internal repeat of HSV (Poffenberger, et al., Proc.

Natl. Acad. Sci. USA, 1981).

The following cases involve insertion of genes that encode protein into herpesviruses: the insertion of HSV glycoprotein C into a naturally occurring deletion mutant of this gene in HSV (Gibson and Spear, J. of Virology, 1983) the insertion of glycoprotein D of HSV 15 type 2 into HSV type 1 (Lee, et al., Proc. Natl. Acad.

Sci. USA, 1982), with no manipulation of promoter sequences since the gene is not 'foreign'; the insertion of hepatitis B surface antigen into HSV under the control of the HSV ICP4 promoter (Shih, et al., 20 Proc. Natl. Acad. Sci. USA, 1984); and the insertion of bovine growth hormone into herpes saimiri virus with an promoter (the promoter did not work in this system and an endogenous upstream promoter served to transcribe the gene) (Desrosiers, et al., 1984). Two additional foreign genes (chicken ovalbumin gene and Epstein-Barr virus nuclear antigen) have been inserted into HSV (Arsenakis and Roizman, 1984), and glycoprotein X of pseudorabies virus has been inserted into HSV (Post, et al., 1985).

These cases of deletion or insertion of genes into herpesviruses demonstrate that it is possible to.

genetically engineer herpesvirus genomes by recombinant DNA techniques. The methods that have been used to insert genes involve homologous recombination between the viral DNA cloned in plasmids and purified viral DNA transfected into the same animal cell. However, the extent to which one can generalize the location of the deletion and the sites for insertion of foreign genes is not known from these previous studies.

One object of the present invention is a vaccine for Marek's disease. Marek's disease virus (MDV) is the causative agent of Marek's disease which encompasses fowl paralysis, a common lymphoproliferative disease of chickens. The disease occurs most commonly in young 15 chickens between 2 and 5 months of age. The prominent clinical signs are progressive paralysis of one or more of the extremities, incoordination due to paralysis of legs, drooping of the limb due to wing involvement, and a lowered head position due to involvement of the neck 20 muscles. In acute cases, severe depression may result.

In the case of highly oncogenic strains, there is characteristic bursal and thymic atrophy. In addition, there are lymphoid tumors affecting the gonads, lungs, liver, spleen, kidney and thymus (Mohanty and Dutta, 1981).

Most chickens are vaccinated against MDV at one day of age to protect the bird against MDV for life. Prior to the present invention, the principal vaccination method for MDV involved using naturally occurring strains of turkey herpesvirus (HVT). It would be advantageous to incorporate other antigens into this vaccination at one day of age,. but efforts to combine conventional vaccines have not proven satisfactory to date due to competition and immunosuppression between pathogens.

The multivalent HVT-based vaccines engineered in this invention represent a novel way to simultaneously vaccinate against a number of different pathogens. For the first time, a recombinant HVT with a foreign gene inserted into a non-essential region of the HVT genome is disclosed.

The types of genetic engineering that have been performed on these herpesviruses consist of cloning parts of the virus DNA into plasmids in bacteria, reconstructuring the virus DNA while in the cloned 15 state so that the DNA contains deletions of certain sequences, and furthermore adding foreign DNA sequences either in place of the deletions or at sites removed from the deletions.

A foreign gene of interest targeted for insertion into the genome of HVT may be obtained from any pathogenic organism of interest. Typically, the gene of interest will be derived from pathogens that in poultry cause diseases that have an economic impact on the poultry industry. The genes may be derived from organisms for which there are existing vaccines, and because of the novel advantages of the vectoring technology the HVT derived vaccines will be superior. Also, the gene of interest may be derived from pathogens for which there is currently .no vaccine but where there is a requirement for control of the disease. Typically, the gene of interest encodes immunogenic polypeptides of the pathogen, and may represent surface proteins, secreted proteins and structural proteins.

8 A relevant avian pathogen that is- a target for HVT vectoring is Infectious Laryngotracheitis virus (ILTV) ILTV is a member of the herpesviridiae family, and this pathogen causes an acute disease of chickens which is characterized by respiratory depression, gasping and expectoration of bloody exudate. Viral replication is limited to cells of the respiratory tract, where in the trachea the infection gives rise to tissue erosion and hemorrhage. In chickens, no drug has been effective in reducing the degree of lesion formation or in decreasing clinical signs. Vaccination of birds with various modified forms of the ILT virus derived by cell

S

passage and/or tedious regimes of administration have 15 conferred acceptable protection in susceptible chickens. Because of the degree of attenuation of current ILT vaccines care must be taken to assure that the correct level of virus is maintained; enough to provide protection, but not enough to cause disease in the flock.

.eaa An additional target for the HVT vectoring approach is Newcastle disease, an infectious, highly contagious and debilitating disease that is caused by the Newcastle disease virus (NDV). NDV is a single-stranded

RNA

virus of the paramyxovirus family. The various pathotypes of NDV (velogenic, mesogenic, lentogenic) differ with regard to the severity of the disease, the specificity and symptoms, but most types seem to infect the respiratory system and the nervous system. NDV primarily infects chickens, turkeys and other avian species. Historically vaccination has been used to prevent disease, but because of maternal antibody interferences, life-span of the bird and route of administration, the producer needs to adapt immunization protocols to fit specific needs.

The therapeutic agent that is delivered by a viral vector of the present invention must be a biological molecule that is a by-product of swinepox virus replication. This limits the therapeutic agent in the first analysis to either DNA, RNA, or protein. There are examples of therapeutic agents from each of these classes of compounds in the form of anti-sense DNA, anti-sense RNA Joshi, et al., J. of Virology, 1991), ribozymes Wachsman, et al., J. of General Virology, 1989), suppressor tRNAs Bhat, et al., Nucleic Acids Research, 1989), interferon-inducing S 15 double stranded RNA and numerous examples of protein therapeutics, from hormones, insulin, to lymphokines, interferons and interleukins, to naturals opiates. The discovery of these therapeutic agents and the elucidation of their structure and 20 function does not make obvious the ability to use them in a viral vector delivery system.

a SUMMARY OF THE INVENTION This invention provides a recombinant herpesvirus of turkeys comprising a foreign DNA sequence encoding a cytokine inserted into an insertion region which comprises a XhoI site within a EcoR1 #9 fragment of a herpesvirus of turkeys viral genome, and the foreign DNA sequence encoding a cytokine which is capable of being expressed in a host cell infected with the herpesvirus of turkeys.

Lastly, this invention provides homology vectors for producing a recombinant herpesvirus of turkeys, host cells, and vaccines and methods for immunization.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1c: Details of HVT Construction and Map Data.

Figure 1A shows BamHI restriction fragment map of the HVT genome. Fragments are numbered in order of decreasing size; letters refer to small fragments whose comparative size has not been determined.

Figure 1B shows BamHI #16 fragment of the HVT genome showing location of 3 -galactosidase gene S 15 insertion in S-HVT-001.

Figure 1C shows BamHI #19 fragment of the HVT genome showing location of P-galactosidase gene insertion.

Legend: B BamHI; X XhoI; H HindIII; P PstI; S Sal; N NdeI; R EcoRI.

Figures 2A-2D: Insertion in Plasmid 191-47.

Figure 2A contains a diagram showing the orientation of DNA fragments assembled in plasmid 191-47. Figures 2A to 2D show the sequences located at each of the junctions between the DNA fragments in plasmid 191-47. (SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, and 27).

Fiures 3A-3B: Details of S-HVT-003 Construction.

Figure 3A shows restriction map of HVT DNA in the region of the BamHI #16 fragment. This fragment is contained within large HindIII fragment. Figure 3A also shows the XhoI site which was first changed to an EcoRI site by use of a "linker" and standard cloning procedures. Figure 3A also shows details of the construction of the beta-gal gene and IBVD gene inserted into the BamHI #16 fragment for use in homologous recombination.

Both genes were under the control of the PRV gX gene promoter (gX).

Figure 3B show the S-HVT-003 genome, including the location of the two inserted foreign genes, -gal and IBDV.

:In Figure 3 H HindIII; B BamHI; X XhoI; 15 R EcoRI; Xb XbaI; Hp HpaI; S SmaI; UL unique long region; US unique short region; IR internal repeat region; TR terminal repeat region.

20 Ficure 4: Western blot indicating the differential expression of the IBDV 32kD antigen in cellular lysates of S-HVT-003 infected cells (32kD present) and S-HVT-001 infected cells (32kD negative).

IBDV specific polypeptides were identified by probing the blot with hyper-immune rat antiserum directed against denatured IBDV virions. This serum reacts primarily with the immunodominant 32kD antigen (IBDV VP3). The lanes on the blot contain: 1) protein molecular weight standards, 2) uninfected CEF cells, 3) S-HVT-001 infected CEF's, 4) 5) 6) S-HVT-003 and 7) IBDV virion polypeptides.

Figure Western blot indicating the differential expression of the 42kD (VP2) antigen in cellular lysates of S-HVT-003 infected cells (42kD present) and S-HVT-001 infected cells (42kD negative).

IBDV specific polypeptides were identified using a VP2 specific rabit anti-peptide antiserum. The lanes contain: 1) protein molecular weight standards, 2) wild-type HVT infected CEF's, 3) S- HVT-001 infected CEF's, 4) S-HVT-003 infected CEF's, 5) S-HVT-003 infected CEF's, and 6) IBDV virion polypeptides.

Fieures 6A-6C: Details of S-HVT-004 Construction.

Figure 6A is a restriction map of HVT DNA in the region of the BamHI #16 fragment. This fragment is contained within a large HindIII fragment.

Shown also is the XhoI site where applicants have made their insertion. Before the insertion, the XhoI was first changed to EcoRI site by use of a "linker" and standard cloning procedures.

Figure 6B provides details of the construction of the #-gal gene and MDV gA gene inserted into the i BamHI #16 fragment for use in homologous recombination. Beta-gal was under the control of the PRV gX gene promoter while the MDV gA gene was under the control of its own promoter.

Figure 6C is of S-HVT-004 genome showing the location of the two inserted foreign genes, #-gal and MDV gA.

In Figure 6, H HindIII; B BamHI; X XhoI; R EcoRI; Xb XbaI; UL unique long region; US unique short region; IR internal repeat region; TR terminal repeat region.

Figures 7A-7B: Detailed description of the P-galactosidase (lacZ) marker gene insertion in homology vector 467- 22.A12. Figure 7A shows a diagram indicating the orientation of DNA fragments assembled in the marker gene. The origin of each fragment .is described in the Materials and Methods section.

Figures 7A and 7B show the DNA sequences located at the junctions between DNA fragments and at the ends of the marker gene (SEQ ID NOs: 28, 29, 31, 32, and 33). Figures 7A and 7B further show the restriction sites used to generate each DNA fragment at the appropriate junction and the location of the lacZ gene coding region. Numbers 15 in parenthesis refer to amino acids, and restriction sites in brackets indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, pseudorabies virus (PRV), lactose operon 2 20 gene (lacZ), Escherichia coli (E.Coli), polyadenylation signal and glycoprotein X (gpX).

Fiqure 8: BamHI, NotI restriction map of the HVT genome.

The unique long (UL) and unique short (US) regions are shown. The long and short region repeats are indicated by boxes. The BamHI fragments are numbered in decreasing order of size. The location of probes P1-P4 are indicated. The origin of each probe is as follows: P1 BamHI P2 BamHI P3 BamHI #13, and P4 4.0 kb BgIII to StuI sub-fragment of HVT genomic XbaI fragment #5 (8.0 kb).

Figure 9: Shows the Procedure for construction of plasmid pSY229.

Figures 10A-10B: Detailed description of the MDV gene cassette insert in Homology Vectors 456-18.18 and 456- 17.22. Figure 10A and 10B show a diagram indicating the orientation of DNA fragments assembled in the cassette and the location of the MDV gA and gB genes. The origin of each fragment is described in the Materials and Methods section.

The sequences located at the junctions between each fragment and at the ends of the marker gene are shown in Figures 10A and 10B, including 15 junction A (SEQ ID NO: 34), junction B (SEQ ID NO: and junction C (SEQ ID NO: 36). The restriction sites used to generate each fragment are indicated at the appropriate junction.

Numbers in parenthesis refer to amino acids, S 20 and restriction sites in brackets indicate the remnants of sites which were destroyed during construction.

Fiqures 11A-11B: Detailed description of the HindIII fragment insert in Homology Vector 556-41.5. The diagram of Figures 11A and 11B show the orientation of DNA fragments assembled in the cassette. The origin of each fragment is described in the Materials and Methods section. Figures 11A and 11B further show the DNA sequences located at the junctions between each DNA fragment of the plasmid and at the ends of the marker gene, including junction A (SEQ ID NO: 37), junction B (SEQ ID NO: 38), and junction C (SEQ ID NO: 39). The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the MDV gD and a portion of the gI gene is also given.

Numbers in parenthesis refer to amino acids, and restriction sites in brackets indicate the remnants of sites which were destroyed during construction.

Figures 12A-12C: Detailed description of the SalI fragment insert in Homology Vector 255-18.B16. Figure 12A shows a diagram indicating the orientation of DNA fragments assembled in the cassette. The origin of each fragment is described in the Materials and Methods section. Figures 12A to 12C further show the DNA sequences located at the junctions between 15 each fragment and at the ends of the marker gene are shown, including junction A (SEQ ID NO: junction B (SEQ ID NO: 41), junction C (SEQ ID NO: junction D (SEQ ID NO: 43), junction E (SEQ ID NO: 44), junction F(SEQ ID NO: 45), junction G 20 (SEQ ID NO: 46), and junction H (SEQ ID NO: 47).

The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the.NDV F and lacZ-NDV "i HN hybrid gene are shown. Numbers in'parenthesis refer to amino acids, and restriction sites in brackets indicate the remnants of sites which were destroyed during construction.

Figures 13A-13B: Show how the unique XhoI site of the BamHI fragment of the HVT genome was converted into a PacI site and a NotI site by insertion of the synthetic DNA sequence at the XhoI site (Nucleotides #1333-1338; SEQ ID NO. 48). Figure 13A shows the Xho site converted into a PacI site to generate Plasmid 654-45.1 (SEQ ID NO. 55) and Figure 13B shows the Xhol site converted into a NotI site to generate Plamid 686-63.Al (SEQ ID NO.

56).

Fiqure 14: Restriction map and open reading frames of the sequence surrounding the insertion site within the unique long of HVT (SEQ ID NO. 48). This map shows the XhoI restriction site (SEQ ID NO. 48; Nucl.

1333-1338) used for insertion of foreign genes.

Also shown are four open reading frames within this sequence. ORF A is interrupted by insertion :0oa o* "of DNA into the XhoI site. The ORF A amino acid sequence (SEQ ID NO. 50; Nucl. 1402 to 602; 267 amino acids) shows no significant sequence 15 identity to any known amino acid sequence in the protein databases..UL 54 (SEQ ID NO. 49; Nucl. 146 to 481; 112 amino acids) and UL55 (SEQ ID NO. 51; 0.0: Nucl. 1599 to 2135; 179 amino acids) show significant sequence identity to the herpes 20 simplex virus type I UL54 and UL55 proteins, respectively. ORF B (SEQ ID NO. 52; Nucl. 2634 to 2308; 109 amino acids) shows no significant sequence identity to any known amino acid sequence S: in the protein databases. Searches were performed s.

on NCBI databases using Blast software.

Fiqure Restriction map of cosmids 407-32.1C1, 672-01.A40, 672-07.C40, and 654-45.1. The overlap of HVT genomic DNA fragments EcoRI #9 and BamHI #10 is illustrated. A unique XhoI site within the EcoRI #9 and BamHI #10 fragments has been converted to a unique PacI site in Plasmid 654-45.1 or a unique NotI site in Plasmid 686-63.Al.

17 DETAILED DESCRIPTION OF THE INVENTION This invention provides a recombinant herpesvirus of turkeys (HVT) comprising a foreign DNA sequence inserted into a non-essential site in the HVT genome.

The foreign DNA sequence is capable of being expressed in a host cell infected with the recombinant HVT and its expression is under the control of a promoter located upstream of the foreign DNA sequence.

As defined herein "a non-essential site in the HVT genome" means a region in the HVT viral genome which is i: not necessary for the viral infection or replication.

15 As defined herein, "viral genome" or "genomic DNA" means the entire DNA which the naturally occurring herpesvirus of turkeys contains. As defined herein, "foreign DNA sequence" or "gene" means any DNA or gene that is exogenous to the genomic DNA.

As defined herein, an "open reading frame" is a segment of DNA which contains codons that can be transcribed into RNA which can be translated into an amino acid sequence and which does not contain a termination codon.

The invention further provides several appropriate insertion sites in the HVT genome useful for constructing the recombinant herpesvirus of the present invention. Insertion sites include the EcoRI #9 fragment and the BamHI #10 fragment of the HVT genome, a preferred insertion site within both of those fragments being a XhoI restriction endonuclease.

Another such site is the BamHI #16 fragment of the HVT genome. A preferred insertion site within the BamHI #16 fragment lies within an open reading frame encoding 18 UL43 protein and a preferred insertion site within that open reading frame in a XhoI restriction endonuclease site.

Yet another insertion site is the HVT US2 gene, with a preferred insertion site within it being a StuI endonuclease site.

This invention provides a recombinant herpesvirus of 10 turkeys comprising a herpesvirus of turkeys viral genome which contains a foreign DNA sequence inserted within the EcoR1 #9 fragment of the herpesvirus of turkeys viral genome, and the foreign DNA sequence is capable of being expressed in a host cell infected with 15 the herpesvirus of turkeys.

In one embodiment, the foreign DNA sequence is inserted within an Open Reading Frame A (ORFA) of the EcoR1 #9 fragment. Insertion of foreign DNA sequences into the XhoI site of EcoRl #9 interrupts ORFA indicated that the entire ORFA region is non-essential for replication of the recombinant.

For purposes of this invention, "a recombinant herpesvirus of turkeys" is a live herpesvirus of turkeys which has been generated by the recombinant methods well known to those of skill in the art, e.g., the methods set forth in DNA TRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUS in Materials and Methods, and the virus has not had genetic material essential for the replication of the herpesvirus of turkeys deleted. The purified herpesvirus of turkeys results in stable insertion of foreign DNA sequences or a gene in the EcoRl #9 fragment or BamHl #10 fragment.

The invention further provides recombinant herpesvirus of turkeys where the foreign DNA sequence encodes a polypeptide which is antigenic in an animal into which the recombinant herpesvirus is introduced.

In one embodiment the polypeptide is a detectable marker. For purposes of this invention, a "polypeptide which is a detectable marker" includes the bimer, trimer and tetramer form of the polypeptide. E. coli B-galactosidase is a tetramer composed of four polypeptides or monomer subunits. In one embodiment 10 the polypeptide is E. coli beta-galactosidase.

Preferably this recombinant herpesvirus of turkeys is designated S-HVT-001, S-HVT-014, or S-HVT-012.

S-HVT-012 has been deposited on October 15, 1992 15 pursuant to the Budapest Treaty on the International Deposit of Microorganism for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, S. Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR. 2382.

S-HVT-014 has been deposited on December 7, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganism for the Purposes of Patent Procedure with the Patent Culture Depository of. the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR. 2440.

In another embodiment the foreign DNA sequence encodes a cytokine. In another embodiment the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN). In a preferred embodiment the recombinant herpesvirus of turkeys is designated S-HVT- 144.

The invention further provides a recombinant herpesvirus of turkeys whose viral genome contains foreign DNA encoding an antigenic polypeptide which is from Marek's disease virus (MDV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV) or infectious bursal disease virus (IBDV).

This invention provides a recombinant herpesvirus of turkeys with a foreign DNA sequence insertion in the 10 EcoR1 #9 fragment which further comprises a foreign DNA sequence encoding the antigenic polypeptide selected from the group consisting of: Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus and. infectious 15 bursal disease virus.

In one embodiment the foreign DNA sequence encoding the antigenic polypeptide is from Marek' s disease virus and encodes Marek's disease virus glycoprotein gA, Marek's disease virus glycoprotein gB or Marek's disease virus glycoprotein gD. In another embodiment the foreign DNA sequences encoding the Marek's disease virus glycoprotein gA, glycoprotein gB or glycoprotein gD are inserted into the unique StuI site of the US2 gene coding region of the herpesvirus of turkeys.

The invention further provides recombinant herpesvirus of turkeys whose genomic DNA contains foreign DNA encoding antigenic polypeptide from Marek's disease virus. Preferably, the antigenic polypeptide is Marek's disease virus glycoprotein gB, gA or gD.

In one embodiment a recombinant HVT containing a foreign DNA sequence encodes IBDV VP2, MDV gA, and MDV gB. Preferably, such recombinant virus is designated S-HVT-137 and S-HVT-143.

The invention further provides recombinant herpesvirus of turkeys whose genomic DNA contains foreign DNA encoding Marek's disease virus glycoprotein gA and further comprising foreign DNA encoding a polypeptide which is a detectable marker. Preferably, this recombinant herpesvirus of turkeys is designated S-HVT- 004.

:The invention further provides recombinant herpesvirus 10 of turkeys whose genomic DNA contains foreign DNA encoding Marek's disease virus glycoprotein gB.

Preferably, this recombinant herpesvirus of turkeys is designated S-HVT-045.

15 An embodiment of a recombinant HVT containing a foreign DNA sequence encoding MDV gB is also provided and this recombinant HVT is designated S-HVT-045. S-HVT-045 has been deposited on October 15, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganism for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR.

2383.

The present invention also provides recombinant HVTs engineered to contain more than one foreign DNA sequence encoding an MDV antigen. For example, a foreign DNA sequence encoding MDV gA and gB can both be vectored into the HVT genome. Furthermore, a recombinant HVT can be constructed to include a foreign DNA sequence encoding MDV gA, gB, and gD.

Recombinant HVT designated S-HVT-046 and S-HVT-047 provide embodiments of a recombinant HVT containing foreign DNA sequence encoding MDV gA and gB; recombinant HVT designated S-HVT-048 and S-HVT-062 provide embodiments of a recombinant HVT containing foreign DNA sequence encoding MDV gA, gB and gD.

S-HVT-062 has been deposited on February 23, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Paten Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession 10 No. VR. 2401.

The present invention provides a recombinant HVT containing a foreign DNA sequence encoding an antigenic polypeptide from Newcastle disease virus (NDV). In 15 such case, it is preferred that the antigenic polypeptide is Newcastle disease virus fusion (F) protein or Newcastle disease virus hemagglutininneuraminidase or a recombinant protein comprising E. coli B-galactosidase fused to Newcastle disease virus hemagglutinin-neuraminidase One example of such a virus is designated S-HVT-007.

The present invention also provides recombinant HVTs engineered to contain one or more foreign DNA sequence encoding an antigenic polypeptide form MDV as well as one or more foreign DNA sequences encoding an antigenic polypeptide from NDV. Preferably, the MDV antigenic polypeptide is MDV gB, gD, or gA and the NDV F or HN.

In one embodiment of the invention, the recombinant HVT contains foreign DNA sequence encoding MDV gB, MDV gA and NDV F. Preferably, this HVT is designated S-HVT- 048.

In one embodiment of the invention, the recombinant HVT contains foreign DNA sequence encoding MDV gB, MDV gA and NDV HN. Preferably, this HVT is designated S-HVT- 049.

For example, a foreign DNA sequence encoding MDV gA and gB can both be vectored into the HVT genome.

Furthermore, a recombinant HVT can be constructed to include a foreign DNA sequence encoding MDV gA, gB, and gD.

Further, in another embodiment the foreign DNA sequence 10 encoding the antigenic polypeptide is from Newcastle disease virus and encodes Newcastle disease virus fusion protein or Newcastle disease virus hemagglutinin-neuraminidase. In another embodiment the foreign DNA sequences encoding the Newcastle disease 15 virus fusion protein or Newcastle disease virus hemagglutinin-neuraminidase are inserted into a Xhol site in EcoR1 #9 of the unique long region of the herpesvirus of turkeys. In a preferred embodiment the recombinant herpesvirus of turkeys is designated S-HVT- 136.

*oo The invention further provides recombinant herpesvirus of turkeys whose genomic DNA contains foreign DNA encoding antigenic polypeptide from Marek's disease virus and further comprising foreign DNA encoding antigenic polypeptide form Newcastle disease virus.

The present invention further provides a recombinant HVT which contains a foreign DNA sequence encoding an antigenic polypeptide from Marek's disease virus glycoprotein gB and Marek's disease virus glycoprotein gA and further comprising foreign DNA encoding Newcastle disease virus fusion protein.

Preferably, this recombinant herpesvirus of turkeys is designated S-HVT-048.

The invention further provides recombinant herpesvirus of turkeys whose genomic DNA contains foreign DNA encoding Marek's disease virus glycoprotein gB and Marek's disease virus glycoprotein gA and further comprising foreign DNA encoding Newcastle disease virus hemagglutinin-neuraminidase (HN) Preferably, this recombinant herpesvirus of turkeys is designated S-HVT- 049.

The invention further provides recombinant herpesvirus 10 of turkeys whose genomic DNA contains foreign DNA encoding Marek's disease virus glycoprotein gB and Marek's disease virus glycoprotein gA and further comprising foreign DNa encoding Newcastle disease virus fusion protein and Newcastle disease virus 15 hemagglutinin-neuraminidase Preferably, this recombinant herpesvirus of turkeys is designated S-HVT- 050.

9 S-HVT-050 has been deposited on February 23, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purpose of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR. 2400.

In yet another embodiment of the invention, the recombinant HVT contains foreign DNA sequence encoding MDV gB, MDV gA, MDV gD, NDV F and NDV HN. Preferably, such recombinant HVT is designated S-HVT-106 or S-HVT 128.

The invention further provides recombinant herpesvirus Further, in one embodiment the foreign DNA sequence encodes the antigenic polypeptide from an infectious laryngotracheitis virus and encodes infectious laryngotracheitis virus glycoprotein gB, infectious laryngotracheitis virus glycoprotein gI or infectious laryngotracheitis virus glycoprotein gD.

In another embodiment the foreign DNA sequence encodes an antigenic polypeptide which is derived or derivable from a group consisting of: MDV gA, MDV gB, MDV gD, NDV HN, NDV F, ILT gB, ILT gI, ILT gD, IBV, IBDV VP2, IBDV VP3, IBDV VP4, avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, 10 avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia virus (agent), Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes, trematodes, poultry 15 mites/lice, poultry protozoa.

The invention further provides a recombinant herpesvirus of turkeys which contains a foreign DNA o' sequence encoding an antigenic polypeptide from infectious laryngotracheitis virus. It is preferred that the antigenic polypeptide is ILTV glycoprotein gB, ILTV gD or ILTV gI.

Also provided are recombinant HVTs which are engineered to contain more than one foreign DNA sequence encoding an ILTV antigen. For example, ILTV gB and gD can be vectored together into the HVT genome, so can ILTV gD and gI, and ILTV gB, gD and gI. Recombinant HVT designated S-HVT-051, S-HVT-052, and S-HVT-138 are embodiments of such recombinant virus.

The present invention also provides a recombinant HVT which contains more than one foreign DNA sequence encoding an antigenic polypeptide from MDV as well as one or more foreign DNA sequences encoding an antigenic polypeptide from ILTV. Preferably, the MDV antigenic polypeptide is MDV gB, gD or gA and the ILTV antigenic polypeptide is ILTV gB, gD or gI.

In one embodiment of the invention, the recombinant HVT contains foreign DNA sequences encoding MDV gB, MDV gA, MDV gD, ILTV gD and ILTV gB. Preferably, this recombinant HVT is designated S-HVT-123.

In another embodiment of this invention, the Srecombinant HVT contains foreign DNA sequences encoding 10 MDV gB, MDV gA, MDV gD, ILTV gland ILTV gD.

Preferably, this recombinant HVT is designated S-HVT- 139 or S-HVT-140.

The invention further provides recombinant herpesvirus 15 of turkeys whose genomic DNA contains foreign DNA encoding Marek's disease virus glycoprotein gB, Mareck's disease virus glycoprotein gA, and Marek's disease virus glycoprotein gD and further comprising foreign DNA which encodes infectious laryngotracheitis virus glycoprotein gD, infectious laryngotracheitis virus glycoprotein gB, and E. coli B-galactosidase.

Preferably, this recombinant herpesvirus of turkeys is designated S-HVT-104.

The invention further provides recombinant herpesvirus of turkeys whose genomic DNA contains foreign DNa encoding infectious bronchitis virus spike protein or infectious bronchitis virus matrix protein.

The present invention further provides a recombinant HVT which contains a foreign DNA sequence encoding an antigenic polypeptide from infectious bronchitis virus (IBV). Preferably, the antigenic polypeptide is IBV spike protein or IBV matrix protein.

The present invention also provides a recombinant HVT which contains one or more foreign DNA sequences encoding an antigenic polypeptide from IBV as well as one or more foreign DNA sequences encoding an antigenic polypeptide from MDV. Preferably, the IBV antigenic polypeptide is IBV spike protein or IBV matrix protein, and the MDV antigenic polypeptide is MDV gB, gD or gA.

One embodiment of such recombinant virus is designated S-HVT-066.

The invention further provides a recombinant herpesvirus of turkeys whose genomic DNA contains foreign DNA encoding antigenic polypeptide from infectious bursal disease virus and further comprising foreign DNA encoding a polypeptide which is a detectable marker.

Further, in one embodiment a foreign DNA sequence Sencoding the antigenic polypeptide is from infectious bursal disease virus. In another embodiment the foreign DNA sequence encodes infectious bursal disease virus VP2 gene. In another embodiment the foreign DNA sequence encodes infectious bursal disease virus VP3 gene. In another embodiment the foreign DNA sequence encodes infectious bursal disease virus VP4 gene.

Preferably, this recombinant herpesvirus of turkeys is S: 25 designated S-HVT-003 or S-HVT-096.

Recombinant HVT designated S-HVT-003 and S-HVT-096 are each an embodiment of a recombinant HVT comprising foreign DNA sequence encoding antigenic polypeptide from IBDV and encoding a detectable marker. S-HVT-003 has been deposited on July 21, 1987 pursuant to the Budapest Treaty on the International Deposit of Microorganism for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR.

2178.

This invention provides a recombinant herpesvirus of turkeys containing a foreign DNA sequence inserted into the EcoR1 #9 fragment herpesvirus of turkeys viral genome wherein the foreign DNA sequence is from an infectious laryngotracheitis virus and encodes infectious laryngotracheitis virus glycoprotein gB, or infectious laryngotracheitis virus glycoprotein gD.

In one embodiment the foreign DNA sequence is from an infectious laryngotracheitis virus and encodes infectious laryngotracheitis virus glycoprotein gD, or laryngotracheitis virus glycoprotein gI.

S" This invention provides a recombinant herpesvirus of 15 turkeys containing a foreign DNA sequence inserted into the EcoR #9 fragment herpesvirus of turkeys viral genome wherein the foreign DNA sequence is from an Newcastle disease virus and encodes a Newcastle disease vius HN or Newcastle disease virus F.

This invention provides a recombinant herpesvirus of turkeys containing a foreign DNA sequence inserted into the EcoRl #9 fragment herpesvirus of turkeys viral genome wherein the foreign DNA sequence is from an 25 infectious bursal virus and encodes an infectious bursal disease virus VP2, VP3, VP4.

This invention provides a recombinant herpesvirus of turkeys containing a foreign DNA sequence inserted into the EcoRl #9 fragment herpesvirus of turkeys viral genome wherein the foreign DNA sequence is from an infectious bronchitis virus and encodes an infectious bronchitis virus matrix protien.

In another embodiment a foreign DNA sequence encodes an antigenic polypeptide which is derived or derivable from a group consisting of: MDV gA, MDV gB, MDV gD, NDV HN, NDV F, ILT gB, ILT gI, ILT gD, IBV, IBDV VP2, IBDV VPD3, IBDV VP4, avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia virus (agent), Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa. In a preferred embodiment the recombinant herpesvirus of turkeys is designated S- HVT-136.

Such antigenic polypeptide may be derived or derivable from the following: feline pathogen, canine pathogen, 15 equine pathogen, bovine pathogen, avian pathogen, porcine pathogen, or human pathogen.

In another embodiment, the antigenic polypeptide of a human pathogen is derived from human herpesvirus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicell-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, human immunodeficiency virus, rabies virus, measles virus, hepatitis B virus and hepatitis C virus.

25 Furthermore, the antigenic polypeptide of a human pathogen may be associated with malaria or malignant tumor from the group consisting of Plasmodium falciparum, Bordetella pertusis, and malignant tumor.

The invention further provides recombinant herpes virus of turkeys whose genomic DNA contains foreign DNA encoding Newcastle disease virus fusion protein and further comprising foreign DNA encoding a recombinant protein, wherein E. coli B-galactosidase is fused to Newcastle disease virus hemagglutinin-neuraminidase

(HN).

The invention further provides recombinant herpesvirus of turkeys whose genomic DNA contains foreign DNA encoding Marek's disease virus glycoprotein gB and Marek's disease virus glycoprotein gA and further comprising foreign DNA encoding Newcastle disease virus hemagglutinin-neuraminidase (HN).

This invention provides a recombinant herpesvirus of turkeys-Marek's disease virus chimera comprising a herpesvirus of turkeys unique long viral genome region and a Marek's disease virus unique short region. In one embodiment the recombinant herpesvirus of turkeys- Marek's disease virus chimera contains a foreign DNA sequence inserted within the EcoRl #9 fragment of the 15 herpesvirus of turkeys viral genome, and the foreign DNA sequence capable of being expressed in a host cell infected with the herpesvirus of turkeys.

In one embodiment the recombinant herpesvirus of turkeys contains a foreign DNA sequence which encodes a polypeptide. The polypeptide may be antigenic in an animal into which the recombinant herpesvirus is introduced.

25 In another embodiment the polypeptide is E. coli betagalactosidase. In another embodiment the foreign DNA sequence encodes a cytokine. In another embodiment the cytokine is chicken mylomonocytic growth factor (cMGF) br chicken interferon (cIFN).

The invention further provides recombinant herpesvirus of turkeys where the foreign DNA sequence encodes a polypeptide which is antigenic in an animal into which the recombinant herpesvirus is introduced.

Further, the recombinant herpesvirus of turkeys further comprises a foreign DNA sequence encoding the antigenic polypeptide selected from the group consisting of: Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus and infectious bursal disease virus.

This invention provides a recombinant herpesvirus.of turkeys wherein the foreign DNA sequence is under control of an endogenous upstream herpesvirus promoter.

In one embodiment the foreign DNA sequence is under control of a heterologous upstream promoter. In another embodiment the promoter is selected from PRV gX, HSV-1 alpha 4, HCMV immediate early, MDV gA, MDV gB, MDV gD, ILT gB, BHV-1.1 VP8 and ILT gD.

15 This invention provides a homology vector for producing a recombinant herpesvirus of turkeys by inserting foreign DNA into the viral genome of a herpesvirus of turkey which comprises a double-stranded DNA molecule consisting essentially of: a) double stranded foreign DNA not usually present within the herpesvirus of turkeys viral genome; b)at one end the foreign DNA, double-stranded herpesvirus of turkeys DNA homologous to the viral genome located at one side of the EcoRL #9 site the coding region of the herpesvirus of turkeys 25 viral genome; and c) at the other end of the foreign DNA, double-stranded herpesvirus of turkeys DNA homologous to the viral genome located at the other side of the EcoR1 #9 fragment of the coding region of the herpesvirus of turkeys viral genome. Examples of the homology vectors are designated 751-87.A8 and 761- 7.A1.

In one embodiment the polypeptide is antigenic in the animal into which the recombinant herpesvirus of turkeys is introduced. In another embodiment the antigenic polypeptide is from a cytokine, Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus, or infectious bronchitis virus. In a preferred embodiment the antigenic polypeptide is a chicken mylomonocytic growth factor (cMGF) or chicken interferon (cIFN), infectious bursal disease virus polyprotein, infectious bursal disease virus VP2 protein, Marek's disease virus glycoprotein gB, Marek's disease virus glycoprotein gA, Marek's disease virus glycoprotein gD, Newcastle disease virus fusion protein, Newcastle disease virus hemagglutininneuraminidase, infectious laryngotracheitis virus glycoprotein gB, infectious laryngotracheitis virus glycoprotein gD, infectious bronchitis virus spike protein, or infectious bronchitis virus matrix protein.

15 In another embodiment the double stranded foreign DNA sequence in the homology vector encodes an antigenic polypeptide derived from an equine pathogen. The antigenic polypeptide of an equine pathogen can derived from equine influenza virus or equine herpesvirus.

Examples of such antigenic polypeptide are equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidaseequine

S

25 herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D.

In another embodiment the double stranded foreign DNA sequence of the homology vector encodes an antigenic polypeptide derived from bovine respiratory syncytial virus or bovine parainfluenza virus. The antigenic polypeptide of derived from bovine respiratory syncytial virus equine pathogen.can derived from equine influenza virus is bovine respiratory syncytial virus attachment protein '(BRSV bovine respiratory syncytial virus .fusion protein (BRSV bovine respiratory syncytial virus nucleocapsid protein (BRSV bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase.

In another embodiment the double stranded foreign DNA sequence in the homology vector encodes a cytokine capable of stimulating human immune response. For example, the cytokine may be, but is not limited to, interleukin-2, interleukin-6, interleukin-12, interferons, granulocyte-macrophage colony stimulating factors, and interleukin receptors.

In one-embodiment of the invention, the double-stranded herpesvirus of turkeys DNA is homologous to DNA 15 sequences present within the BamHI #16 fragment of the herpesvirus of turkeys genome. Preferably, the doublestranded herpesvirus of turkeys DNA is homologous to DNA sequences present within the open reading frame encoding UL 43 protein of the herpesvirus of turkeys genome. Preferably, this homology vector is designated 172-29.31.

For purposes of this invention, a "homology vector" is a plasmid constructed to insert foreign DNA in a 25 specific site on the genome of a herpesvirus of turkeys.

In one embodiment of the invention, the double-stranded herpesvirus of turkeys DNA is homologous to DNA sequences present within the EcoRl #9 fragment of the herpesvirus of turkeys genome. Preferably, this homology vector is designated 172-63.1.

In one embodiment of the invention, the double-stranded herpesvirus of turkeys DNA is homologous to DNA sequences present within the US2 gene coding region of the herpesvirus of turkeys genome. Preferably, this homology vector is designated 435-47.1.

In another embodiment the foreign DNA sequence encodes a screenable marker. Examples of screenable markers, inlcude but are not limited to: E. coli B-galactosidase or E. coli B-glucuronidase.

The invention further provides a vaccine which comprises an effective immunizing amount of a recombinant herpesvirus of turkeys of the present invention and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird against Marek's disease virus which comprises an 15 effective immunizing amount of the recombinant Oo *O herpesvirus of turkeys and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird against Newcastle disease virus which comprises an effective immunizing amount of the recombinant herpesvirus of turkeys and a suitable carrier.

o This invention provides a vaccine useful for immunizing a bird against infectious laryngotracheitis virus which S. 25 comprises an effective immunizing amount of the recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird against infectious bronchitis virus which comprises an effective immunizing amount of the recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird against infectious bursal disease virus which comprises an effective immunizing amount of the recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a multivalent vaccine useful for immunizing a bird against Marek's disease virus and Newcastle disease virus which comprises an effective immunizing amount of the recombinant herpesvirus of turkeys.

This invention provides a multivalent vaccine useful for immunizing a bird against Marek's disease virus and infectious laryngotracheitis virus which comprises an effective immunizing amount of the recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a multivalent vaccine useful for immunizing a bird against Marek's disease virus and infectious bronchitis virus which comprises an effective immunizing amount of the recombinant herpesvirus.of turkeys and a suitable carrier.

4 S This invention provides a multivalent vaccine useful for immunizing a bird against Marek's disease virus and infectious bursal disease virus which comprises an 25 effective immunizing amount of the recombinant herpesvirus of turkeys and a suitable carrier.

The present invention also provides a method of immunizing a fowl. For purposes of this invention, this includes immunizing a fowl against infectious bursal disease virus, Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus, or infectious bronchitis virus. The method comprises administering to the fowl an effective immunizing dose of the vaccine of the present invention. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneous, intraperitoneal or intravenous injection. Alternatively, the vaccine may be administered intranasally or orally.

This invention provides a host cell infected with the recombinant herpesvirus of turkey. In one embodiment the host cell is an avian cell.

For purposes of this invention, a "host cell" is a cell 10 used to propagate a vector and its insert. Infecting the cell was accomplished by methods well known to those skilled in the art, for example, as set forth in DNA TRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUS in Materials and Methods. Methods for constructing, selecting and purifying recombinant herpesvirus of turkeys are detailed below in This invention provides a method of distinguishing chickens or other poultry which are vaccinated with the 20 above vaccine from those which are infected with a naturally-occurring Marek's disease virus which comprises analyzing samples of body fluids from chickens or other poultry for the presence of glycoprotein gG and at least one other antigen normally expressed in chickens or other poultry infected by a naturally-occurring Marek's disease virus, the presence of those antigens normally expressed in infected chickens but the absence of glycoprotein gG being indicative of vaccination with the above vaccine and not infection with a naturally-occurring Marek's disease virus.

This invention provides a recombinant herpesvirus of turkeys which expresses foreign DNA sequences is useful as vaccines in avian or mammalian species including but not limited to chickens, turkeys, ducks, feline, canine, bovine, equine, and primate, including human.

This vaccine may contain either inactivated or live recombinant virus.

For purposes of this invention, an "effective immunizing amount" of the recombinant feline herpes virus of the present invention is within the range of 103 to 109 PFU/dose. In another embodiment the immunizing amount is 10 5 to 10 7 PFU/dose. In a preferred embodiment the immunizing amount is 106 PFU/dose.

The method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneous, intraperitoneal or intravenous injection.

Alternatively, the vaccine may be administered intranasally or orally.

Suitable carriers for the recombinant virus are well known to those skilled in the art and include but are not limited to proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as hydrolyzed proteins, lactose, etc.

Preferably, the live vaccine is created by taking tissue culture fluids and adding stabilizing agents such as stabilizing, hydrolyzed proteins. Preferably, the inactivated vaccine uses tissue culture fluids directly after inactivation of the virus.

This invention is further illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS: Materials and Methods PREPARATION OF HERPESVIRUS OF TURKEYS STOCK SAMPLES.

Herpesvirus of turkeys stock samples were prepared by infecting tissue culture cells at a multiplicity of infection of 0.01 PFU/cell in Dulbecco's Modified Eagle 10 Medium (DMEM) containing 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components are obtained from Irvine Scientific or an equivalent supplier, and hereafter are referred to as complete DME medium) plus 1% fetal bovine serum. After cytopathic effect was complete, the medium and cells were harvested and the cells were pelleted at 3000 rpm for minutes in a clinical centrifuge. Infected cells were resuspended in complete medium containing fetal bovine serum, 10% DMSO and stored frozen at S 20 70 0

C.

PREPARATION OF HERPESVIRUS OF TURKEY DNA. All manipulations of herpesvirus of turkey (HVT) were made using strain FC-126 (ATCC #584-C) For the preparation of HVT viral DNA from the cytoplasm of infected cells, primary chicken embryo fibroblasts were infected at a MOI sufficient to cause extensive cytopathic effect before the cells overgrew. All incubations were carried out at 39 0 C in a humidified incubator with

CO

2 in air. Best DNA yields were obtained by harvesting monolayers which were maximally infected, but showing incomplete cell lysis (typically 5-7 days).

Infected cells were harvested by scraping the cells into the medium using a cell scraper (Costar brand).

The cell suspension was centrifuged at 3000 rpm for minutes at 5 0 C in a GS-3 rotor (Sorvall Instruments).

The resultant pellet was resuspended in cold PBS ml/Roller Bottle) and subjected to another centrifugation for 10 minutes at 3000 rpm in the cold.

After decanting the PBS, the cellular pellet was resuspended in 4 ml/roller bottle of RSB buffer (10 mM Tris pH 7.5, 1 mM EDTA, and 1.5 mM MgCl 2 (Nonidet P-40";Sigma) was added to the sample to a final concentration of 0.5% minutes with occasional mixing. The sample was centrifuged for 10 minutes at 3000 rpm in the cold to pellet the nuclei and remove cellular debris. The supernatant fluid was carefully transferred to a 15 ml Corex centrifuge tube. Both EDTA (0.5M pH 8.0) and SDS (sodium dodecyl sulfate; stock 20%) were added to the sample to final concentrations of 5 mM and respectively. One hundred Al of proteinase-K (10 mg/ml; Boehringer Mannheim) was added per 4 ml of sample, mixed, and incubated at 45 0 C for 1-2 hours. After this period, an equal volume of water-saturated phenol was added to the sample and gently mixed by hand. The sample was spun 20 in a clinical centrifuge for 5 minutes at 3000 rpm to separate the phases. NaAc was added to the aqueous phase to a final concentration of 0.3M (stock solution 3M pH and the nucleic acid precipitated at -70 0

C

for 30 minutes after the addition of 2.5 volumes of cold absolute ethanol. DNA in the sample was pelleted by spinning for 20 minutes to 8000 rpm in an HB-4 rotor at 5 0 C. The supernatant was carefully removed and the DNA pellet washed once with 25 ml of 80% ethanol. The DNA pellet was dried briefly by vacuum (2-3 minutes), and resuspended in 50 Al/roller bottle of infected cells of TE buffer (10 mM Tris pH 7.5, 1 mM EDTA).

Typically, yields of viral DNA ranged between 5-10 ig/roller bottle of infected cells. All viral DNA was stored at approximately 100C.

POLYMERASE.FILL-IN REACTION. DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KC1, 5 mM MgCi 2 and 400 micromolar each of the four deoxynucleotides. Ten units of Klenow DNA polymerase (BRL) were added and the reaction was allowed to proceed for 15 minutes at room temperature. The DNA was then phenol extracted and ethanol precipitated as above.

DNA SEQUENCING. Sequencing was performed using the USB Sequenase Kit and 35 S-dATP (NEN). Reactions using both 10 the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, compressed areas were resolved on formamide gels.

Templates were double-stranded plasmid subclones or single stranded M13 subclones, and primers were either made to the vector just outside the insert to be sequenced, or to previously obtained sequence.

Sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with Superclone and 20 Supersee programs from Coral Software.

MOLECULAR BIOLOGICAL TECHNIQUES. Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels, ligation, phosphorylation with kinase, treatment with phosphatase, growth of bacterial cultures, transformation of bacteria with DNA, and other molecular biological methods are described by Maniatis et al (1982) and Sambrook et al (1989). The polymerase chain reaction (PCR) was used to introduce restriction sites convenient for the manipulation of various DNAs.

The procedures used are described by Innis et al (1990). In general amplified fragments were less than 500 base pairs in size and critical regions of amplified fragments were confirmed by DNA sequencing.

Except as noted, these techniques were used with minor variation.

SOUTERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis et al.

(1982). DNA was blotted to nitrocellulose filters (S&S in 20X SSC (1X ssc 0.15M NaC1, 0.015M sodium citrate, pH and prehybridized in hybridization solution consisting of 30% formamide, 1X Denhardt's solution (0.02% polyvinylpyrrolidone (PVP), 0.02% 10 bovine serum albumin (BSA), 0.02% Ficoll), 6X SSC, mM NaH 2 PO,, pH 6.8, 200 tg/ml salmon sperm DNA for 4-24 hours at 55 0 C. Labeled probe DNA was added that had been labeled by nick translation using a kit from Bethesda Research Laboratories (BRL) and one "P-labeled nucleotide. The probe DNA was separated from the unincorporated nucleotides by NACS column (BRL) or on a Sephadex G50 column (Pharmacia). After overnight hybridization at 55 0 C, the filter was washed once with 2X SSC at room temperature followed by two washes with 20 0.1X SSC, 0.1% sodium dodecyl sulfate (SDS) for minutes at 550C. The filter was dried and autoradiographed.

9* cDNA CLONING PROCEDURE. cDNA cloning refers to the methods used to convert RNA molecules into DNA molecules following state of the art procedures.

Applicants' methods are described in (Gubler and Hoffman, 1983). Bethesda Research Laboratories (Gaithersburg, MD) have designed a cDNA Cloning Kit that is very similar to the procedures used by applicants, and contains a set of reagents and protocols that may be used to duplicate our results.

For cloning virus mRNA species, a host cell line sensitive to infection-by the virus was infected at plaque forming units per cell. When cytopathic effect was evident, but before total destruction, the medium was removed and the cells were lysed in 10 mis lysis buffer (4 M guanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0, 0.5% N-lauroyl sarcosine, 0.1 M beta-metcaptoethanol). The cell lysate was poured into a sterilized Dounce homogenizer and homogenized on ice 8-10 times until the solution was homogenous. For RNA purification, 8 mis of cell lysate were gently layered over 3.5 mls of CsCl solution (5.7 M CsCi, 25 mM sodium citrate pH 7.0) in 10 Beckman SW41 centrifuge tube. The samples were centrifuged for 18 hrs at 200 C at 36000 rpm in a Beckman SW41 rotor. The tubes were put on ice and the supernatants from the tubes were carefully removed by aspiration to leave the RNA pellet undisturbed. The pellet was resuspended in 400 Ml glass distilled water, and 2.6 mis of guanidine solution (7.5 M guanidine-HCL, 25 mM sodium citrate pH 7.0, 5 mM dithiothreitol) were added. The 0.37 volumes of 1 M acetic acid were added, followed by 0.75 volumes of cold ethanol and the sample 20 was put at -20° C for 18 hrs to precipitate RNA. The precipitate was collected by centrifugation in a Sorvall centrifuge for 10 min a 40 C at 10000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, recentrifuged at 13000 rpm, and the supernatant saved. RNA was re-extracted from the pellet 2 more times as above with 0.5 ml distilled water, and the supernatants were pooled. A 0.1 volume of 2 M potassium acetate solution was added to the sample followed by 2 volumes of cold ethanol and the sample was put at -20° C for 18 hrs. The precipitated RNA was collected by centrifugation in the SS34 rotor at 40 C for 10 min at 10000 rpm. The pellet was dissolved in 1 ml distilled water and the concentration taken by absorption at A260/280. The RNA was stored at -70 0

C.

mRNA containing polyadenylate tails (poly-A) was selected using oligo-dT cellulose (Pharmacia #27 5543- Three mg of total RNA was boiled and chilled and applied to the 100 mg oligo-dT cellulose column in binding buffer (0.1 M Tris pH 7.5, 0.5 M LiC1, 5mM EDTA pH 8.0, 0.1% lithium dodecyl sulfate). The retained poly-A RNA was eluted from the column with elution buffer (5mM Tris pH 7.5, ImM EDTA pH 8.0, 0.1% sodium dodecyl sulfate). This mRNA was reapplied to an oligodT column in binding buffer and eluted again in elution buffer. The sample was precipitated with 200 mM sodium acetate and 2 volumes cold ethanol at -20 0 C for 18 hrs.

S" The RNA was resuspended in 50 Al distilled water.

S" Ten fg poly-A RNA was denatured in 20 mM methyl mercury 15 hydroxide for 6 min at 22°C. 9-mercaptoethanol was added to 75 mM and the sample was incubated for 5 min at 22 0 C. The reaction mixture for first strand cDNA synthesis in 0.25 ml contained 1 Ag oligo-dT primer (P- L Bio-chemicals) or 1 Mg synthetic primer, 28 units 20 placental ribonuclease inhibitor (Bethesda Research Labs #5518SA), 100 mM Tris pH 8.3, 140 mM KCl, S...MgCl 2 0.8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia), 100 microcuries 3 p-labeled dCTP (New England Nuclear #NEG-013H), and 180 units AMV reverse transcriptase (Molecular Genetics Resources #MG 101). The reaction was incubated at 42 0 C for 90 min, and then was terminated with 20mM EDTA pH 8.0. The sample was extracted with an equal volume of phenol/chloroform and precipitated with 2 M ammonium acetate and 2 volumes of cold ethanol -20 0 C for 3 hrs. After precipitation and centrifugatioA, the pellet was dissolved in 100 Al distilled water. The sample was loaded onto a 15 ml G-100 Sephadex column (Pharmacia) in buffer (100 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 100 mM NaC1). The leading edge of the eluted DNA fractions was pooled, and DNA was concentrated by lyophilization until the volume was about 100 g1, then the DNA was precipitated with ammonium acetate plus ethanol as above.

The entire first strand sample was used for second strand reaction which followed the Gubler and Hoffman (1983) method except that 50 /g/ml dNTP's, 5.4 units DNA polymerase I (Boerhinger Mannheim #642-711), and 100 units/ml E. coli DNA ligase (New England Biolabs #205) in a total volume of 50 microliters were used.

After second strand synthesis, the cDNA was phenol/chloroform extracted and precipitated. The DNA was resuspended in 10 pl distilled water, treated with 1 Ag RNase A for 10 min at 22 0 C, and electrophoresed through a 1% agarose gel (Sigma Type II agarose) in mM Tris-acetate pH 6.85. The gel was stained with ethidium bromide, and DNA in the expected size range was excised from the gel and electroeluted in 8 mM Tris-acetate pH 6.85. Electroeluted DNA was lyophilized to about 100 microliters, and precipitated with ammonium acetate and ethanol as above. The DNA was resuspended in 20 Al water.

Oligo-dC tails were added to the DNA to facilitate cloning. The reaction contained the DNA, 100 mM 25 potassium cacodylate pH 7.2, 0.2 mM dithiothreitol, 2mM CaC,, 80 Amoles dCTP, and 25 units terminal deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 1l. After 30 min at 37 0 C, the reaction was terminated with 10mM EDTA, and the sample was phenol/chloroform extracted and precipitated as above.

The dC-tailed DNA sample was annealed to 200 ng plasmid vector pBR322 that contained oligo-dG tails (Bethesda Research Labs #5355 SA/SB) in 200 pl of 0.01 M Tris pH 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65 0 C for 2 min and then 57 0 C for 2 hrs. Fresh competent E. coli DH-1 cells were prepared and transformed as described by Hanahan (1983) using half the annealed cDNA sample in twenty 200 Al aliquots of cells. Transformed cells were plated on L-broth agar plates plus 10 jg/ml tetracycline. Colonies were screened for the presence of inserts into the ampicillin gene using Ampscreen (Bethesda Research Labs #5537 UA), and the positive colonies were picked for analysis.

DNA TRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUS. The method is based upon the polybrene- DMSO procedure of Kawai and Nishizawa (1984) with the following modifications. Generation of recombinant HVT virus is dependent upon homologous recombination 15 between HVT viral DNA and the plasmid homology vector containing the desired foreign DNA flanked by the appropriate herpesvirus cloned sequences.

Transfections were carried out in 6 cm plates (Corning plastic) of 50% confluent primary chick embryo fibroblast (.CEF) cells. The cells were plated out the day before in CEF growth media (1X F10/199, 5% fetal calf serum, 2% glutamine, 1% non-essential amino acids, and 2% penicillin/streptomycin) containing 4 Ag/ml Spolybrene (stock 4 mg/ml in 1X HBSS). For 25 cotransfections into CEF cells, 5 pg of intact HVT DNA, and suspended in 1 ml of CEF media containing 30 g/ml polybrene (stock 4 mg/ml in IX HBSS). The DNApolybrene suspension (1 ml) was then added to a 6 cm plate of CEF cells from which the media had been aspirated, and incubated at 39 0 C for 30 minutes. The plates were rocked periodically during this time to redistribute the inoculum. After this period, 4 ml of CEF growth media was added directly to wash plate, and incubated an additional 2.5 hours a 39°C. At this time, the media was removed from each plate, and the cells shocked with 2 ml of 30% DMSO (Dimethyl Sulfoxide, J.T. Baker Chemical Co.) in IX HBSS for 4 minutes at room temperature. The 30% DMSO was carefully removed and the monolayers washed once with 1X HBSS at room temperature. The cells were then incubated at 39 0 C after the addition of 5 mis of CEF growth media. The next day, the media was changed to remove any last traces of DMSO and to stimulate cell growth. Cytopathic effect from the virus becomes apparent within 6 days. Generation of a high titer stock (80%-90% CPE) can usually be made within 1 week from this date. HVT stock samples were prepared by resuspending the infected cells in CEF growth media containing 20% fetal calf serum, 10% DMSO and stored at 15 PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS

FROM

SUBGENOMIC DNA FRAGMENTS. The ability to generate herpesviruses by cotransfection of cloned overlapping subgenmoic fragments has been demonstrated for pseudorabies virus (Zijl et al., 1988). If deletions and/or insertions are engineered directly into the subgenomic fragments prior to the cotransfection, this procedure results in a high frequency of viruses containing the genomic alteration, greatly reducing the amount of screening required to purify the recombinant 25 virus. This procedure was used to construct recombinant

HVT.

A library of subclones containing overlapping

HVT

subgenomic fragments was generated as follows. HVT DNA was obtained from the American Type Culture Collection (FC-126("Calnek)). It was sheared and then size selected on a glycerol gradient as described by van Zijl et al.,(19 8 8 with 40-50 kb fragments chosen as the insert population. The pooled fractions were diluted twofold with TE, one-tenth volume of 3M NaAc and 2.5 volumes of ethanol were added, and the DNA was precipitated at 30K rpm in a Beckman SW41 rotor for 1 hr. The sheared fragments were given blunt ends by initial treatment with T4 DNA polymerase, using low DNTP concentrations to promote 3' overhang removal, followed by treatment with Klenow polymerase to fill in recessed 3' ends. These insert fragments were then ligated to a pWE15 (Strategene) cosmid vector, which had been digested with BamHI, treated with calf intestinal phosphatase, and made blunt by treatment with Klenow polymerase. The ligated mixture was then packaged using Gigapack XL packaging extracts (Stratagene). Ligation and packaging was as recommended by the manufacturer.

S

Published restriction maps for the enzymes BamHI, 15 HindIII, and XhoI permitted the use of subcloned Sfragments as specific probes to screen the cosmid library for subclones spanning the genome. Probes were generated from subcloned restriction fragments. The fragments were then labeled using a non-radioactive system (Genius, Boehringer Mannheim). Screening was facilitated by picking colonies to media followed by growth overnight. Sets of five filters and a master plate were stamped from microtiter dish and again grown overnight. Glycerol was added to the wells to 15% and 25 the plates were frozen at -20 0 C to provide stock cultures of each colony. Filters were BioRad Colony Lift Membranes and were treated and hybridized per manufacturer's instructions, and washed in 0.1X SSC, 0.1% SDS, 65 0 C. Clones which hybridized with the nonradioactive probe were detected according to the Genius kit directions.

Colonies were selected for further analysis on the basis of their hybridization to two or more of the specific probes. These were then digested with BamHI, and compared to published maps of HVT (Buckmaster et al., 1988). The three cosmids (407-32.2C3,407-32.IG7, 49 and 407-32.5G6) were obtained in this manner. A detailed description of each clone is given below. It was found that chloramphenicol amplification (Maniatis et al.,1982) was necessary to achieve reasonable yields of DNA from these clones. In addition, one cosmid clone (407-32.5G6) was unstable and had to be grown from the original frozen stock in order to obtain satisfactory DNA preparations.

The pWE15 vector allows the inserts to be excised with NotI. However, four NotI sites are present in the HVT genome, so that inserts spanning these sites cannot be excised with NotI. Two of the NotI sites are present in the BamHI #2 fragment of HVT, this fragment was 15 cloned directly in pSP64. The other two sites are present in the unique short region within the BamHI #1 fragment. This fragment was cloned directly in the vector. The three sheared cosmids and the two BamHI fragments cover all but a small portion of the ends of the HVT genome. Because these regions are repeated in the internal portions of the genome, all of the genetic information is available.

A StuI site within the HVT US2 gene was established as 25 a useful site for foreign DNA insertion utilizing the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSES (see Example The HVT US2 gene is located within the BamHI #1 fragment which contains five StuI sites. To facilitate the use of this site for insertion of foreign DNA by the StuI site within the US2 gene was converted to a unique HindIII site. This was accomplished by partially digesting the BamHI #1 subclone with StuI, and then inserting a 10 kb fragment conferring kanomycin resistance (Neo") into the site using HindIII linkers. The kanomycin resistance gene allowed positive selection of recombinant clones. The Neo' fragment was removed by digestion with HindIII followed by religation generating clone 430-84.215.

DNA was prepared for reconstruction experiments by restriction digestion with enzymes which cut the subclones outside or flanking the HVT insertions. In some instances, one cosmid in a reconstruction was used undigested. Digested DNAs were extracted once with phenol and precipitated with ethanol. DNA was resuspended at a concentration of 0.5 to 1 ug/ml. Viral reconstruction experiments were performed using Lipofectin (BRL) to mediate transfection. Two to three 15 micrograms each of subclone were added to 0.5 ml of MEM media (Earle's salts) supplemented with 1% nonessential amino acids and 2% penicillin/Streptomysin Controls consisted of MEM+ with no DNA, with several ug of HVT DNA, or with 4 out of 5 of the subclones. Separately, 30 pl of the Lipofectin were added to another 0.5 ml. of MEM+. These two mixtures were then combined and incubated at RT for 15 minutes.

Chick embryo fibroblast (CEF) cells were prepared for 25 transfection in the following manner. CEFs (Spafas) were grown in 6 well dishes at 39 0 C in F10/M199 (1:1) media containing 1% non-essential amino acids, 2% penicillin/streptomycin, and 5% fetal calf serum Cells were transfected at a confluence of 90 95%. For transfection, wells were aspirated and rinsed 3 times with MEM+, and then incubated 4 hours at 39 0

C

with the 1 ml lipofectin/DNA mixture above. One ml more of CEF+ was then added to the wells, and cells were incubated overnight and fed with CEF+. Plates were then examined daily for the appearance of plaques.

Lipofectin with control HVT DNA resulted in the appearance of plaques within 5 days. When only four of the five subclones were used, no plaques were obtained.

When the five overlapping genomic fragments of HVT were used to reconstruct the virus, plaques appeared anywhere from 5 to 19 days after the initial lipofection. In the case of plaques appearing late, plaques were not initially seen on the infected monolayer, and it was only after passaging the monolayer and replating on a larger surface that plaques appeared. After passaging, plaques generally appeared within 3 days. Recombinant viruses were plaque purified approximately three and then analyzed for insertion of foreign DNAs.

15 BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. When the foreign gene encoded the enzyme 0-galactosidase, the plaques that contained the gene were visualized more easily. The chemical Bluogal" (Bethesda Research Labs) was incorporated at the level of 200-300 pg/ml into the agarose overlay during the plaque assay, and the -plaques that expressed active P-galactosidase turned blue.. The blue plaques were then picked and purified by further blue plaque isolations. Other foreign genes were inserted by homologous recombination such that 25 they replaced the -galactosidase gene; in this instance non-blue plaques were picked for purification of the recombinant virus.

SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT EVT USING BLACK PLAQUE ASSAYS. To analyze expression of foreign antigens expressed by recombinant HVT viruses, monolayers of CEF cells are infected with recombinant HVT, overlaid with nutrient agarose media and incubated for 4-5 days at 39 0 C. Once plaques have developed, the agarose overlay is removed from the dish, the monolayer rinsed Ix with PBS, fixed with 100% methanol for minutes at room temperature and the cells air dried.

After re-hydrating the plate with PBS, the primary antibody is diluted to the appropriate dilution with PBS and incubated with the cell monolayer for 2 hours to overnight at room temperature. Unbound antibody is then removed from the cells by washing three times with PBS at room temperature. An alkaline phosphatase conjugated secondary antibody is diluted with PBS and incubated with the cells for 2 hours at room temperature. Unbound secondary antibody is then removed by washing the cells three times with PBS at room temperature. Next, the monolayer is rinsed in color development buffer (100mM Tris pH 9.5/ 100mM NaCl/ 5mM MgCl2), and then incubated 10 minutes to overnight at room temperature with freshly prepared 15 substrate solution (0.3 mg/ml Nitro Blue tetrazolium 0.15 mg/ml 5-Bromo-4-Chloro-3-Indolyl Phosphatase in color development buffer.) Finally, the reaction is stopped by replacing the substrate solution with TE (10mM Tris, pH7.5/ 1 mM EDTA). Plaques expressing the correct antigen will stain black.

PLAQUE HYBRIDIZATION PROCEDURE FOR ASSESSING THE PURITY OF RECOMBINANT EVT STOCKS. When no suitable immunological reagent exists to detect the presence of 25 a particular antigen in a recombinant HVT virus, plaque hybridization can be used to assess the purity of a stock. Initially, CEF cell monolayers are infected with various dilutions of the viral stocks to give 100 plaques/1 0 cm.dish, overlaid with nutrient agarose media and incubated for 4-5 days at 39 0 C. Once plaque development occurs, the position of each plaque is marked on bottom of the dish. The agarose overlay is then removed, the plate washed with PBS, and the remaining CEF monolayer is transferred to a NC membrane or BioRad nylon membrane pre-wetted with PBS (making note of the membrane position relative to the dish).

Cells contained on the NC membranes are then lysed by placing the membranes in 1.5 mis of 1.5M NaCi and NaOH for five minutes. The membranes are neutralized by placing them in 1.5 mis of 3M Sodium acetate (pH 5.2) for five minutes. DNA from the lysed cells is then bound to the NC membranes by baking at 80 0 C for one hour. After this period the membranes are prehybridized in a solution containing 6X SSC, 3% skim milk, 0.5% SDS, salmon sperm DNA (50 pg/ml) for one hour at 650C. Radio-labeled probe DNA (alpha 32P-dCTP) is then added and the membranes incubated at 65 0

C

overnight (-12 hours). After hybridization the NC membranes are washed two times (30 minutes each) with 2X SSC at 65 0 C, followed by two additional washes at 650C with 0.5X SSC. The NC membranes are then dried 15 and exposed to X-ray film (Kodak X-OMAT,AR) at -70 0

C

for 12 hours. Positive signals are then aligned with the position of the plaques on the dish and purity of the stock is recorded as the percentage of positive plaques over the total.

CONSTRUCTION OF HOMOLOGY VECTOR FOR INSERTION OF THE BETA-GALACTOSIDASE GENE INTO HVT US2 GENE. The betagalactosidase (lacZ) gene was inserted into the HVT EcoRI 7. fragment at the unique StuI site. -The marker 25 gene is oriented in the same direction as the US2 gene.

A detailed description of the marker gene is given in Figures 7A and 7B. It is constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and sambrook et al, 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures'7A and 7B. Fragment 1 is an approximately 413 base pair Sail to BamHI restriction sub-fragment of the PRV BamHI restriction fragment 10 (Lomniczi et al., 1984). Fragment 2 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (Ferrari et al., 1985).

Fragment 3 is an approximately 754 base pair NdeI to SalI restriction sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi et al., 1984).

RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS: Chicken spleens were dissected from 3 week old chicks from SPAFAS, Inc., washed, and disrupted through a syringe/needle to release cells After allowing stroma and debri to settle out, the cells were pelleted and washed twice with PBS. The cell pellet was treated with a hypotonic lysis buffer to lyse red blood cells, and splenocytes were recovered and washed twice with PBS. Splenocytes were resuspended at 5 x 106 cells/ml in RPMI containing 5% FBS and g. /ml Concanavalin A and incubated at 39 for 48 hours.

15 Total RNA was isolated from the cells using guanidine isothionate lysis reagents and protocols from the Promega RNA isolation kit (Promega Corporation, Madison WI). 4Ag of total RNA was used in each 1st strand reaction containing the appropriate antisense primers and AMV reverse transcriptase (Promega Corporation, Madison WI). cDNA synthesis was performed in the same tube following the reverse transcriptase reaction, using the appropriate sense primers and Vent® DNA polymerase (Life Technologies, Inc. Bethesda, MD).

SUBGENOMIC CLONE 172-07.BA2. Plasmid 172-07.BA2 was constructed for the purpose of generating recombinant HVT. It contains an approximately 25,000 base pair region of genomic HVT DNA. It may be used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This plasmid may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining two restriction fragments from the following sources. The first fragment is an approximately 2999 base pair BamHI to BamHI restriction fragment of pSP64 (Promega). The second fragment is the approximately 25,000 base pair BamHI #2 fragment of HVT (Buckmaster et al.',1988).

HOMOLOGY VECTOR 172-29.31. The plasmid 172-29.31 was constructed for the purpose of inserting foreign DNA into HVT. It contains a unique XhoI restriction enzyme site into which foreign DNA may be inserted. When a plasmid containing a foreign DNA insert at the XhoI site is used according to the DNA COTRANSFECTION

FOR

GENERATING RECOMBINANT HERPESVIRUSES or the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS a virus containing the foreign DNA 15 will result. This plasmid may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining two restriction fragments from the following sources. The first fragment is an approximately 2999 base pair BamHI to BamHI restriction fragment of pSP64 (Promega). The second fragment is the approximately 3300 base pair BamHI #16 fragment of HVT (Buckmaster et al., 1988).

The complete sequence of the BamHI #16 fragment is given in SEQ ID NO:3. Note that the fragment was cloned such that the UL43 ORF is in the opposite transcriptional orientation to the pSP64 -lacatamase gene.

HOMOLOGY VECTOR 172-63.1. The plasmid 172-63.1 was constructed for the purpose of inserting foreign DNA into HVT. It contains a unique XhoI restriction enzyme site into which foreign DNA may be inserted. When a plasmid containing a foreign DNA insert at the XhoI site is used according to the DNA COTRANSFECTION

FOR

GENERATING RECOMBINANT HERPESVIRUSES or the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS a virus containing the foreign DNA will result. This plasmid may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining two restriction fragments from the following sources. The first fragment is an approximately 2999 base pair EcoRI to EcoRI restriction fragment of pSP64 (Promega). The second fragment is the approximately 5500 base pair EcoRI #9 fragment of HVT. Note that the EcoRI fragment was cloned such that the unique XhoI site is closest to the unique HindIII site in the pSP64 vector.

HOMOLOGY VECTORS 255-18.B16. The plasmid 255-18.B16 was constructed for the purpose of inserting the NDV HN and F genes into HVT. The NDV HN and F genes were 15 inserted as a Sail fragment into the homology vector 172-29.31 at the XhoI site. The NDV HN and F genes were inserted in the same transcriptional orientation the UL43 ORF in the parental homology vector. A detailed description of the Sall fragment is shown in Figures 12A-12C. The inserted Sal fragment may be constructed utilizing standard recombinant DNA oo techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining, restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 12A, 12B and 12C. Fragment 1 is an approximately 416 base pair Sail to BamHI restriction sub-fragment of the PRV BamHI restriction fragment 10 (Lomniczi et al., 1984). Fragment 2 is an approximately 3009 base pair BamHI to PvuII fragment of the plasmid pJF751 (Ferrari et al., 1985). Fragment 3 is an approximately 1200 base pair Avail to EcoRI restriction fragment of full length NDV HN cDNA.

Fragment 4 is an approximately 179 base pair EcoRI to PvuII restriction fragment of the plasmid pSP64 (Promega). Fragment 5 is an approximately 357 base pair SmaI to BamHI restriction sub-fragment of the HSV-1 BamHI restriction fragment N. Fragment 6 is an approximately 1812 base pair BamHI to Pstl restriction fragment of the full length NDV F cDNA. Fragment 7 is an approximately 235 base pair PstI to Scal restriction fragment of the plasmid pBR322.

SUBGEMOMIC CLONE 378-50.BA1. Cosmid 378-50.BA1 was constructed for the purpose of generating recombinant HVT. It contains an approximately 29,500 base pair region of genomic HVT DNA. It may be used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This cosmid may be constructed by joining two restriction fragments from the following sources. The first fragment is an oo*oo approximately 8164 base pair BamHI to BamHI restriction fragment of pWE15 (Stratagene) The second fragment is the approximately 29,500 base pair BamHI #1 fragment of HVT (Buckmaster et al., 1988).

•SUBGEMOMIC CLONE 407-32.1C1. Cosmid 407-32.1C1 was oo constructed for the purpose of generating recombinant HVT. It contains an approximately 38,850 base pair region of genomic HVT DNA (see Figure This region includes BamHI fragments 11, 7, 8, 21, 6, 18, approximately 1250 base pairs of fragment 13, and approximately 6,700 base pairs of fragment 1. It may be used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This cosmid maybe constructed as described above in the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. It was isolated from the sheared DNA library by screening with the probes P1 and P4 (described in Figure A bacterial strain containing this cosmid has been deposited on March 3, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. 75428.

SUBGEMOMIC CLONE 407-32.2C3. Cosmid 407-32.2C3 was constructed for the purpose of generating recombinant HVT. It contains an approximately 40,170 base pair region of genomic HVT DNA (see Figure This region includes BamHI fragments 10, 14, 19, 17, 5, and approximately 2,100 base pairs of fragment 2. It may be used in conjunction with other subgenomic clones 15 according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This cosmid may be S. constructed as described above in the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. It was isolated from the.sheared DNA library by screening with the probes P1 and P2 (described in Figure A bacterial strain containing this cosmid has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms 25 for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. 75430.

SUBGEMOMIC CLONE 407-32.5G6. Cosmid 407-32.5G6 was constructed for the purpose of generating recombinant HVT. It contains an approximately 40,000 base pair region of genomic HVT DNA (see Figure This region includes BamHI fragments 9, 3, 20, 12, 16, 13, approximately 1,650 base pairs of fragment 2, and approximately 4,000 base pairs of fragment 11. It may be used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING

RECOMBINANT

HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This cosmid may be constructed as described above in the PROCEDURE

FOR

GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. It was isolated from the sheared DNA library by screening with the probes P2 and P3 (described in Figure A bacterial strain containing this cosmid has been deposited on March 3, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent i Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession S 15 No. 75427.

HOMOLOGY VECTOR 435-47.1. The plasmid 435-47.1 was Sconstructed for the purpose of inserting foreign DNA into HVT. It contains a unique HindIII restriction enzyme site into which foreign DNA may be inserted.

When a plasmid containing a foreign DNA insert at the HindIII site is used according to the DNA COTRANSFECTION FOR GENERATING RECOMBINANT

HERPESVIRUSES

or the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM. OVERLAPPING SUBGENOMIC FRAGMENTS a virus containing the foreign DNA will result. This plasmid may be constructed utilizing standard recombinant

DNA

techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining two restriction fragments from the following sources. The first fragment is an approximately 2999 base pair EcoRI to EcoRI restriction fragment of pSP64 (Promega). The second fragment is the approximately 7300 base pair EcoRI #7 fragment of HVT. Note that the HindIII site of the pSP64 vector was removed by digesting the subclone with HindIII followed by a Klenow fill in reaction and religation.

A

synthetic HindIII linker (CAAGCTTG) was then inserted into the unique StuI site of the EcoRI #7 fragment.

SUBGEMOMIC CLONE 437-26.24. Plasmid 437-26.24 was constructed for the purpose of generating recombinant HVT. It contains an approximately 13,600 base pair region of genomic HVT DNA. It may be used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This plasmid may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining two restriction fragments from the following sources. The first fragment is an 15 approximately 2970 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). The second fragment is the approximately 13,600 base pair BamHI to StuI sub-fragment of the BamHI #2 fragment of HVT (Buckmaster et al., 1988). Note that the BamHI #2 fragment contains five Stul sites, the site utilized in this subcloning was converted to a HindIII site as described in the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS.

25 SUBGEMOMIC CLONE 437-26.26. Plasmid 437-26.26 was constructed for the purpose of generating recombinant HVT. It contains an approximately 15,300 base pair region of genomic HVT DNA. It may be used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This plasmid may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining two' restriction fragments from the following sources. The first fragment is an approximately 2970 base pair HindIII to BamHI 61 restriction fragment of pSP64 (Promega). The second fragment is the approximately 15,300 base pair BamHI to StuI sub-fragment of the BamHI #2 fragment of HVT (Buckmaster et al., 1988). Note that the BamHI #2 fragment contains five StuI sites, the site utilized in this subcloning was converted to a HindIII site as described in the PROCEDURE FOR GENERATING

RECOMBINANT

HERPESVIRUS FROM OVERLAPPING SUBGENOMIC

FRAGMENTS.

HOMOLOGY VECTORS 456-18.18 and 456-17.22. The plasmids 456-18.18 and 456-17.22 were constructed for the purpose of inserting the MDV gA and gB genes into HVT.

The MDV genes were inserted as a cassette into the homology vector 435-47.1 at the unique HindIII site.

15 The MDV genes were inserted at the blunt ended HindIII site as a blunt ended PstI to EcoRI fragment (see Figures 10A and 10B). The HindIII and EcoRI sites were blunted by the Klenow fill in reaction. The PstI site was blunted by the T4 DNA polymerase reaction. Note 20 that the MDV cassette was inserted in both orientations. Plasmid 456-18.18 contains the MDV genes inserted in the opposite transcriptional orientation to the US2 gene in the parental homology vector. Plasmid 456-17.22 contains the MDV genes inserted in the same transcriptional orientation as the US2 gene in :the parental homology vector. A detailed description of the MDV cassette is given in Figures 10A and 10B. It may be constructed utilizing standard recombinant

DNA

techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 10A and 10B. Fragment 1 is an approximately 2178 base pair PvuII to EcoRV restriction sub-fragment of the MDV EcoRI 6.9 KB genomic restriction fragment (Ihara et al., 1989). Fragment 2 is an approximately 3898 base pair SalI to EcoRI genomic MDV fragment (Ross, et al., 1989).

HOMOLOGY VECTOR 528-03.37. The plasmid 528-03.37 was constructed for the purpose of inserting the infectious laryngotracheitis (ILT) virus gD gene into HVT. The gD gene followed by the PRV gX poly adenylation signal was inserted as a cassette into the homology vector 435- 47.1 at the unique HindIII site. The cassette may be constructed utilizing standard recombinant

DNA

techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources. The first fragment is an approximately 2060 base pair EcoRI to BclI restriction sub-fragment of the ILT KpnI genomic restriction fragment #8 (10.6 KB). The second fragment is an approximately 754 base pair NdeI to Sall restriction S: 15 sub-fragment of the PRV BamHI restriction fragment #7 S" (Lomniczi et al., 1984). Note that the fragments are oriented such that BcllI and NdeI sites are contiguous.

20 HOMOLOGY VECTOR 528-11.43. The plasmid 528-11.43 was constructed for the purpose of inserting the infectious laryngotracheitis (ILT) virus gB gene Grifin, 1991) into HVT. The gB gene was inserted as an EcoRI fragment into the homology vector 435-47.1 at the unique HindIII site. The gB gene was inserted at the blunt ended HindIII site as a blunt ended EcoRI fragment. The HindIII and EcoRI sites were blunted by the Klenow fill in reaction. The gB gene was inserted in the same transcriptional orientation as the US2 gene in the parental homology vector. The EcoRI fragment may be obtained as a 3.0 KB ILT virus genomic fragment.

HOMOLOGY VECTOR 518-46.B3. The plasmid 518-46.B3 was constructed for the purpose of inserting foreign DNA into HVT. It contains a unique HindIII restriction enzyme site into which foreign DNA may be inserted.

When a plasmid containing a foreign DNA insert at the HindIII site is used according to the DNA COTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES or the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS a virus containing the foreign DNA will result. This plasmid may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining three restriction fragments from the following sources. The first fragment is an approximately 1649 base pair PvuI to Sall restriction S fragment of pSP64 (Promega). The second fragment is an approximately 1368 base pair Pvul to Sall restriction fragment of pSP65 (Promega). The third fragment is the approximately 3400 base pair XhoI to XhoI fragment of 15 plasmid 437-47.1.

HOMOLOGY VECTOR 535-70.3. The plasmid 535-70.3 was constructed for the purpose of inserting the MDV gB, and gA genes and the NDV F gene into HVT. The F gene 20 was inserted as a cassette into homology vector 456- 17.22 at the HindIII site located between the MDV gA and gB genes (see Junction B, Figure 10A). The F gene is under the control of the HCMV immediate early promoter and followed by the HSV-1 TK poly adenylation signal. The F gene was inserted in the same transcriptional orientation as the US2 gene in the parental homology vector. The cassette may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources. The first fragment .is an approximately 1191 base pair PstI to AvaII restriction sub-fragment of the HCMV genomic XbaI E fragment (D.R.

Thomsen, et al., 1981). The second fragment is an approximately 1812 base pair BamHI to PstI restriction fragment of the full length NDV F cDNA clone (Bl strain). The last fragment is an approximately 784 base 64 pair SmaI to SmaI restriction sub-fragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

HOMOLOGY VECTOR 549-24.15. The plasmid 549-24.15 was constructed for the purpose of inserting the MDV gB, and gA genes and the NDV HN and F genes into HVT. The HN and F genes were inserted as a cassette into homolgy vector 456-17.22 at the HindIII site located between the MDV gA and gB genes (see Junction B, Figure 10A). The HN and F genes are under the control of the PRV gpX and HCMV immediate early promoters respectively. The HN and F genes are followed by the PRV gX poly and HSV-1 TK adenylation -signals respectively. The cassette may be constructed utilizing S 15 standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources. The first fragment is an approximately 413 base pair SalI to BamHI restriction sub-fragment of the PRV BamHI 20 fragment #10 (Lomniczi, et al., 1984) The second 6 5 fragment is an approximately 1811 base pair AvaII to NaeI restriction fragment of the full length NDV HN cDNA clone (Bl strain). The third fragment is an approximately 754 base pair NdeI to SalI restriction sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984). The fourth fragment is an approximately 1191 base pair PstI to AvaII restriction sub-fragment of the HCMV genomic XbaI E fragment (D.R.

Thomsen, et al., 1981). The fifth fragment is an approximately 1812 base pair BamHI to PstI restriction fragment of the full length NDV F cDNA clone (Bl strain). The last fragment is an approximately 784 base pair SmaI to SmalI restriction sub-fragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

HOMOLOGY VECTOR 549-62.10. The plasmid 549-62.10 was constructed for the purpose of inserting the MDV gB, and gA genes and the NDV HN gene into HVT. The HN gene was inserted as a cassette into homolgy vector 456- 17.22 at the HindIII site located between the MDV gA and gE genes (see Junction B, Figure 10A) The HN gene is under the control of the PRV gpX promoter and followed by the PRV gX poly adenylation signal. The HN gene was inserted in the same transcriptional orientation as the US2 gene in the parental homology vector. The cassette may be constructed utilizing 10 standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources. The first fragment is an approximately 413 base pair SalI to BamHI restriction sub-fragment of the PRV BamHI fragment #10 (Lomniczi, et al., 1984) The second fragment is an approximately 1811 base pair Avail to NaeI restriction fragment of the full length NDV HN cDNA clone (Bl strain). The last fragment is an approximately 754 base pair NdeI to Sail restriction 20 sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984).

S".0 SUBGENOMIC CLONE 550-60.6. Plasmid 550-60.6 was constructed for the purpose of generating recombinant HVT. It contains an approximately 12,300 base pair region of genomic HVT DNA. It may be used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT

HERPESVIRUS

FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This plasmid may be constructed utilizing standard recombinant

DNA

techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining two restriction fragments from the following sources. The first fragment is an approximately 4176 base pair EcoRV to BamHI restriction fragment of pBR322. The second fragment is the approximately 12,300 base pair sub-fragment of the BamHI #2 fragment of HVT (Buckmaster et al., 1988).

This fragment was generated in the following manner.

Plasmid 437-26.26 was linearized with HindIII and then resected with the ExoIII Mung Bean Deletion Kit (Stratagene). Samples from the 3 and 4 minute reactions were combined and digested with BamHI resulting in a population of fragments containing the desired 12,300 base pair sub-fragment. This population was cloned into the pBR322 fragment and the resulting clones were 10 screened for the appropriate size and restriction map.

Fortuitously the resected sub-fragment that generated clone 550-60.6 ended in the nucleotides GG which :generated a second BamHI site when ligated to the EcoRV site (ATCC) of pBR322. A bacterial strain containing this plasmid has been deposited on March 3, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, 20 Rockville, Maryland 20852 U.S.A. under ATCC Accession No. 75429.

HOMOLOGY VECTORS 566-41.5. The plasmid 566-41.5 was constructed for the purpose of inserting the MDV gA, gB and gD genes into HVT. The MDV gD gene was inserted as a HindIII fragment into the homology vector 456-17.22 at the HindIII site located between MDV gA and gB (see Figures 10A and 10B) The MDV gene was inserted in the same transcriptional orientation as gA and gB in the parental homology vector. A detailed description of the HindIII fragment containing the MDV gD gene is shown in Figures 11A and 11B. Note that a herpesvirus polyadenation signal was added to the gD gene cassette.

The inserted HindIII fragment may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources with 67 the synthetic DNA sequences indicated in Figures 11A and 11B. Fragment 1 is an approximately 784 base pair SmaI to SmaI restriction sub-fragment of the HSV-1 BamHI restriction fragment Q (McGeoch et al., 1988).

Note that this fragment is oriented such that the polyadenylation sequence (AATAAA) is located closest to junction B. Fragment 2 is an approximately 2177 base pair Sail to NcoI sub-fragment of the MDV BglII 4.2 KB genomic restriction fragment (Ross, et al., 1991).

10 1 0 HOMOLOGY VECTOR 567-72.1D. The plasmid 567-72.1D was constructed for the purpose of inserting the MDV gB, gA, and gD genes and the infectious bronchitis virus (IBV) matrix and spike genes into HVT. The IBV genes were inserted as a cassette into homolgy vector 566- 41.5 at the unique NotI site located upstream of the MDV gD gene (see Junction C, Figure 11B). The IBV spike and matrix genes are under the control of the HCMV immediate early and PRV gpX promoters 20 respectively. The IBV spike and matrix genes are followed by the HSV-1 TK and PRV gX poly adenylation signals respectively. The IBV genes were inserted in the same transcriptional orientation as the US2 gene in the parental homology vector. The cassette. may be constructed utilizing standard recombinant

DNA

techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources. The first fragment is an approximately 413 base pair Sall to BamHI restriction sub-fragment of the PRV BamHI fragment #10 (Lomniczi, et al., 1984) The second fragment contains amino acids 1 to 223 of the IBV matrix gene. The coding region was obtained from a cDNA clone of the Arkansas strain of IBV. The third fragment is an approximately 754 base pair NdeI to Sail restriction sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984).

The fourth fragment is an approximately 1191 base pair 68 PstI to Avail restriction sub-fragment of the HCMV genomic XbaI E fragment Thomsen, et al., 1981).

The fifth fragment contains amino acids 4 to 1162 of the IBV spike gene. The coding region was obtained from a cDNA clone of the Arkansas strain of IBV. The last fragment is an approximately 784 base pair SmaI to SmaI restriction sub-fragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

0 HOMOLOGY VECTOR 603-57.F1. The plasmid 603-57.F1 was constructed for the purpose of inserting the IBDV VP2 gene into HVT. The IBDV VP2 gene was inserted as a cassette into homolgy vector 435-47.1 at the unique HindIII site. The VP2 gene is under the control of the HCMV immediate early promoter and is followed by the HSV-1 TK poly adenylation signal. The VP2 gene was inserted in the same transcriptional orientation as the US2 in the parental homology vector. The cassette may be constructed utilizing standard recombinant

DNA

techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources. The first fragment is an approximately 1191.base pair PstI to AvalI restriction sub-fragment of the HCMV genomic Xbal E fragment

(D.R.

Thomsen, et al., 1981). The second fragment is an approximately 1081 base pair BclI to BamHI restriction sub-fragment of the full length IBDV cDNA clone (see SEQ ID NO:1). Note that the BclI site was introduced into the cDNA clone directly upstream of the VP2 initiator methionine by converting the sequence

CGCAGC

to TGATCA. The first and second fragments are oriented such that Avail and BclI sites are contiguous. The third fragment is an approximately 784 base pair SmaI to SmaI restriction sub-fragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

HOMOLOGY VECTOR 633-13.27. The plasmid 633-13.27 was 69 constructed for the purpose of inserting the MDV gB, gA and gD genes and the NDV HN and F genes into HVT. The HN and F genes are under the control of the PRV gpX and HCMV immediate early promoters respectively. The HN and F genes are followed by the PRV gX poly and HSV-1 TK adenylation signals respectively. All five genes were inserted in the same transcriptional orientation as the US2 gene in the parental homology vector. The genes were inserted in the following order MDV gA, NDV 10 HN, NDV F,MDV gD, and MDV gB.

9 HOMOLOGY VECTOR 634-29.16. The plasmid 634-29.16 was constructed for the purpose of inserting the ILT virus gB and gD genes into HVT. The lacZ marker gene followed by the ILT gB and gD genes inserted as a cassette into the homology vector 172-29.31 at the unique XhoI site.

The cassette may be constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989) by joining restriction fragments 20 from the following sources. The first fragment is an approximately 4229 base pair Sail to Sail restriction fragment derived from the lacZ marker gene described above and shown in Figures 7A and 7B. The second fragment is an approximately 2060 base pair EcoRI to BclI restriction sub-fragment of the ILT KpnI genomic restriction fragment #8 (10.6 KB). The third fragment is an approximately 754 base pair Ndel to SalI restriction sub-fragment of the PRV BanHI restriction fragment #7 (Lomniczi et al., 1984). Note that the second and third fragments are oriented such that Bell and Ndel sites are contiguous. The fourth fragment is the 3.0 KB ILT virus genomic EcoRI fragment containing the gB gene. All three genes are in the same transcriptional orientation as the UL43 gene.

SUBGENOMIC CLONE 415-09.BA1. Cosmid 415-09.BA1 was constructed for the purpose of generating recombinant HVT. It contains an approximately 29,500 base pair BamHI #1 fragment of genomic HVT DNA. It was used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT

HERPESVIRUS

FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT. This cosmid was constructed by joining two restriction fragments (Sambrook, et al., 1989) from the following sources.

The vector is an approximately 4430 base pair BamHI to 10 BamHI restriction fragment of pSY1005 derived from pHC79 (Bethesda Research Labs, Inc.) and (Stratagene, Inc.). The first fragment is the approximately 29,500 base pair BamHI #1 fragment of the HVT genome (Buckmaster et al., 1988).

SUBGENOMIC CLONE 672-01.A40. Cosmid 672-01.A40 was constructed for the purpose of generating recombinant HVT. It was isolated as a subclone of cosmid 407-32.1C1 (see Figures 8 and 15). Cosmid 672-01.A40 contains an 20 approximately 14,000 base pair NotI to AscI subfragment and an approximately 1300 base pair AscI to BamHI subfragment of cosmid 407-32.1C1. The cosmid was constructed by joining restriction fragments (Sambrook, et al., 1989) from the following sources. The vector is an approximately 2700 base pair NotI to BamHI fragment constructed from pNEB193 (New England Biolabs, Inc.) which contains a NotI linker inserted into the SmaI site. Fragment 1 is an approximately 15,300 base pair region of genomic HVT DNA. This region includes BamHI fragments 11 and 7, and approximately 1250 base paris of fragment 13. It was used in conjunction with other subgenomic clones according to the PROCEDURE

FOR

GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant

HVT.

SUBGENOMIC CLONE 654-45.1. Plasmid 654-45.1 was 71 constructed for the purpose of generating recombinant HVT. It was isolated as an AscI subclone of cosmid 407- 32.1C1 (see Figures 8 and 15). The cosmid was constructed by joining restriction fragments (Sambrook, et al., 1989) from the following sources. The vector is an approximately 2000 base pair AscI fragment constructed from a 2000 base pair AatII to PvuII fragment of pNEB 193 (New England Bilabs, Inc.) blunt ended with Klenow DNA polymerase and AscI linkers inserted. Fragment 1 is an approximately 8600 base pair AscI to AscI fragment of genomic HVT DNA. This region includes BamHI fragments 10 and 21, and approximately 1100 base pairs of fragment 6 and S: approximately 1300 base pairs of fragment 7. The XhoI site (Nucleotide #1339-1344; SEQ ID NO. 48) has been converted to a unique PacI site using synthetic DNA linkers. The PacI site was used in insertion and expression of foreign genes in HVT. (See Figure 13A) It was used in conjunction with other subgenomic clones 20 according to the PROCEDURE FOR GENERATING

RECOMBINANT

HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinant HVT.

SUBGENOMIC CLONE 686-63.Al. Plasmid .686-63.A1 was constructed for the purpose of generating recombinant HVT. It was isolated as an AscI subclone of cosmid 407-32.1C1 (see Figure 8, 15). The cosmid was constructed by joining restriction fragments (Sambrooks, et al., 1989) from the following sources.

The vector is an approximately 2000 base pair AscI fragment constructed from a 2000 base pair AatII to PvuII fragment of pNEB193 (New England Biolabs, Inc.) blunt ended with Klenow DNA polymerase and AscI linkers inserted. Fragment 1 is an approximately 8600 base pair AscI to AscI fragment of genomic HVT DNa. This region includes BamHI fragments 10 and 21, and approximately 1100 base pairs of fragment 6 and approximately 1300 base pairs of fragment 7. The XhoI site (Nucleotide #1339-1344; SEQ ID NO. 48) has beenconverted to a unique NotI site using synthetic DNA linkers. The NotI site was used for the insertion and expression of foreign genes in HVT. (See Figure 13B). It was used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING

SUBGENOMIC

FRAGMENTS for the construction of recombinant

HVT.

SSUBGENOMIC CLONE 672-07.C40. Cosmid 672-07.C40 was constructed for the purpose of generating recombinant HVT. It was isolated as a subclone of cosmid 407-32.1C1 (see Figures 8 and 15). Cosmid 672-07.C40 contains an S 15 approximately 1100 base pair BamHI to AscI subfragment and an approximately 13,000 base pair AscI .to NotI g subfragment of cosmid 407-32.1C1. The cosmid was constructed by joining restriction fragments (Sambrook, et al., 1989) from the following sources. The vector 20 is an approximately 2700 base pair NotI to BamHI fragment constructed from pNEB193 New England Biolabs, Inc.) which contains a NotI linker inserted into the SmaI site. Fragment 1 is an approximately 14,100 base pair region of genomic HVT DNA. This region includes BamHI fragments 6 and 18, and an approximately 2600 base pair BamHI to NotI fragment within BamHI fragment It was used in conjunction with other subgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING

SUBGENOMIC

FRAGMENTS for the construction of recombinant

HVT.

SUBGENOMIC CLONE 706-57.A3. Plasmid 706-57.A3 was constructed for the purpose .of generating recombinant HVT. Plasmid 706-57.A3 contains the IBDV VP2 gene inserted into the PacI site of plasmid 654-45.1. The IBDV VP2 gene uses the IBRV VP8 promoter and ILTV US3 polyadenylation signal. The cosmid was constructed 73 utilizing standard recombinant DNA techniques (Sambrook, et al., 1989). The first fragment is a 208 base pair HindIII to BamHI fragment coding for the IBRV VP8 promoter (Carpenter, et al., 1991)). The second fragment is an approximately 1626 base pair fragment coding for the IBDV VP2 gene derived by reverse transcription and polymerase chain reaction (Sambrook, et al., 1989) of IBDV standard challenge strain

(USDA)

genomic RNA (Kibenge, et al., 1990). The antisense 10 primer used for reverse transcription and PCR was CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3' (SEQ ID NO.

53). The sense primer used for PCR was CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3' (SEQ ID NO.

54). The DNA fragment generated by PCR was cloned into 15 the PCR-Direct m vector (Clontech Laboratories, Inc., Pali Alto, CA). The IBDV VP2 fragment was subcloned next tot he VP8 promoter using Bcll sites generated by the PCR primers. The DNA sequence at this junction adds amino acids methionine, aspartate and glutamine 20 before the antive initiator methionine of VP2. The DNA fragment contains the coding sequence from amino acid 1 to amino acid 536 of the IBDV polyprotein (SEQ ID NO: 2) which includes the entire coding sequence of the VP2 protein. The third fragment is an approximately 494 base pair fragment coding for the ILTV US3 polyadenylation signal.

SUBGENOMIC CLONE 711-92.1A. Plasmid 711-92.1A was constructed for the purpose of generating recombinant HVT. Plasmid 711-92.1A contains the ILTV gD and gI genes inserted- into the PacI site of plasmid 654-45.1.

The ILTV gD and gI genes use their respective endogenous ILTV promoters and single shared endogenous polyadenylation signal. The plasmid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989). The first fragment is an approximately 3556 base pair Sall to HindIII restriction subfragment of the ILTV Asp718I genomic fragment #8 (10.6 kb).

SUBGENOMIC CLONE 717-38.12. Plasmid 717-38.12 was constructed for the purpose of generating recombinant HVT. Plasmid 717-38.12 contains the NDV HN and F genes inserted into the PacI site of plasmid 654-45.1. The NDV HN gene uses the PRV gX promoter and the PRV gX polyadenylation signal. The NDV F gene uses the HCMV immediate early promoter and the HSV TK polyadenylation signal. The plamid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989).

The first fragment is an approximately 413 base pair Sall to BamHI restriction subfragment of the PRV BamHI fragment #10 (Lomniczi, et al., 1984). The second fragment is an approximately 1811 base pair AvaII to 01 NaeI restriction fragment of the full length NDV HN cDNA clone (Bl strain). The third fragment is an approximately 754 base pair NdeI to Sall restriction 20 subfragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984). The fourth fragment is an approximately 1191 base pair PstI to AvalI restriction subfragment of the HCMV genomic Xbal E fragment (D.R.

Thomsen, et al., 1981). The fifth fragment is an approximately 1812 base pair BamHI to PstI restriction fragment of the full length NDV F cDNA clone (Bl strain; SEQ ID NO: 12). The sixth fragment is an approximately 784 base pair SmaI to SmaI restriction subfragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

SUBGENOMIC CLONE 721-38.1J. Cosmid 721-38.1J was constructed for the purpose of inserting the MDV gA, gD, and gB genes into the unique short of HVT and for the purpose of generating recombinant HVT. Cosmid 721- 38.1J contains the MDV gA, gD and gB genes inserted into a Stul site in the HVT US2 gene converted to a unique HindIII site within the BamHI #1 fragment of the unique short region of HVT. This region of the HVT BamHI #1 fragment containing the MDV genes was derived from S-HVT-062. Cosmid 721-38.1J was constructed by a partial restriction digest with BamHI of S-HVT-062 DNA and isolation of an approximately 39,300 base pair fragment. The cosmid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989) by joining restriction fragments from the following sources. The vector is an approximately 8200 base pair BamHI fragment from cosmid vector pWE15. The first fragment is an approximately 900 base pair BamHI fragment from the repeat region of the HVT genome. The second fragment is an approximately 15,500 base pair 15 BamHI to StuI subfragment of BamHI #1 of HVT. The third fragment is an approximately 8400 base pair cassette containing the MDV gA, gD, and gB genes (see figures and 11) The fourth fragment is an approximately 14,500 base pair HindIII to BamHI subfragment of the BamHI #1 20 of HVT.

SUBGENOMIC CLONE 722-60.E2. Cosmid 722-60.E2 was constructed for the purpose of inserting the MDV gA, gD, and gB genes and the NDV HN and F genes into the unique short of HVT and for the purpose of generating recombinant HVT. Cosmid 722-60.E2 contains the MDV gA, gD and gB genes and the NDV HN and F genes inserted into a StuI site in the HVT US2 gene converted to a unique HindIII site within the BamHI #1 fragment of the unique short region of HVT. All five genes were inserted in the same transcriptional orientation as the HVT US2 gene. This region of the HVT BamHI #1 fragment containing the MDV and NDV genes was derived from S- HVT-106. Cosmid 722-60.E2 was constructed by a partial restriction digest with BamHI of S-HVT-106 and isolation of an approximately 46,300 base pari fragment. The cosmid was constructed utilizing 76 standard recombinant DNA techniques (Sambrook, et al., 1989) by joining restriction fragments from the following sources. The vector is an approximately 6100 base pair BamHI fragment from cosmid vector pSY1626 derived from pHC79 (Bethesda Research Labs, Inc.) and (Strategene, Inc.). The first fragment is an approximately 900 base pair BamHI fragment from the repeat region of the HVT genome. The second fragment is an approximately 15,500 base pair BamHI to StuI subfragment of BamHI #1 of HVT. The third fragment is an approximately 15,400 base pair cassette containing the MDV gA gene, (Figures 10A and 10B, SEQ ID NO: 8), the PRV gX promoter (Lomniczi et al., 1984), the NDV HN gene (SEQ ID NO: 10), the PRV gX polyadenylation site S 15 (Lomniczi et al., 1984), the HCMV immediate early promoter Thomsen, et al., 1981), the NDV F gene (SEQ ID NO: 12), the HSV TK polyadenylation site (McGeoch, et al., 1985), the MDV gD gene (Figures 11A and the approximately 450 base pair ILTV US3 20 polyadenylation site, and the MDV gB gene (Figures and 10B). The fourth fragment is an approximately 14,500 base pair StuI to BamHI subfragment of the BamHI #1 of HVT.

SUBGENOMIC CLONE 729-37.1. Plasmid 729-37.1 was constructed for the purpose of generating recombinant SHVT. Plasmid 729-37.1 contains the ILTV gD and gB genes inserted into the NotI site of plasmid 686-63.A1. The ILTV gD and gB genes use their respective endogenous ILTV promoters, and the ILTV gD and gB gene are each followed by a PRV gX polyadenylation signals. The ILTV gD and gB gene cassette was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989). The first fragment is an approximately 2052 base pair Sall to XbaI restriction subfragment of the ILTV Asp718I genomic fragment #8 (10.6 kb). The second fragment is an approximately 572 base pair XbaI to 77 Asp718I restriction subfragment of the PRV BamHI restriction fragment #7 (Lomniczi et al., 1984). The third fragment is an approximately 3059 base pair EcoRI to EcoRI restriction fragment of ILTV genomic DNA. The fourth fragment is an approximately 222 base pair EcoRI to Sail restriction subfragment of the PRV BamHI restriction fragment #7 (Lomniczi et al., 1984).

SUBGENOMIC CLONE 739-27.16. Cosmid 739-27.16 was constructed for the purpose of constructing achimeric HVT/MDV virus containing the HVT genes of the unique long region and the MDV type 1 genes of the unique short region. Cosmid 739-27.16 contains the complete unique short region of MDV type 1. This region 15 contians the entire SmaI B fragment and two SmaI K fragments. Cosmid 739-27.16 was constructed by a partial restriction digest with SmaI of MDV DNA and isolation of an approximately 29,000 to 33,000 base pair fragment. The cosmid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989) by joining restriction fragments from the following sources. The vector is an approximately 8200 Sbase pair BamHI fragment (made blunt-ended with Lenow DNa polymerase) from cosmid vector pWE15. The first fragment is an approximately 4050 base pair SmaI K fragment from the short internal repeat region of the S" MDV genome. The second fragment is an approximately 21,000 base pair fragment SmaI B of MDV. The third fragment is an approximately 3,650 base pair Sina K fragment from the short terminal repeat region of the MDV genome (Fukuchi, et al., 1984, 1985).

SUBGENOMIC CLONE 751-87.A8. Plasmid 751-87.A8 was constructed for the purpose of generating recombinant HVT. Plasmid 751-87.AB contains the chicken myelomonocytic growth factor (cGMF) gene inserted into the PacI site of plasmid 654-45.1. The cMGF gene uses the HCMy immediate early promoter and HSV-1 TK polyadenylation signal. The cosmid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989). The following fragments were inserted into the PacI site of HVT subgenomic clone 654-45.1. The first fragment is an approximately 1191 base pair PstI to AvaII restriction subfragment of the HCMV genomic XbaI E fragment Thomsen, et al., 1981). The second fragment is an approximately 640 base pair fragment coding for the cMGF gene (58) derived by reverse transcription and polymerase chain reaction (PCR) (Sambrook, et al., 1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. The antisense primer used for reverse transcription and PCR was 5'-CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3' (SEQ ID NO: 57). The sense primer used for PCR was GAGCGGATCCTGCAGGAGGAGACACAGAGCTG-3' (SEQ ID NO: 58).

The cMGF fragment was subcloned next to the HCMV IE promoter using BamHI sites generated by the PCR primers. The DNA fragment contains the coding sequence from amino acid 1 to amino acid 201 of the cMGF protein (58) which includes a 23 amino acid leader sequence at the amino terminus and 178 amino acids of the mature cMGF protein. The third fragment is -an approximately 25 784 base pair SmaI to SmaI restriction subfragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

SUBGENOMIC CLONE 761-07.A1. Plasmid 761-07.Al was constructed for the purpose of generating recombinant HVT. Plasmid 761-07.A1 contains the chicken interferon gene inserted into the PacI site of plasmid 654-45.1.

The chicken interferon gene uses the HCMV immediate early promoter and HSV-1 TK polyadenylation signal. The cosmid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989). The following fragments were inserted into the PacI site of HVT subgenomic clone 654-45.1. The first fragment is an approximately 1191 base pair PstI to AvaII restriction subfragment of the HCMV genomic XbaI E fragment (D.R.

Thomsen, et al., 1981). The second fragment is an approximately 577 base pair fragment coding for the chicken interferon gene (59) derived by reverse transcription and polymerase chain reaction (PCR) (Sambrook, et al., 1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. The antisense primer used for reverse transcription and PCR was 5' -TGTAGAGATCTGGCTAAGTGCGCGTGTTGCCTG- 3' (SEQ ID NO: 59). The sense primer used for PCR was 5' TGTACAGATCTCACCATGGCTGTGCCTGCAAGC-3' (SEQ ID NO: The chicken interferon gene fragment was subcloned next to the HCMV IE promoter using BglII sites generated by the PCR primers. The DNA fragment contains the coding sequence from amino acid 1 to amino acid 193 of the "*chicken interferon protein (59) which includes a 31 amino acid signal sequence at the amino terminus and 20 162 amino acids of the mature protein encoding chicken interferon. The third fragment is an approximately 784 base pair SmaI to SmaI restriction subfragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985) 1985).

EXAMPLE 1 S-HVT-001 S-HVT-001 is a herpesvirus of turkeys (HVT) that contains the E. coli f-galactosidase gene inserted into the unique long region of the HVT genome. The restriction enzyme map of HVT has been published (T.

Igarashi, et al., 1985). This information was used as a starting point to engineer the insertion of foreign genes into HVT. The BamHI restriction map of HVT is shown in Figure IA. From this data, several different regions of HVT DNA into which insertions of foreign S" genes could be made were targeted. The foreign gene chosen for insertion was the E. coli f-galactosidase (lacZ) gene which was used in PRV. The promoter was the PRV gpX promoter. The lacZ gene was inserted into the unique long region of HVT, specifically into the hoI site in the BamHI #16 (3329bp) fragment, and was 20 shown to be expressed in an HVT recombinant by the formation of blue plaques using the substrate Bluogal" (Bethesda Research Labs). Similarly, the lacZ gene has been inserted into.the Sall site in the-repeat region contained within the BamHI #19 (900 bp) fragment.

These experiments show that HVT is amenable to the procedures described within this application for the insertion and expression of foreign genes in herpesviruses. In particular, two sites for insertion of foreign DNA have been identified (Figs. 1B and 1C).

EXAMPLE 2 S-HVT-003 S-HVT-003 is a herpesvirus of turkeys (HVT) that contains the E. coli 0-galactosidase (lacZ) gene and the infectious bursal disease virus (IBDV) strain S40747 large segment of RNA (as a cDNA copy) (SEQ ID NO: 1) inserted into the unique long region of the HVT genome. This IBDV DNA contains one open reading frame that encodes three proteins (5'VP2-VP4-VP3 (SEQ ID NO: two of which are antigens to provide protection against IBDV infections of chickens. Expression of the genes for both f-galactosidase and the IBDV polyprotein are under the control of the pseudorabies virus (PRV) gpX gene promoter. S-HVT-003 was made by homologous recombination. S-HVT-003 was deposited on July 21, 1987 pursuant to the Budapest Treaty on the International Deposit of Microorganism for Purposes of ooo Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2178.

The IBDV genes were cloned by the cDNA CLONING co0 20 PROCEDURE. Clones representing the genome of IBDV were screened by SOUTHERN BLOTTING OF DNA procedure against blots containing authentic IBDV RNA. Positive clones were then characterized by restriction mapping to identify. groups of clones. Two such clones were S 25 identified, that together were found to represent the entire coding region of the IBDV large segment of RNA (3.3 kb dsRNA). One cDNA clone (2-84) contained an approximately 2500 base pair fragment representing the first half of the IBDV gene. The second clone (2-40) contained an approximately 2000 base pair fragment representing the distal half of the IBDV gene. Plasmid 2-84/2-40, representing the entire IBDV gene, was constructed by joining clone. 2-84 and 2-40 at a unique PvuII site present in the overlapping sequences. The IBDV genome can be obtained from plasmid 2-84/2-40 as an approximately 3400 base pair SmaI to HpaI fragment.

Confirmation of the nature of the proteins encoded by 82 the IBDV gene was obtained by expressing the clone (2- 84/2-40) in E. coli and detecting VP3 antigen using antiserum made against purified IBDV capsid proteins on Western blots. The cDNA of the IBDV large segment of RNA encoding the IBDV antigens show one open reading frame that will henceforth be referred to as the IBDV gene. The sequence of an Australian IBDV strain has been published which bears close homology to applicants' sequence (Hudson et al,1986). Comparison of the amino acid differences between the two viruses revealed 29 amino acid changes within the 1012 amino acid coding region. There were only 3 amino acid differences deduced for VP4 and only 8 in VP3. In contrast, VP2 contained 18 amino acid changes, 14 of which were clustered between amino acids 139 to 332.

For insertion into the genome of HVT, the coding region •for the IBDV gene was cloned between the PRV gpX promoter and the HSV TK poly-A signal sequence, 20 creating plasmid 191-23. To aid in the identification of HVT recombinants made by homologous recombination containing the IBDV gene, the gpX promoted IBDV fragment from plasmid 191-23 was inserted behind (in tandem to) a lacZ gene controlled by a gpX promoter.

The resultant plasmid, 191-47, contains the E.coli lacZ gene and the IBDV gene under the control of individual PRV gpX promoters. In constructing plasmid 191-47, various DNA fragments were joined by recombinant DNA techniques using either naturally occurring restriction sites or synthetic linker DNA. Details concerning the construction of these genes contained in plasmid 191-47 can be seen in Figures 2A, 2B, 2C and 2D.

The first segment of DNA (Segment 1, Figure 2A) contains the gpX promoter region including the residues encoding the first seven amino acids of the gpX gene, and was derived from a subclone of the PRV BamHI 9 9* *9 83 fragment as an approximately 800 base pair Sall to BamHI fragment. The second segment of DNA (Segment 2, Figure 2A) contains the E. coli P-galactosidase coding region from amino acid 10 to amino acid 1024 and was derived from the plasmid pJF751 (obtained from Jim Hoch, Scripps Clinic and Research Foundation) as an approximately 3300 base pair BamHI to Ball fragment followed by an approximately 40 base pair Ava I to Sma I fragment. The third segment of DNA (Segment 3, Figure 2A) contains the gpX poly A signal sequence and was derived from a subclone of the PRV BamHI #7 fragment as an approximately 700 base pair NdeI to StuI fragment. Segment three was joined to segment two by ligating the NdeI end which had been filled in according to the POLYMERASE FILL-IN REACTION, to the SmaI site. The fourth segment of DNA (Segment 4, Figure 2A) contains the gpX promoter (TATA box and cap site) and was derived from a subclone of the PRV BarHI fragment as an approximately 330 base pair Nael to 20 AluI fragment. Additionally, segment four contains approximately 36 base pairs of HSV TK leader sequence as a PstI to BglII fragment in which the PstI site has been joined to the Alul site through the use of a synthetic DNA linker (McKnight and Kingbury, 1982). DNA segments four through six were inserted as a unit into the unique Kpn I site of segment three which is located 3' of the gpX poly A signal sequence. The fifth segment of DNA (Segment Figure 2A) contains the entire coding region of the IBDV large segment of RNA (cDNA clone) as an approximately 3400 base pair SmaI to HpaI fragment.

The SmaI site of segment five was fused to the BglII site of segment four which had been filled in according to the POLYMERASE FILL IN REACTION. Expression of the IBDV gene (5'VP2-VP4-VP3 is under the control of the gpX promoter (segment but utilizes its own natural start and stop codons. The sixth segment of DNA (Segment 6, Figure 2A) contains the HSV TK poly-A signal sequence as an approximately 800 base pair SmaI fragment (obtained from Bernard Roizman, Univ. of Chicago). The HpaI site of segment five was fused to the SmaI site of segment six through the use of a synthetic DNA linker.

In summary, the construct used to create S-HVT-003 (plasmid 191-47) contains to the PRV promoter, the gpX TATA box, the gpX cap site, the first seven amino acids of gpX, the E. coli f-galactosidase (lacZ) gene, the PRV poly-A signal sequence, the PRV gpX promoter, the gpX TATA box, the-gpX cap site, a fusion within the gpX untranslated 5' leader to the IBDV gene, 15 IBDV start codon, a fusion within the IBDV untranslated 3' end to HSV TK untranslated 3' end, and the TK poly-A signal sequence. The cassette containing these genes S"was engineered such that it was flanked by two EcoRI restriction endonuclease sites. As a result, an o 20 approximately 9100 base pair fragment containing both oo" lacZ gene and the IBDV gene can be obtained by digestion with EcoRI. Henceforth, the 9161 base pair EcoRI fragment will be referred to as the IBDV/lacZ cassette. The following procedures were used to construct S-HVT-003 by homologous recombination. The IBDV/IacZ cassette was inserted into the unique XhoI site present within a subclone of the HVT BamHI #16 fragment. To achieve this, the XhoI site was first changed to an EcoRI site through the use of an EcoRI linker. This site had previously been shown to be nonessential in HVT by the insertion of lacZ (S-HVT- 001). It was also shown that the flanking homology regions in BamHI #16 were efficient in homologous recombination. Shown in Figures 3A and 3B, the genomic location of the BamHI #16 fragment maps within the unique long region of HVT. The BamHI #16 fragment is approximately 3329 base pairs in length (SEQ ID NOs: 3, 4, 5, 6, and HVT DNA was prepared by the PREPARATION OF HERPESVIRUS DNA procedure.

Cotransfections of HVT DNA and plasmid DNA into primary chick embryo fibroblast (CEF) cells were done according to the DNA TRANSFECTION FOR GENERATING

RECOMBINANT

HERPESVIRUS. The recombinant virus resulting from .the cotransfection stock was purified by three successive rounds of plaque purification using the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure. When 100% of the plaques were blue, the DNA was analyzed for the presence of the IBDV gene by the SOUTHERN BLOTTING

OF

DNA procedure. Southern blots, probing EcoRI digested S-HVT-003 DNA with an IBDV specific nick translated :probe (plasmid 2-84/2-40), confirmed the presence of 15 the 9100 base pair EcoRI fragment. This result confirmed that S-HVT-003 contained both the lacZ gene and- the IBDV gene incorporated into its genome.

o Additional Southern blots, using a probe specific for BamHI #16, confirmed that the homologous recombination 20 occurred at the appropriate position in BamHI #16 and that no deletions were created. -No differences in the growth of S-HVT-003 compared to wild type virus (S-HVT- 000) were observed in vitro.

Expression of IBDV specific proteins from S-HVT-003 were assayed in vitro using the WESTERN BLOTTING PROCEDURE. Cellular lysates were prepared as described in PREPARATION OF HERPESVIRUS CELL LYSATES. Briefly, the proteins contained in the cellular lysates of S- HVT-003 were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with either an antiserum made against denatured purified IBDV capsid proteins or antiserum made against a synthetic peptide corresponding to a predicted imuno dominant region of the IBDV 40 kd (VP2) capsid protein.

The filters were washed and treated with protein A to detect the position of the bound antibodies.

Figure 4 shows the results obtained using the antiserum made against denatured purified IBDV capsid proteins, which have been shown by the applicants to react primarily with VP3 (32 kd protein) As seen, S-HVT-003 produces a protein which is immunologically indistinguishable from the authentic VP3 protein from intact IBDV virions. Moreover, the polyprotein appears to be processed correctly, producing a VP3 species that comigrates with the authentic VP3 protein. Recent evidence using an Australian IBDV stain indicates that VP4 is involved in the processing of the precursor polyprotein into mature VP2 and VP3 protein species (Jagadish, et al., 1988). Figure 5 shows the results obtained using a rabbit antiserum raised against a S 15 synthetic peptide that is homologous to a 14 amino acid region of the IBDV VP2 (40 kd) capsid protein. As seen, S-HVT-003 produces a protein that is O immunologically indistinguishable from the authentic viral VP2 protein. In addition, the VP2 protein 20 produced from S-HVT-003. :comigrates with the 40 kd Sspecies of VP2 isolated from intact IBDV virions. This species represents a major component of infectious (complete) viral particles.

In summary, analysis of the expression of IBDV specific proteins from S-HVT-003 has shown that the polyprotein is processed in CEF cell culture, producing proteins of the appropriate size that react to immunological reagents specific for either VP2 or VP3 proteins on Western blots.

The following set of experiments was carried out in chickens to analyze the in vivo expression of the IBDV genes contained within S-HVT-003 as determined by seroconversion data, serum neutralization results, and protection from IBDV challenge.

87 The first experiment was designed to show the seroconversion of chickens to -IBDV upon being vaccinated with S-HVT-003. Eleven 11-week-old chickens, seronegative to HVT and IBDV were obtained from SPAFAS Inc. Six birds were vaccinated subcutaneously in the abdominal region with 0.5 ml of a cellular suspension of CEF cells containing S-HVT-003 (40,000 PFU/ml). Serum samples were obtained every seven days for eight weeks for all birds in this study.

On day 28 (4th week), three of these birds received a boost of S-HVT-003, while the other three birds Sreceived 0.5 ml of an inactivated IBDV vaccine Sinoculated subcutaneously in the cervical region.

Three additional birds were given only the inactivated vaccine on day 28. Two birds served as contact controls and received no vaccinations. On day 56, all birds were sacrificed and necropsied. Table 1 show the results of the serum neutralization assay against IBDV.

No detectable SN activity was observed in the birds 20 given only S-HVT-003. Additionally, only one of the three birds that were given only the inactivated vaccine demonstrated low but detectable SN activity.

SN titers were also detected in one of the three birds that received the S-HVT-003 followed by the inactivated IBDV vaccine boost; these titers were at a much higher level than with the inactivated IBDV vaccine alone.

These results suggest that S-HVT-003 is priming the chicken for a secondary response against IBDV. In vitro analysis of the serum samples by WESTERN BLOTTING confirmed the seroconversion of the chickens to IBDV upon vaccination with S-HVT-003 both prior to and after boosts administered on day 28.

88 TABLE 1

DAY

Vaccine Group Bird No. 28 31 35 38 2 49 HVT-003 265 <2 <2 <2 <2 <2 <2 HVT-003 266 <2 <2 <2 <2 <2 <2 267 <2 <2 <2 <2 <2 <2 HVT-003 260 <2 <2 <2 <2 <2 <2 IBDV3 264 <2 <2 <2 1:64 1:256 1:512 269 <2 <2 <2 <2 <2 <2 C 261 <2 <2 <2 <2 <2 <2 IBDV8 262 <2 <2 <2 <2 1:4 1:4 20 263 <2 <2 <2 <2 <2 <2 C 270 <2 <2 <2 <2 <2 <2 271 <2 <2 <2 <2 <2 <2 a Commercial In the second experiment, twenty five 1-day old SPF :ooo chicks were vaccinated with S-HVT-003 (20 with 0.2ml subcutaneously and 5 by bilateral eyedrop). Twenty chicks were kept as controls. On days four and seven postinfection, five vaccinates and two control birds were bled, sacrificed and their spleens removed for virus isolation. Spleen cell suspensions were made by standard method, and -1 x 106 cells in 3 ml of chick embryo fibroblast (CEF) growth media were inoculated directly onto secondary cells. Cultures were incubated for 6-7 days and then scored for cytopathic effects (CPE) as determined by observing cell morphology. The cultures were passed a second time, and again scored for CPE. The results are shown in Table 2. All nonvaccinated control birds remained negative for.HVT for both day 4 and 7 spleen cell isolations. Four out of the five birds vaccinated with S-HVT-003 were positive for HVT at day 4 for both the first and second passages. One 89 bird did not produce virus, this may represent a vaccination failure. Five out of five birds were positive for HVT on day 7 at both passage one and two. Overall, the vector recovery experiment demonstrates that S-HVT-003 replicates as well as wild type HVT virus in vivo and that insertion of the IBDV/lacZ cassette into the XhoI site of BamHI #16 does not result in detectable attenuation of virus.

Subsequent experiments examining the recovered virus by the BLUOGAL SCREEN FOR RECOMBINANT

HERPESVIRUS

procedure confirmed the in vivo stability of S-HVT- 003, by demonstrating P-galactosidase expression in 100% of the viruses.

o* o *ooo* o *oe TABLE 2 Harvest Date Day 4 Day 7 Sample P1 P2 P1 P2 N N2 N3 N4 T T 2 2+ 2+ T 3 2+ 2+ T 4 4+ T 5 3+ 3+ 15 T 6 2+ contaminated T 7 T 8 T 8 T 9 T10 N control, T vaccinated CPE ranged from negative to At days 0, 4, 7, 14, 21, and 27 postinfection, blood samples were obtained from the rest of the chickens for determining serum ELISA titers against IBDV and HVT antigens as well as for virus neutralizing tests against IBDV. Additionally, at 21 days postinfection five control and fourteen vaccinated chicks were challenged with virulent IBDV by bi-lateral eyedrop 3 -'EIDso). All birds were sacrificed 6-days post challenge and bursa to body weight ratios were calculated. A summary of the results is shown in tables 3 and 4, respectively. As presented in Table 3, no antibodies were detected against HVT antigens by ELISA prior to 21-27 days post vaccination. In chickens, the immune response during the first two weeks post hatch is both immature and parentally suppressed, and therefore these results are not totally unexpected. In contrast, IBDV ELISA's were negative up to day 21 post-vaccination, and were only detectable after challenge on day 27. The ELISA levels seen on 91 day 27 post-vaccination indicate a primary response to IBDV. Table 4 comparing the Bursa-to-Body weight ratios for challenged controls and vaccinated/challenged groups show no significant differences. Vaccination with S-HVT-003 under these conditions did not prevent infection of the vaccinated birds by IBDV challenge, as indicated by the death of four vaccinated birds following challenge.

TABLE 3 ELISA VN Sample Group HVT IBDV IBDV C-0 0 0 <100 C-4 0 0 nd T-4 0 0 nd C-7 0 0 <100 T-7 0 0 <100 C-14 0 0 nd T-14 (n=14) 0 0 <100 C-21 0 0 nd T-21 (n=14) 1 0 <100 C-27 0 0 nd CC-27 0 5 nd 15 CT-27 (n-10) 3.2 2 nd C=control T=vaccinated CC=challenged control CT=Challenged vaccinated.

ELISA titers are GMTs and they range from 0-9.

TABLE 4 Sample Group Body wt. Bursa wt. BBR Control 258.8 1.5088 0.0058 Challenge 209 0.6502 0.0031 30 Control Challenge 215.5 0.5944 0.0027 Treated Values are mean values. Body weights are different in .control group because challenged birds did not feed well. Four challenged-treated birds died.

A third experiment was conducted repeating Experiment 2 but using immunologically responsive chicks (3 weeks of age). Six three week old SPF leghorn chickens were vaccinated intraperitoneally with 0.2ml of S-HVT-003 (one drop in each eye). Serum samples were obtained every seven days for six-weeks and the birds were challenged with the virulent USDA standard challenge IBDV virus on day 43 post-vaccination. Six days post challenge, the control, vaccinated-challenged, and challenged groups were sacrificed and bursas were harvested for probing with anti-IBDV monoclonal antibodies (MAB) (provided by Dr. David Snyder, Virginia-Maryland Regional College of Veterinary Medicine). Bursal homogenates were prepared by mixing 1 ml of 0.5% NP40 with one bursa. Bursa were then ground and briefly sonicated. Supernatants from the homogenates were reacted with the R63 MAB which had been affixed to 96-well Elisa plates via a protein A linkage. After incubation, a biotin labeled preparation of the R63 MAB was added. After washing, an avidin-horse radish peroxidase conjugate was added 15 and incubated. Tests were developed with Tris-malcate buffer (TMB) H 2 0 2 substrate. The test. results are presented in Table 5. The data show the presence of high levels of IBDV antigen in all bursa in the vaccinate-challenged group and in the challenged group.

20 No IBDV antigen was detected in the controls. IBDV specific antigen could be detected at dilutions of over 1/1000, and there does not appear to be differences between vaccinated and non-vaccinated challenged groups. HVT titers as determined by ELISA were first detectable at day 7 in four out of the six birds vaccinated. By day 14, six out of six vaccinated birds showed titers to HVT. All six birds continued to show HVT titers throughout the experiment. No IBDV SN titers were seen prior to the challenge. In contrast, analysis of these same serum samples by the WESTERN BLOTTING procedure demonstrated the seroconversion of chickens vaccinated with S-HVT-003 to IBDV prior to administration of the virus challenge. The level of response, however, remains small unless boosted by challenge. Comparison between the vaccinated/challenged and challenged only groups clearly demonstrates that the level of reactivity by Western blots is much higher in the vaccinated/challenged group. These results show that S-HVT-003 is seroconverting vaccinated birds to IBDV, and suggest that the level of IBDV specific expression are not high enough to induce a neutralizing response in the birds.

S-HVT-003 shows the merit of the vaccine approach the applicants have invented. HVT has been engineered to simultaneously express the foreign antigens (Pgalactosidase and IBDV antigens) that are recognized in the host by an immune response directed to these proteins.

*0**oo 0 TABLE Serology: Herpes/IBDV ELISA titer Bleed Date 11/10 11/14 11/24 12/1 12/8 12/15 12/22 Bird# 11/3 Vaccinated and Challenged 0* 5 so0.

0*000: 0.

221 41 42 43 44 0/0 0/0 0/0 0/0 0/0 0/0 7/0 4/0 3/0 0/0 1/0 0/0 5/0 4/0 2/0 5/0 5/0 1/0 6/0 1/0 1/0 5/0 1/0 1/0 5/0 1/0 5/0 5/0 2/0 1/0 5/0 1L/0 5/0 5/0 1/0 1/0 5/0 1/0 5/0 3/0 1/0 1/0 3/3 1/3 3/2 3/2 2/4 1/3 0/0 0/0 0/0 0/0

C*

Control 28 0/0 38 0/0 73 0/0 0/0 Challenged only 0/0 74 0/0 39 0/0 72 0/0 Maximum titer level is 9 0/3 0/3 0/3.

Examle 3 S-HVT-004 S-HVT-004 is a recombinant herpesvirus of turkeys that contains the Marek's disease virus (MDV) glycoprotein A (gA) gene inserted into the long unique region, and the -galactosidase (lacZ) gene also inserted in the long unique region. The MDV antigen is more likely to elicit the proper antigentic response than the HVT equivalent antigen.

The MDV gA (SEQ ID NOS: 8 and 9) gene was cloned by standard DNA cloning gA procedures. An EcoRI restriction fragment had been reported to contain the MDV gA gene (Isfort et al., 1984) and this fragment was identified by size in the DNA clones. The region of the DNA reported to contain the gA gene was sequenced by applicants and found to contain a glycoprotein gene as expected. The DNA from this gene was used to find the corresponding gene in HVT by the SOUTHERN BLOTTING OF DNA procedure, and a gene in HVT was identified that contained a very similar sequence. This gene is the same gene previously called gA (Isfort et al., 1984).

For insertion into the genome of HVT, the MDV gA gene was used intact because it would have good herpesvirus signal sequences already. The lacZ gene was inserted into the XhoI fragment in BamHI fragment #16, and the MDV gA gene was inserted behind lacZ as shown in Figures 6A and 6B. Flanking regions .in BamHI #16 were used for the homologous recombination. HVT DNA and plasmid DNA were co-transfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUS procedure into primary chick embryo fibroblast (CEF) cells. The virus from the transfection stock was purified by successive plaque purifications using the 97 BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure.

At the end of this procedure, when l00k of the plaques were blue, the DNA was analyzed for the presence of the MDV gA gene. S-HVT-004 is a recombinant virus that contains both the P-galactosidase gene and the MDV gA gene incorporated into the genome.

Figure 6C shows the structure of S-HVT-004.

98 Example 4 NEWCASTLE DISEASE VIRUS Newcastle disease virus (NDV) is closely related to PI- 3 in overall structure. Hemagglutinin (HN) and fusion genes of PI-3 was engineered for expression in IBR (ref). Similarly hemagglutinin (HN) and fusion (F) genes was cloned from NDV for use in the herpesvirus delivery system (Herpesvirus of turkeys, HVT).

The procedures that was utilized for construction of herpesvirus control sequences for expression have been applied to NDV.

S INFECTIOUS BRONCHITIS VIRUS Infectious bronchitis virus (IBV) is a virus of chickens closely related in overall structure to TGE.

20 Major neutralizing antigen of TGE was engineered for expression in PRV (ref). Similarly major neutralizing antigens was cloned from three strains of IBV: Massachusetts (SEQ.ID NOs: 14 and 15), Connecticut

(SEQ

ID NOs: 18 and 19), and Arkansas-99 (SEQ ID NOs: 16 and 25 17) for use in a herpesvirus delivery system (HVT).

The procedures that was utilized for the construction of herpesvirus control sequences for expression have been applied to IBV.

EXAMPLE S-HVT-045 S-HVT-045 is a recombinant herpesvirus of turkeys that contains the Marek's disease virus (MDV) glycoprotein B (gB) gene inserted into the short unique region. The MDV antigen is more likely to elicit the proper antigenic response than the HVT equivalent antigen. S- HVT-045 has been deposited on October 15, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, 15 Maryland 20852 U.S.A. under ATCC Accession No. VR 2383.

The MDV gB gene was cloned by standard DNA cloning procedures. The MDV gB gene was localized to a 3.9 kb 20 EcoRI-SallI fragment using an oligonucleotide probe based on the HSV gB sequence in a region found to be :conserved among known herpesvirus gB genes. The restriction map 3.9 kb EcoRI-SalI fragment is similar to the published map (Ross et al., 1989).

For insertion into the HVT genome, the MDV gB was used intact because it would have good herpesvirus signal sequences already. The MDV gB gene was inserted into a cloned 17.15 kb BamHI-EcoRI fragment derived from the HVT BamHI #1 fragment. The site used for insertion was the StuI site within HVT US2, previously utilized for the construction of S-HVT-012. The site was initially altered by insertion of a unique HindIII linker, and the MDV gB gene was inserted by standard DNA cloning procedures. Flanking regions in the 17.15 kb BamHI- EcoRI fragment were used, together with the remaining cloned HVT fragments using the PROCEDURE FOR GENERATING 100 RECOMBINANT HERPESVIRUSES FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The virus obtained from the transfection stock was plaque purified and the DNA was analyzed for the presence of the MDV gB gene. S-HVT-045 is a recombinant virus that contains the MDV gB gene incorporated into the genome at the StuI site in HVT US2 gene.

TESTING OF RECOMBINANT S-HVT-045 Two studies were conducted to demonstrate the effectiveness of these recombinant HVT/MDV viruses in protecting against challenge with virulent Marek's disease virus. In Study A, one-day-old specific pathogen free (SPF) chicks were vaccinated with either S-HVT-045 or S-HVT-046. Seven days post-vaccination, vaccinated chicks, and non-vaccinated, control chicks were challenged with the highly virulent MD-5 strain of Marek's disease virus. Following a 6-week post- 20 challenge observation period for clinical signs typical of Marek's disease, all chicks were necropsied and examined for lesions diagnostic of Marek's disease.

The results, in Table 6, show that. both recombinant viruses gave complete protection against a challenge S 25 that caused Marek's disease in 90% of non-vaccinated control chicks.

In a second study, one-day-old chicks were vaccinated either with S-HVT-045 or S-HVT-047. A third group of chicks were vaccinated with a USDA-licensed, conventional vaccine comprised of HVT and SB-1 viruses.

Five days post-vaccination, the vaccinated chicks and a group of non-vaccinated, control chicks were challenged with virulent Marek's virus, strain RB1B.

The chicks were observed for 8 weeks for cl\nical signs of Marek's disease, then necropsied and observed for Marek's lesions. This study demonstrated the ability 101 of HVT-045 and HVT-047 to provide 100t protection against' challenge (Table 1) The commercial vaccine gave 96!k protection, and 79!k of the non-vaccinated chicks developed Marek's disease.

TABLE 6 EFFICACY OF RECOMBINANT HVT/MDV VIRUSES TO PROTECT SUSCEPTIBLE CHICKS AGAINST VIRULENT MAREK'S DISEASE VIRUS Marek' s Protection a.

a a a a. a a.

Vaccine Group S -HVT -045.

MD-5 Challenge 20/20 R]B1B-Challenge 24/24 S -HVT -046 S -HVT -047

HVT&

Controls a Commercial 20/20 Not Tested Not Tested Not Tested 24/24 24/25 5/24 2/20 102 Example 6 S-HVT-012 S-HVT-012 is a recombinant herpesvirus of turkeys that contains the E. coli f-galactosidase (lacZ) gene inserted into the short unique region. The lacZ gene was used to determine the viability of this insertion site in HVT [ATCC F-126 ("Calnek")] S-HVT-012 has been deposited on October 15, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure on with the Patent Culture Depository of the American Type Culture S 15 Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2382.

For insertion into the genome of HVT, the 3galactosidase gene was introduced into the unique StuI site of the.cloned EcoRI fragment #7 of HVT, the fragment containing the StuI site within the US2 gene of HVT (as described in Methods and Materials).

Flanking regions of EcoRI fragment #7 were used for homologous recombination. HVT DNA and plasmid DNA were 25 co-transfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF) cells. A blue virus obtained from the transfection stock was purified by successive plaque purifications using the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure. At the end of this procedure, when 100% of the plaques were blue, the DNA was analyzed for the presence of the lacZ gene. S-HVT-012 is a recombinant virus that contains the lacZ gene incorporated into the genome at the StuI site within the US2 gene of HVT.

S-HVT-012 may be formulated as a vaccine in the same 103 manner as S-HVT-045. When administered to chickens, such a vaccine provides protection against Marek's disease virus.

Example 7 Sites for Insertion of Foreign DNA into HVT In order to define appropriate insertion sites, a library of HVT BamHI and EcoRI restriction fragments was generated. Several of these restriction fragments (BamHI fragments #16 and #13, and EcoRI fragments #6, and #9 (see figure were subjected to restriction mapping analysis. One unique restriction site was identified in each fragment as a potential insertion site. These sites included XhoI in BamHI fragments #13 and #16, and EcoRI fragment #9 and SalI in EcoRI fragment #6 and StuI in EcoRI fragment A 20 -galactosidase (lacZ) marker gene was inserted in each of the potential sites. A plasmid containing such a foreign DNA insert may be used according to the DNA COTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES to CONSTRUCT a HVT containing the foreign DNA. For 25 this procedure to be successful it is important that :the insertion site be in a region non-essential to the replication of the HVT and that the site be flanked with HVT DNA appropriate for mediating homologous recombination between virus and plasmid DNAs. The plasmids containing the lacZ marker gene were utilized in the DNA COTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES. The generation of recombinant virus was determined by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. Three of the five sites were successfully used to generate a recombinant virus. In each case the resulting virus was easily purified to 100%, clearly defining an appropriate site for the insertion of 104 foreign DNA. The three homology vectors used to define these sites are described below.

Example 7A Homologv Vector 172-29.31 The homology vector 172-29.31 contains the HVT BamHI #16 fragment and is useful for the insertion of foreign DNA into HVT. Plasmid 172-29.31 contains a unique XhoI restriction site into which foreign DNA may be cloned.

XhoI site in homology vector 172-29.31 may be used to insert foreign DNA into HVT by the construction of at least three recombinant HVT (see examples 1-3).

The homology vector 172-29.31 was further characterized :by DNA sequence analysis. The complete sequences of the BamHI #16 fragment was determined. Approximately 2092 base pairs of the adjacent BamHI #13 fragment was 20 also determined (see SEQ ID NO: This sequence indicates that the open reading frame coding for HVT glycoprotein A (gA) spans the BamHI #16 BamHI #13 junction. The HVT gA gene is homologous to the HSV-1 glycoprotein C The XhoI site interrupts an ORF 25 which lies directly upstream of the HVT gA gene. This ORF shows amino acid sequence homology to the PRV p43 and the VZV gene 15. The PRV and VZV genes are the homologues of HSV-1 UL43. Therefore this ORF was designated as HVT UL43 (SEQ ID NO: It should be noted that the HVT UL43 does not exhibit direct homology to HSV-1 UL43. Although HVT UL43 is located upstream of the HVT gC homologue it is encoded on the same DNA strand as HVT gA, where as the HSV-1 UL43 is on the opposite strand relative to HSV-1 gC. The XhoI site interrupts UL43 at approximately amino acid 6, suggesting that the UL43 gene is non-essential for HVT replication.

105 Example 7B Homology Vector 435-47.R17 The homology vector 435-47.R17 contains the HVT EcoRI #7 fragment and is useful for the insertion of foreign.

DNA into HVT. Plasmid 435-47.R17 contains a unique HindIII restriction site into which foreign DNA may be cloned. The HindIII restriction site in plasmid results from the insertion of a HindIII linker into the naturally occurring StuI site of EcoRI fragment #7.

HindIII site in homology vector 435-47.R17 may be used to insert foreign DNA into HVT by the construction of at least 25 recombinant HVT.

DNA sequence analysis at the StuI indicated that this fragment contains open reading frames coding for US2, and US3. The StuI site interrupts US2 at approximately amino acid 124, suggesting that the US2 20 gene is non-essential for HVT replication.

Example 7C o Homoloqy Vector 172-63.1 The homology vector 172-63.1 contains the HVT EcoRI #9 fragment and is useful for the insertion of foreign DNA into HVT. Plasmid 172-63.1 contains a unique Xhol restriction site into which foreign DNA may be cloned.

XhoI site in homology vector 172-63.1 may be used to insert foreign DNA into HVT by the construction of S- HVT-014 (see example 8).

106 Example 8 S-HVT-014 S-HVT-014 is a recombinant herpesvirus of turkeys that contains the E. coli P-galactosidase (lacZ) gene inserted into the long unique region. The lacZ gene was used to determine the viability of this insertion site in HVT [ATCC F-126 ("Calnek")].

For insertion into the genome of HVT, the 0galactosidase gene was introduced into the unique Xhol site of the cloned EcoRI fragment #9 (as described in Methods and Materials) The XhoI site within the EcoRI #9 fragment of the HVT genome is the same site as the Xhol site within the BamHI #10 fragment used for construction recombinant herpesvirues of turkeys described in Examples 16 through 19. Flanking regions 20 of EcoRI fragment #9 were used :for homologous recombination. HVT DNA and plasmid DNA were cotransfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF) cells. A blue virus 25 obtained from the transfection stock was purified by successive plaque purifications using the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure. At the end of this procedure when 100% of the plaques were blue. S-HVT-014 is a recombinant virus that contains the lacZ gene incorporated into the genome at the XhoI site within the EcoRI #9 fragment of HVT.

S-HVT-014 may be formulated as a vaccine in the same manner as S-HVT-045. When administered to chickens, such a vaccine provides protection against Marek's disease virus.

107 Example 9 S-HVT-005 S-HVT-005 is a recombinant herpesvirus of turkeys that contains the E. coli -galactosidase (lacZ) gene inserted into the long unique region. The lacZ gene was used to determine the viability of this insertion site in HVT [ATCC F-126 ("Calnek")].

For insertion into the genome of HVT, the 3galactosidase gene was introduced into an approximately 1300 base pair deletion of the XhoI #9 fragment of HVT.

The deletion which lies between the unique MluI and 15 EcoRV sites removes the complete coding region of the HVT gA gene (see SEQ ID NO: Flanking regions of hoI fragment #9 were used for homologous recombination. HVT DNA and plasmid DNA were cotransfected according to the DNA TRANSFECTION FOR 20 GENERATING RECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF) cells. A blue virus obtained from the transfection stock was purified by successive plaque purifications using the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure. At the 25 end of this procedure, when 100% of the plaques were blue, the DNA was analyzed for the presence of the lacZ gene. S-HVT-005 is a recombinant virus that contains the lacZ gene incorporated into the genome in place of the deleted gA gene of HVT.

S-HVT-005 may be formulated as a vaccine in the same manner as S-HVT-045. When administered to chickens, such a vaccine provides protection against Marek's disease virus.

108 Example Marek's Disease Vaccines Recombinant HVT expressing glycoproteins from Marek's Disease Virus make superior vaccines for Marek's Disease. We have constructed several recombinant HVT expressing MDV glycoproteins: S-HVT-004 (Example 3), S-HVT-045 (Example S-HVT-046 (Example 10A), S-HVT- 047 (Example 10B), S-HVT-062 (Example Example 10A S-HVT-046 S-HVT-046 is a recombinant herpesvirus of turkeys that 15 contains the Marek's disease virus (MDV) glycoprotein B (gB) and glycoprotein A (gA) genes inserted into the short unique region. The MDV genes are inserted in the same transcriptional orientation as the US2 gene. The Ge. MDV antigens are more likely to elicit the proper 20 antigenic response than the HVT equivalent antigen.

too@ S-HVT-046 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic 25 clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 *5 with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 456-17.22 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.

109 Example 10B S-HVT-047 S-HVT-047 is a recombinant herpesvirus of turkeys that contains the MDV gB and gA genes inserted into the short unique region. The MDV genes are inserted in the opposite transcriptional orientation as the US2 gene.

The MDV antigens are more likely to elicit the proper antigenic response than the HVT equivalent antigen.

S-HVT-047 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 S 15 with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 456-17.18 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.

Example 10C S-HVT-062 S-HVT-062 is a recombinant herpesvirus of turkeys that contains the MDV gB, glycoprotein D and gA genes inserted into the short unique region. The MDV genes are inserted in the same transcriptional orientation as the US2 gene. The MDV antigens are more likely to elicit the proper antigenic response than the HVT equivalent antigen. S-HVT-062 has been deposited on February 23, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.

under ATCC Accession No. VR 2401.

S-HVT-062 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC 110 DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindIII, 556-60.6 with BamHI and HindIII, and 456-17.22 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.

TESTING OF RECOMBINANT HVT EXPRESSING MDV ANTIGENS Two studies were conducted to demonstrate the effectiveness of these recombinant HVT/MDV viruses in protecting against challenge with virulent Marek's disease virus. In Study 1, one-day-old specific 15 pathogen free (SPF) chicks were vaccinated with either S-HVT-045, S-HVT-046, or S-HVT-047. Five days postvaccination, vaccinated chicks, and non-vaccinated, control chicks were challenged with MDV. Following a 6week post-challenge observation period for clinical signs typical of Marek's disease, all chicks were necropsied and examined for lesions diagnostic of Marek's disease. The results, in Table 7, show these recombinant viruses gave complete protection against a challenge that caused Marek's disease in 84% of nonvaccinated control chicks.

In the second study, one-day-old chicks were vaccinated with S-HVT-062. Two more groups of chicks were vaccinated with a USDA-licensed, conventional vaccines comprised of HVT and a combination HVT and SB-1 viruses. Five days post-vaccination, the vaccinated chicks and a group of non-vaccinated, control chicks were challenged with MDV. The chicks were observed for 111 8 weeks for clinical signs of Marek's disease, then necropsied and observed for Marek's lesions. This study demonstrated the ability of S-HVT-062 to provide 100% protection against challenge (Table 7) The commercial vaccines gave 81% and 95% protection, respectively and 100% of the non-vaccinated chicks developed Marek's disease.

*9 112 TABLE 7 EFFICACY OF RECOMBINANT HVT/MDV VIRUSES AGAINST VIRULENT MAREK'S VIRUS CHALLENGE Study Vaccine Group Dose-3 Protection b 1 S-HVT--045 1 S-HVT-046 1 S-HVT-047 1 Controls 1 HVT/SB-1 2 S-HVT-062 2 S-HVT-062 2 Controls 2 1i~JrC 2 HVT/SB-1c 2.2 X 103 2.2 X 103 2.2 X 103 24/24 (100t) 20/20 (100%1) 24/24 (100k) 7/44 24/25 32/32 (100%) 22/22 (100%) 7. 5 X 102 1. 5 X 103 0/20 (0k) 7. 5 X 102 7. 5 X 102 17/21 (81%) 21/22 PFU/0.2 ml.

bNo. protected/Total; Challenge 5 days postvaccination.

cCommercial vaccine.

113 Example 11 Bivalent Vaccines Against Newcastle Disease and Marek's Disease Recombinant HVT expressing proteins from NDV make bivalent vaccines protecting against both Marek's Disease and Newcastle disease. Several recombinant HVT expressing NDV proteins were constructed S-HVT-007 (Example 11A), S-HVT-048 (Example 11B), S-HVT-049 (Example 11C) S-HVT-050 (Example 11D),and S-HVT-106 (Example 11E) Example 11A S-HVT-007 S-HVT-007 is a recombinant herpesvirus of turkeys that contains a E. coli lacZ NDV HN hybrid protein gene under the control of the PRV gX promoter and the NDV F gene under the control of the HSV-1 a4 promoter inserted into the long unique region. The NDV genes are inserted in the same transcriptional orientation as the UL43 gene.

To construct S-HVT-007, HVT DNA and the plasmid 255- 25 18.B16 were co-transfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF) cells. A blue virus obtained from the transfection stock was purified by successive plaque purifications using the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure.

At the end of this procedure, when 100% of the plaques were blue.

114 Example 11B S-HVT-048 S-HVT-048 is a recombinant herpesvirus of turkeys that contains the MDV gB and gA genes and the NDV F gene under the control of the HCMV immediate early promoter inserted into the short unique region. The MDV and NDV genes are inserted in the same transcriptional orientation as the US2 gene.

S-HVT-048 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32. 5G6 with NotI, 407-32.1C1 S 15 with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 535-70.3 uncut. Insertion of -the appropriate DNA was confirmed by southern blot analysis.

Example 11C S-HVT-049 S-HVT-049 is a recombinant herpesvirus of turkeys that contains the MDV gB and gA genes and the NDV HN gene S under the control of the PRV gX promoter inserted into 25 the short unique region. The MDV and NDV genes are inserted in the same transcriptional orientation as the US2 gene.

S-HVT-049 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 withBamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 549-62.10 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.

115 Example 11D S-HVT-050 S-HVT-050 is a recombinant herpesvirus of turkeys that contains the MDV gB and gA genes and the NDV HN (SEQ ID NOs: 10 and 11) and F (SEQ ID NOs: 12 and 13) genes.

The NDV genes are under the control of the PRV gX and HCMV immediately promoters respectively. All four genes are inserted into the short unique region in the same transcriptional orientation as the US2 gene.

S-HVT-050 was constructed according to the PROCEDURE •FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC S. DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 15 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 •with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 549-24.15 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis. S-HVT-050 has been deposited on February 23, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.

S 25 under ATCC Accession No. VR 2400.

Example 11E S-HVT-106 S-HVT-106 is a recombinant herpesvirus of turkeys that contains the MDV gA, gB, gD genes and the NDV HN and F genes. The NDV genes are under the control of the PRV gX and HCMV immediately promoters respectively. All five genes are inserted into the short unique region in the same transcriptional orientation as the US2 gene.

S-HVT-106 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC 116 DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 633-13.27 uncut.

TESTING OF RECOMBINANT HVT EXPRESSING NDV ANTIGENS Two studies were conducted to demonstrate the effectiveness of these recombinant HVT/MDV/NDV viruses in protecting against challenge with virulent Newcastle and Marek's disease viruses. In Study 1, one-day-old specific pathogen free (SPF) chicks were vaccinated with either S-HVT-048, S-HVT-049, S-HVT-050, or a S* 15 USDA-licensed, conventional vaccine comprised of NDV B1/B1 virus. Three weeks post-vaccination, vaccinated chicks, and non-vaccinated, control chicks were challenged with NDV. Birds were then observed for clinical signs of disease. The results, in Table 8, show these recombinant viruses (S-HVT-048 and S-HVT- S• 050) gave complete protection against :a challenge that caused Newcastle disease in 100% of non-vaccinated control chicks. Recombinant virus S-HVT-049 gave 2 partial protection against Newcastle-disease.

In the second study, one-day-old chicks were vaccinated with S-HVT-050. Two more groups of chicks were vaccinated with a USDA-licensed, conventional vaccines comprised of HVT and a combination HVT and SB-1 viruses. Five days post-vaccination, the vaccinated chicks and a group of non-vaccinated, control chicks were challenged with MDV. The chicks were observed for 8 weeks for clinical signs of Marek's disease, then necropsied and observed for Marek's lesions. This study demonstrated th6 ability of S-HVT-050 to provide protection greater than the commercial Marek's disease vaccines.

117 TABLE 8 EFFICACY OF RECOMBINANT HVT/MDV/NDV VIRUSES AGAINST- VIRULENT NEWCASTLE AND MAREK' S DISEASE VIRUS

CHALLENGE

Protection (c Vaccine Group Study Dose' NDVb MVDV a a a 1 151 15 2 2 30 a b

C

d S-HVT-048 4.0 X 104 19/1,9 (100) S-HVT-049 3.0 X 104 4/20 S-HVT-050 1.5 X 104 20/20 (100) Controls 0/20 (0) NDV El/Bid 18/18 (100) S-HVT-050 7.5 X 102 13/14 (93) S-HVT-050 1. 5 X 103 16/17 (94) Controls 5/23 (22) HVTd 20/26 (77) HVT/SB- 1d 10/12 (83) PFU/0.2 ml.

No. protected/Total; Challenge 3 weeks post-vaccination.

No. protected/Total; Challenge 5 days post-vaccination.

Commercial vaccine.

118 Example 12 Bivalent Vaccines Against Infectious Larvnqotracheitis and Marek's Disease Recombinant HVT expressing glycoproteins from ILT virus make bivalent vaccines protecting against both Marek's disease and infectious laryngotracheitis. Several recombinant HVT expressing ILT virus glycoproteins S- HVT-051 (Example 12A), S-HVT-052 (Example 12B), and S- HVT-104 (Example 11C) were constructed.

°Example 12A S-HVT-051 15 S-HVT-051 is a recombinant herpesvirus of turkeys that contains the ILT virus gB gene inserted into the short unique region. The ILT gene is inserted in the same transcriptional orientation as the US2 gene.

S-HVT-051 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 25 with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 528-11.34 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.

Example 12B S-HVT-052 S-HVT-052 is a recombinant herpesvirus of turkeys that contains the ILT virus gD gene inserted into the short unique region. The ILT gene is inserted in the opposite transcriptional orientation as the US2 gene.

119 S-HVT-052 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and 528-03.37 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.

Example 12C S-HVT-104 S-HVT-104 is a recombinant herpesvirus of turkeys that contains six foreign genes. The MDV gA, gB, and gD 15 genes are inserted in the unique short region in the same transcriptional orientation as the US2 gene. An E. coli lacZ marker gene and the ILT gB and gD genes are inserted in BamHI #16 region in the same transcriptional orientation as the UL43 gene.

To construct S-HVT-104, DNA from S-HVT-062 and the S- plasmid 634-29.16 were co-transfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure into primary chick embryo "fibroblast (CEF) 25 cells.

TESTING OF RECOMBINANT HVT EXPRESSING ILT ANTIGENS The following study was conducted to demonstrate the effectiveness of these recombinant HVT/ILT viruses in protecting against challenge with virulent Infectious Laryngotracheitis virus. One-day-old specific pathogen free (SPF) chicks were vaccinated with either S-HVT- 051, S-HVT-052, a combination of S-HVT-051 and S-HVT- 052, or a USDA-licensed, conventional vaccine comprised of ILT virus. Two to three weeks post-vaccination, vaccinated chicks, and non-vaccinated, control chicks 120 were challenged with ILT. Birds were then observed for clinical signs of disease. The results, in Table 9, show these recombinant viruses (S-HVT-051 and S-HVT- 052) gave protection against challenge with ILT virus comparable to a commercial ILT vaccine.

Animals vaccinated with the vaccines described here may be easily differentiated from animals infected with virulent ILT. This is accomplished by testing the suspect birds for antibodies to any ILT antigens other than gB or gD. Examples of such antigens are ILT glycoproteins C, E, and G. Vaccinated, uninfected birds will be negative for these antigens whereas infected birds will be positive.

e 121.

TABLE 9 EFFICACY OF RECOMBINANT HVT/ILT VIRUSES AGAINST VIRULENT rINFECTIOUS LARYNGOTRACHEITIS VIRUS CHALLENGE Vaccine Group Dosea Protection b S -HVT -051 S -HVT -052 S-HVT-051 S -HVT -052 2.1 X 103 1.7 X 103 2.1 X 103 1. 7 X 103 28/30 (93%) 29/29 (100%) 24/24 (100%) 2/30 29/30 (97%) Controls ILTc PFU/0. 2 ml.

*No.protected/Total; Challenge 2-3 weeks postvaccination.

CCommercial vaccine.

122 Example 13 Bivalent Vaccines Against Infectious Bursal Disease and Marek's Disease Recombinant HVT expressing proteins from IBDV make bivalent vaccines protecting against both Marek's Disease and infectious bursal disease. Several recombinant HVT expressing IBDV proteins were constructed. These viruses include S-HVT-003 (example 2) and S-HVT-096.

too S-HVT-096 is a recombinant herpesvirus of turkeys that contains the IBDV VP2 gene, under the control of the 15 HCMV immediate early promoter, inserted into the short unique region. The IBDV gene is inserted in the same transcriptional orientation as the US2 gene.

S-HVT-096 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindIII, 556-60.6 1. 25 with BamHI, and 602-57.Fl uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.

S-HVT-096 was assayed for expression of VP2 by black plaque and western blot analysis. Both assays indicated that the virus was expressing high levels of protein which reacts specifically with an IBDV neutralizing monoclonal antibody. This virus will be useful as a vaccine against infectious bursal disease.

123 Example 14 Bivalent Vaccines Against Infectious Bronchitis and Marek's Disease S-HVT-066 is a recombinant herpesvirus of turkeys that contains the MDV gB, gD and gA genes and the IBV spike and matrix genes. The IBV spike and matrix genes are under the control of the HCMV immediate early and PRV gX promoters respectively. All five genes are inserted into the short unique region. The MDV and IBV genes are inserted in the same transcriptional orientation as the US2 gene.

15 S-HVT-066 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindII, 556-60.6 with BamHI, and 567-72.1D uncut.. Insertion of the appropriate DNA was confirmed by southern blot analysis.

S-HVT-066 was assayed for expression of the IBV spike protein by black plaque and western blot analysis.

Both assays indicated that the virus was expressing high levels of protein which reacts specifically with an IBV neutralizing monoclonal antibody. This virus will be useful as a vaccine against infectious bronchitis.

124 Example Vaccines utilizing HVT to express antigens from various pathoqens.

Anticipate that antigens from the following pathogens may also be utilized to develop poultry vaccines: Chick anemia virus (agent), Avian encephalomyelitis virus, Avian reovirus, Avian paramyxoviruses, Avian influenza virus, Avian adenovirus, Fowl pox virus, Avian coronavirus, Avian rotavirus, Salmonella spp E. coli, S• Pasteurella spp, Haemophilus spp, Chlamydia spp, Mycoplasma spp, Campylobacter spp, Bordetella spp, Poultry nematodes, cestodes, trematodes, Poultry S: 15 mites/lice, Poultry protozoa (Eimeria spp, Histomonas spp, Trichomonas spp) Example 16 Trivalent vaccines against Infectious Laryngotracheitis, Marek's Disease and Newcastle's Disease and bivalent vaccines against Infectious Laryngotracheitis and Marek's Disease are described.

Superior protection against Infectious Laryngotracheitis is achieved with a vaccine combining S-HVT-123 (expressing ILTV gB and gD) with S-HVT-138, 139, or 140 (expressing ILTV gD and gI).

Example 16A S-HVT-123 S-HVT-123 is a recombinant herpesvirus of turkeys that contains the ILT virus gB and gD genes inserted into an XhoI site converted to a NotI site in the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figures 13B and 15; SEQ ID NO: 48). S-HVT-123 further contains the MDV gA, gD, and gB genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The 125 ILTV genes and the MDV genes each use their own respective promoters. S-HVT-123 is useful as a vaccine in poultry against Infectious Laryngotracheitis and Marek's Disease.

S-HVT-123 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 721-38.1J uncut, 729-37.1 with AscI.

Example 16B S-HVT-138 S* S-HVT-138 is a recombinant herpesvirus of turkeys that contains the ILT virus gD and gI genes inserted into a unique XhoI site converted-to a PacI site in the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figures 13A and 15). The ILTV gD and gI genes are in the opposite transcriptional orientation to the open reading frame (ORF A) within the Ecorl #9 (BamHI #10) fragment of the HVT genome (Figure 14; SEQ ID NOs: 48, 50). The ILTV gD and gI genes are expressed as overlapping transcripts from endogenous ILTV promoters, and share their own endogenous polyadenylation signal.

S-HVT-138 is useful as a vaccine in poultry against Infectious Laryngotracheitis and Marek's Disease.

S-HVT-138 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 With BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 711-92.1A uncut, 415-09.BA1 with BamHI.

126 Sera from S-HVT-138 vaccinated chickens reacts on Western blots with ILTV gI protein indicating that the S-HVT-138 vaccine expressed the ILTV protein and does elicit an immune response in birds. S-HVT-138 vaccinated chickens were protected from challenge by virulent infectious laryngotracheitis virus.

Example 16C S-HVT-139 S-HVT-139 is a recombinant herpesvirus of turkeys that contains the ILT virus gD and gI genes inserted into a unique XhoI site converted to a PacI site in the EcoRi #9 (BamHI #10) fragment of the HVT genome. The ILTV gD and gI genes are in the opposite transcriptional 15 orientation to the open reading frame (ORF A) within *"the EcoRI #9 (BamHI #10) fragment of the HVT genome (Figure 13A and 15; SEQ ID NO: 48, 50). S-HVT-139 further contains the MDV gA, gD, and gB genes are o* inserted into the unique StuI site converted into a HindIII site in the HVT US2 gene.-The ILTV gD and gI genes are expressed as overlapping transcripts from their won respective endogenous ILTV promoters, and the MDV genes are also expressed. from their own endogenous promoters. S-HVT-139 is',useful as a vaccine in poultry against Infectious Laryngotracheitis and Marek's Disease.

S-HVT-139 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 711-92.1A uncut, 721-38.1J uncut.

Example 16D S-HVT-140 127 S-HVT-140 is a recombinant herpesvirus of turkeys that contains the ILT virus gD and gI genes inserted into a unique XhoI site converted to a PacI site in the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figures 13A and 15). The ILTV gD and gI genes are in the opposite transcriptional orientation to the open reading frame (ORF A) within the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figure 14; SEQ ID NO: 48, 50). S-HVT-140 further contains the MDV gA, gD, and gB genes and the NDV F and HN genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The ILTV gD and gI genes are expressed as overlapping S" transcripts from their own respective endogenous ILTV promoters, and the MDV genes are also expressed from 15 their own respective endogenous MDV promoters. The NDV F gene is transcribed from the HCMV immediate early promoter, and the NDV HN gene is transcribed from the to.* PRV gX promoter. S-HVT-140 is useful as a vaccine in poultry against Infectious Laryngotracheitis, Marek's S 20 Disease, and Newcastle's Disease.

S-HVT-140 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 711-92.1A uncut, 722-60.E2 uncut.

Example 17 Trivalent vaccines against Infectious Bursal Disease, Marek's Disease and Newcastle's Disease and bivalent vaccines against Infectious Bursal Disease and Marek's Disease are described.

Examle 17A HVT-126 128 S-HVT-126 is a recombinant herpesvirus of turkeys that contains the IBDV VP2 gene inserted into an XhoI site converted to a PacI site in the EcoRl #9 (BamHI fragment in the HVT genome (Figures 13A and 15). The IBDV gene is in the same transcriptional orientation as the open reading frame (ORF A) within the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figure 14; SEQ ID NO: 48, 50). The IBDV VP2 gene is expressed from an IBRV VP8 promoter. S-HVT-126 is useful as a vaccine in poultry against Infectious Bursal Disease and Marek's Disease.

S-HVT-126 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING :i 15 SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 415-09.BA1 with BamHI.

Example 17B HVT-137 S-HVT-137 is a recombinant herpesvirus of turkeys that contains.the IBDV VP2 gene inserted into a'uniqe XhoI 25 site converted to a PacI site in the EcoRl #9 (BamHI #10) fragment in the HVT genome (Figures 13A and The IBDV gene is in the same transcriptional orientation as the open reading frame (ORF A) within the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figure 14; SEQ ID NO: 48, 50). S-HVT-137 further contains the MDV gA, gD, and gB genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The IBDV VP2 gene is expressed from an IBRV VP8 promoter. The MDV genes are expressed from their own respective endogenous MDV promoters. S-HVT- 137 is useful as a vaccine in poultry against Infectious Bursal Disease and Marek's Disease.

129 S-HVT-137 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 721-38.1J uncut.

Example 17C HVT-143 S-HVT-143 is a recombinant herpesvirus of turkeys that contains the IBDV VP2 gene inserted into a unique XhoI site converted to a PacI site in the EcoR1 #9 (BamHI #10) fragment of the HVT genome (Figures 13 A and 15 The IBDV gene is in the same transcriptional i orientation as the open reading frame (ORF A) within the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figure 14; SEQ ID NO: 48, 50). S-HVT-143 further contains the MDV gA, gD, and gB genes and the NDV F and HN genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The IBDV VP2 gene is expressed from an IBRV VP8 promoter. The MDV genes are expressed from their own respective endogenous MDV promoters. The NDV-F gene is transcribed 25 from the HCMV immediate early promoter, and the NDV HN gene is transcribed from the PRV gX promoter. S-HVT-143 is useful as a vaccine in poultry against Infectious Bursal Disease, Marek's Disease, and Newcastle's Disease.

S-HVT-143 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with Notl, 672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 722-60.E2 uncut.

130 Example 18 HVT-128 S-HVT-128 is a recombinant herpesvirus of turkeys that contains the NDV HN and F genes inserted into a unique XhoI site converted to a PacI site in the EcoR1 #9 (BamHI #10) fragment of the HVT genome (Figures 13A and S-HVT-128 further contains the MDV gA, gD, and gB genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The NDV HN gene is expressed from the PRV gX promoter and the NDV F gene is expressed from the HCMV immediate early promoter.

The MDV genes are expressed from the endogenous MDV promoters. S-HVT-128 is useful as a vaccine in poultry .*against Newcastle's Disease and Marek's Disease.

S-HVT-128 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 20 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, and 717- 38.12 uncut. To a mixture of these six cosmids was added a limiting dilution of a recombinant HVT virus containing the MDV gA, gD, and gB genes inserted into 25 the unique short region (see HVT-062) and the PRV gX promoter-lacZ gene inserted into an Xhol site converted to a NotI site in the EcoRl #9 (BamHI #10) fragment within the unique long region of HVT. A recombinant virus S-HVT-128 was selected which was lac Z negative.

Example 18B HVT-136 S-HVT-136 is a recombinant herpesvirus of turkeys that contains the NDV HN and F genes inserted into an XhoI site converted to a PacI site in the EcoRl #9 (BamHI fragment within the unique long region of HVT.

(Figure 14; SEQ ID NOs: 48 and 50) The NDV HN gene is 131 expressed from the PRV gX promoter and the NDV F gene is expressed from the HCMV immediate. early promoter. S- HVT-136 is useful as a vaccine in poultry against Newcastle's disease and Marek's disease.

S-HVT-136 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, and 717- 38.12 uncut, and 415-09.BA1 with BamHI.

HVTIMDV recombinant virus vaccine S-HVT-145 is a recombinant virus vaccine containing MDV *CC* 20 and HVT genomic sequences which protects against Marek's disease is produced by combining cosmids of MDV genomic DNA containing genes coding for the relevant protective antigens of virulent MDV serotype 2 and cosmids of.HVT genomic DNA according to the PROCEDURE 25 FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The resulting virus is a vaccine that ahs the protective immune respnse to virulent MDV serotype 2 and the attenuated growth characteristics of the HVT. In one embodiment, a chimeric virus vaccine containing the MDV genes of the unique short and the HVT genes of the unique long is useful as a vaccine against Marek's disease in chickens. The MDV protective antigens withinthe unique short (gD, gE, and gI) elicit a protective immune response to MDV, while the virulence elements present in the unique long of MDV (55,56, 57) are replaced by the attenuating uniuqe long sequences of HVT. The result is an attenuated 132 virus vaccine which protects against Marek's disease.

Multivalent protection against Marek's disease, infectious laryngotracheitis, infectious vursal disease, Newcastle's dises, or another poultry pathogen is achieved by inserting the ILTV gB,gD, and gI genes, the IBDV VP2 gene, the NDV HN and F genes, or an antigen gene froma poultry pathogen into an XhoI site converted to a PacI site or NotI site in the EcoRl #9 (BamHI #10) fragment within the uniuqe long region of HVT/MDV recombinant virus (Figures 13 and A cosmid was constructed containing the entir MDV unique short region. MDV genomic DNa contains several SmaI sites in the uniuqe long and internal and terminal 15 repeats of the virus, but no SmaI sites wihin the unique short of the virus. The entire unique short region of MDV was isolated by a partial restriction digestion of MDV genomic DNa with SmaI. A DNA fragment approximately 29,000 to 33,000 base pairs was isolated 20 and cloned into a blunt ended site of.the cosmid vector To generate HVY-145, a recombinant HVT/MDV chimeric virus, the cosmid containing the MDV unique short region was combined with cosmids containing the HVT unique long region according to the PROCEDURE FOR S 25 GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI, and 739-27.16 with NotI.

The resulting virus vaccine provides superior protection against Marek's disease or as a multivalent vaccine against Marek's disease and infectious laryngotracheitis, infectious bursal disease, Newcastle's disease, or another poultry pathogen. This vaccine is superior because expression of MDV genes in the HVT/MDV chimera vaccine is safer and provides 133 better protection against Marke's disease than vaccines presently available containing HVT and MDV type 1 (SB- 1) or HVT alone. Secondly, one can demonstrate expression of the MDV glycoprotein gens in the absence of the homologous HVT genes for both diagnostic and regulatory purposes. This is useful since antibodies to an MDV glycoprotein will cross react with the homologous HVT glycoprotein. Finally, a recombinant HVT/MDV virus which contains a single copy of each glycoprotein gene is more stable that a recombinant virus containing two copies of a homologous glycoprotein gene from HVT and MDV which may delete by homologous recombination.

15 In an alternative embodiment, cosmids containing MDV protective antigen genes from the unique long (MDV gB and gC) are combined with cosmids containing HVT gene sequences from the unique short and the unique long, effecitvely avoiding the MDV virulence genes at the 20 unique long/internal repeat junction and the unique long/terminal repeat junction 56, and 57).

SB-1 strain is an MDV serotype 1 with attenuated pathogenicity. Vaccination with a combination of HVT 25 and SB-1 live viruses protects against virulent MDV challenge better than vaccination with either virus alone. In an alternative embodiment of the present invention, a recombinant virus vaccine comprises protective antigen genes of the virulent MDV serotypes 2 combined with the attenuating genes of the nonvirulent MDV serotypes 1 and 3, such as SB-1 and HVT.

The genomic DNA corresponding to the unique long region is contributed by the SB-1 serotype. The genomic DNA corresponding to the unique short region is contributed by the HVT serotype. Three major glycoprotein antigens (gB, gA and gD) from the MDV serotype 2 are inserted into the unique short region of the virus.

134 The recombinant virus is constructed utilizing HVT subgenomic clones 672-01.A40, 672-07.C40 and 721-38.1J to reconstruct the unique short region. Subgenomic clone 721-38.1J contains an insertion of the MDV gB, gA, and gD genes. A large molar excess of these clones is cotransfected with a sub-infectious dose of Sb-1 genomic DNA. To determine the appropriate subinfectious dose, transfection of the SB-1 is titrated down to a dose which no longer yields virus plaques in cell culture. Such a dose contains sub-genomic fragments spanning the unique long region of SB-1 which recombine withthe HVT unique short subgenomic clones.

Therefore, a virus resulting from recombination between overlapping homologous regions of the SB-1 and HVT 15 subgenomic fragments is highly favored. Alternatively, SB-1 genomic fragments from the unique long region are .:...subcloned into cosmid vectors. A recombinant virus containing the Sb-1 unique long the HVT unique short with the MDV, gB, gA, and gD genes were produced using 20 the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. This procedure is also used with an HVT subgenomic clone to insert antigen genes from other avian pathogens including but not limited to infectious laryngotracheitis virus, 25 Newcastle's disease virus and infectious bursal disease virus.

Example Recombinant HVT expressing chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN) are useful as vaccines against Marek's disease virus and are also useful to enhance the immune response against other diseases of poultry. Chicken myelomonocytic growth factor (cMGF) is related to mammalian G-CSF and interleukin-6 protein and chicken interferon (cIFN) is homologous to marmalian type 1 interferon 135 (59) interferon. When used in combination with vaccines described in previous examples, S-HVT-144 or HVT expressing cIFN are useful to provide enhanced mucosal, humoral, or cell mediated immunity against avian disease-causing viruses including, but not limited to, Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus.

Recombinant HVT expressing cMGF or cIFN are useful provide enhanced immunity against avian disease causing organismsdescribed in Example Example 20A S-HVT-144 15 S-HVT-144 is a recombinant herpesvirus of turkeys that *contains the chicken myelomonocytic growth factor (cMGF) gene inserted into an XhoI site converted to a PacI site in the EcoRl #9 fragment within the unique long region of HVT. The cMGF gene is in the opposite S* 20 transcriptional orientation to the open reading frame (ORF A) within the EcoRl #9 fragment of the HVT genome (Figure 14; SEQ ID NOs: 48 and 50). The cMGF gene is expressed from a human cytomegalovirus immediate early promoter. S-HVT-144 is useful as a vaccine in poultry S 25 against Marek's Disease.

S-HVT-144 was constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 751-87.A8 with Asc I, 415-09.BA1 with BamHI.

Example 20B Recombinant HVT expressing chicken interferon 136 A recombinant herpesvirus of turkeys contains the chicken interferon (cIFN) gene inserted into an XhoI site converted to a PacI site in the EcoRl #9 fragment within the unique long region of HVT. The cIFN gene is expressed from a human cytomegalovirus immediate early promoter. Recombinant HVT expressing cIFN is useful as a vaccine in poultry against Marek's Disease.

Recombinant HVT expressing cIFN is constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 15 with NotI, 761-07.A1 with Asc I, 415-09.BA1 with BamHI.

o Recombinant HVT expressing avian cytokines is combined with HVT expressing genes for avian disease antigens to enhance immune response. Additional cytokines that are 20 expressed in HVT and have immune stimulating effects include, but not limited to, transforming growth factor •beta, epidermal growth factor family, fibroblast growth factors, hepatocyte growth factor, insulin-like growth factors, B-nerve growth factor, platelet-derived growth 25 factor, vascular endothelial growth factor, interleukin 1, IL-1 receptor antagonist, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, IL-6 soluble receptor, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, interleukin 13, angiogenin, chemokines, colony stimulating factors, granulocyte-macrophage colony stimulating factors, erythropoietin, interferon, interferon gamma, leukemia inhibitory factor, oncostatin M, pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor, tumor necrosis factors, and soluble TNF receptors. These cytokines are 137 from avian species or other animals including humans, bovine, equine, feline, canine or porcine.

Example 20C Recombinant HVT expressing Marek's disease virus genes and chicken interferon gene.

A recombinant herpesvirus of turkeys contains the chicken interferon (cIFN) gene inserted into an Xhol site converted to a PacI site in the EcoRl #9 fragment within the unique long region of HVT and further contains the MDV gA, gD, and gB genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The cIFN gene is expressed from an human 15 cytomegalovirus immediate early promoter. The MDV genes are expressed from the endogenous MDV promoters.

Recombinant HVT expressing cIFN and MDV gA, gB, and gD is useful as a vaccine with an enhanced immune response in poultry against Marek's Disease.

Recombinant HVT expressing MDV genes and the cIFN gene is constructed according to the PROCEDURE FROM GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of "o 25 subgenomic clones and enzymes are used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672- 07.C40 with NotI, 672-01.A40 with NotI, 761-07.A1 with Asc I, 721-38.1J uncut.

Example 20D Recombinant HVT expressing Marek's disease virus genes, Newcastle disease virus genes and chicken interferon gene.

A recombinant herpesvirus of turkeys contains the chicken interferon (cIFN) gene inserted into an XhoI site converted to a PacI site in the EcoR1 #9 fragment within the unique long region of HVT and further 138 contains the MDV gA, gD, and gB genes and NDV HN and F genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The cIFN gene is expressed from an human cytomegalovirus immediate early promoter. The MDV genes are expressed from the endogenous MDV promoters. The NDV HN gene is under the control of the PRV gX promoter, and the NDV F gene is under the control of the HCMV immediate early promoter.

Recombinant HVT expressing cIFN and MDV gA, gB, and gD is useful as a vaccine with an enhanced immune response in poultry against Marek's Disease and Newcastle disease.

Recombinant HVT expression MDV genes, NDV genes and 15 cIFN is constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes are used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 20 672-07.C40 with NotI, 672-01.A40 with NotI, 761-07.A1 with Asc I, 722-60.E2 uncut.

*set*: Example 20E Recombinant HVT expressing Marek's disease virus genes and chicken 25 myelomonocytic growth factor gene.

A recombinant herpesvirus of turkeys contains the chicken myelomonocytic growth factor (cMGF) gene inserted into and XhoI site converted to a PacI site in the EcoRl #9 fragment within the unique long region of HVT and further contains the MDV gA, gD, and gB genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The cMGF gene is expressed from a human cytomegalovirus immediate early promoter. The MDV genes are expressed from the endogenous MDV promoters. Recombinant HVT expression cMGF and MDV gA, gB, and gD is useful as a vaccine with 139 an enhanced immune response in poultry against Marek's Disease.

Recombinant HVT expressing the cMGF gene and MDV genes is constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes are used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672- 07.C40 with NotI, 672-01.A40 with NotI, 751-87.A8 with Asc I, 721-38.1J uncut.

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SExample 20F Recombinant HVT expressing Marek's disease virus genes, Newcastle disease 15 virus genes and chicken myelomonocytic

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growth factor gene.

A recombinant herpesvirus of turkeys contains the chicken myelomonocytic growth factor (cGMF) gene 20 inserted into an XhoI site converted to a PacI site in the EcoR1 #9 fragment within the unique long region of HVT and further contains the MDV gA, gD, and gB genes and NDV HN and F genes inserted into a unique StuI site converted into a HindIII site in the HVT US2 gene. The cGMF gene is expressed from an human cytomegalovirus immediate early promoter. The MDV genes are expressed from the endogenous MDV promoters. The NDV HN gene is under the control of the PRV gX promoter, and the NDV F gene is under the control of the HCMV immediate early promoter. Recombinant HVT expressing cIFN and MDV gA, gB and gD is useful as a vaccine with an enhanced immune response in poultry against Marek's Disease and Newcastle disease.

Recombinant HVT expressing MDV genes, NDV genes and the cGMF gene is constructed according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING 140 SUBGENOMIC FRAGMENTS. The following combination of subgenomic clones and enzymes are used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672- 07.C40 with NotI, 672-01.A40 with NotI, 751-87.A8 uncut, 722-60.E2 uncut.

Example 21 Recombinant herpesvirus of turkeys expressing antigens from disease causing microorganisms Recombinant herpesvirus of turkeys (HVT) is useful for expressing antigens from disease causing microorganisms from animals in addition to avian species. Recombinant HVT is useful as a vaccine in animals including but not 15 limited to humans, equine, bovine, porcine, canine and feline.

e o Recombinant HVT is useful as a vaccine against equine diseases when foreign antigens from diseases or disease 20 organisms are expressed :in the HVT 'vector, -including but not limited to: equine influenza, equine herpesvirus-1 and equine herpesvirus-4. Recombinant HVT is useful as a vaccine against bovine diseases when foreign antigens from the following diseases or disease organisms are expressed in the HVT vector, including, but not limited to: bovine herpesvirus type 1, bovine viral diarrhea virus, bovine respiratory syncytial virus, bovine parainfluenza virus. Recombinant HVT is useful as a vaccine against swine diseases when foreign antigens from the following diseases or disease organisms are expressed in the HVT vector, including but not limited to: pseudorabies virus, porcine reproductive and respiratory syndrome (PRRS/SIRS), hog cholera virus, swine influenza virus, swine parvovirus, swine rotavirus. Recombinant HVT is useful as a vaccine against feline or canine diseases when foreign antigens from the following diseases or disease organisms are 141 expressed in the HVT vector, including but not limited to feline herpesvirus, feline leukemia virus, feline immunodeficiency virus and Dirofilaria immitis (heartworm). Disease causing microorganisms in dogs include, but are not limited to canine herpesvirus, canine distemper, canine adenovirus type 1 (hepatitis), adenovirus type 2 (respiratory disease) parainfluenza, Leptospira canicola, icterohemorragia, parvovirus, coronavirus, Borrelia burgdorferi, canine herpesvirus, Bordetella bronchiseptica, Dirofilaria immitis (heartworm) and rabies virus.

Example 22 Human vaccines using recombinant herpesvirus of turkeys as a vector Recombinant herpesvirus of turkeys (HVT) is useful as a vaccine against human diseases. For example, human influenza is a rapidly evolving virus whose neutralizing viral epitopes are rapidly changing. A 20 useful recombinant HVT vaccine is: one in which the influenza neutralizing epitopes are quickly changed to protect against new strains of influenza. Human influenza HA and NA genes are cloned using.polymerase chain reaction into the recombinant HVT. Recombinant HVT is useful as a vaccine against other human diseases when foreign antigens from the following diseases or disease organisms are expressed in the HVT vector: hepatitis B virus surface and core antigens, hepatitis C virus, human immunodeficiency virus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, measles virus, hantaan virus, pneumonia virus, rhinovirus, poliovirus, human respiratory syncytial virus, retrovirus, human T-cell leukemia virus, rabies virus, mumps virus, malaria (Plasmodium falciparum), Bordetella pertussis, Diptheria, Rickettsia prowazekii, 142 Borrelia bergdorferi, Tetanus toxoid, malignant tumor antigens, Recombinant HVT expressing human cytokines is combined with HVT expressing genes for human disease antigens to enhance immune response. Additional cytokines, including, but not limited to,transforming growth factor beta, epidermal growth factor family, fibroblast growth factors, hepatocyte growth factor, insulin-like growth factors, B-nerve growth factor, platelet-derived Sgrowth factor, vascular endothelial growth factor, interleukin 1, IL-1 receptor antagonist, interleukin 2, interleukin 3, interleukin 4, interleukin interleukin 6, IL-6 soluble receptor, interleukin 7, S 15 interleukin 8, interleukin 9, interleukin interleukin 11, interleukin 12, interleukin 13, angiogenin, chemokines, colony stimulating factors, granulocyte-macrophage colony stimulating factors, erythropoietin, interferon, interferon gamma, leukemia 20 inhibitory factor, oncostatin M, pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor, tumor necrosis factors, and soluble TNF receptors from human and other animals are expressed in HVT and have immune stimulating effects.

Example 23 Improved production of a recombinant herpesvirus of turkeys vaccine.

Cytokines, such as interferons and interleukins, inhibit the replication of viruses in cell culture and in the animal. Inhibition of the production of cellular interferon or interleukin improves the growth of recombinant HVT in cell culture. Chicken interferon (cIFN) expressed from a recombinant swinepox vector was added to chick embryo fibroblast (CEF) cell cultures and infected with S-HVT-012 which expresses 6galactosidase. cIFN added to the cell culture media 143 reduced both the expression of G-galactosidase and S- HVT-012 titer in a dose dependent manner. This result indicates that growth of HVT is limited by exogenous addition of chicken interferon. Several strategies are utilized to improve growth of HVT in CEF cells by removing or inactivating chicken interferon activity in the CEF cells.

In one embodiment, a chicken interferon neutralizing antibody is added to the culture medium to inhibit the chicken interferon activity and improve the growth of recombinant HVT in CEF cell culture. The anti-cIFN antibody is derived from mouse or rabbit sera of animals injected with chicken interferon protein, S 15 preferably the cIFN is from a recombinant swinepox virus expressing chicken interferon.

Poxviruses secrete cytokine-inhibiting proteins as an immune evasion strategy. One type of poxvirus immune 20 evasion mechanism involves poxvirus soluble receptors for interleukins, interferon, or tumor necrosis factors which inactive the cytokines and allow viral replication In an embodiment of the invention, fowlpox virus is useful as a source. of- chicken interferon-inhibiting proteins and other immune evasion proteins. Conditioned media from FPV infected CEF cell cultures is added to the HVT infected CEF cells to inhibit interferon activity and increase the HVT titer.

In a further embodiment, the recombinant chicken interferon inhibiting protein or another poxvirus immune evasion protein is expressed in a vector in combination with an HVT vaccine composition to increase the HVT titer.

Chicken embryo fibroblast cells have been engineered to express foreign genes in a further embodiment, an interferon-negative CEF cell line is constructed by 144 the introduction of a vector expressing a gene encoding antisense RNA for chicken interferon into the CEF cell line. Recombinant HVT grown in an interferon-negative CEF cell line demonstrate improved virus titers compared to HVT grown in an interferon producing CEF cell line. In a further embodiment, a chicken myelomonocytic growth factor (cMGF) -positive CEF cell line is constructed by the introduction of a vector expressing the cMGF gene into the CEF cells.

Recombinant HVT grown in a cMGF-positive CEF cell line demonstrates improved virus titers compared to HVT grown in a cMGF negative CEF cell line.

Recombinant HVT of the present invention is-useful as 15 a vaccine against Marek's disease and against other diseases as outlined in previous examples. An ****increased efficiency in growth of recombinant HVT in CEF cells is useful in production of the vaccine.

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145 References 1. Buckinaster et al., J. Gen. Virol. 69:2033, 1988.

2. F.A. Ferrari et al., Journal of Bacteriologyl161, 556-562, 1985.

3. U. Gubler and B.J Hoffman, Gene 25, 263-269.

4. D. Hanahan, Mol ecular Biology 166, 557-580, 1983.

P.J. Hudson et al., Nucleic Acid Research 14, 5001-5012, 1986.

15 6. T. Igarashi et al., 1ath International Herpesvirus Workshop, Abstract No. 17, Ann Arbor, Michigan, August 1985.

7. T. Ihara et al., Virus Genes 3, 127-140, 1989.

8. M. A. Innis et al., PCR Protocols A Guide to Methods and Applications, 84-91, Academic Press, Inc., San Diego, 1990.

9. R.J. Isfort et al., 9th International Herpesvirus Workshop, Abstract No. 146, Seattle, Washington, August 1984.

M.N. Jagadish et al., J. of Virol. 62, 1084-1087, 1988.

11. Kawai and Nishizawa Mo). arnd Cell Bio. 4, 1172- 1174, 1984.

12. B. Lomnicz i et al., Journal of Virology 49, 970- 979 1984.

13. Maniatis et Molecular CloninqT. Cold Suriria 146 14. D.J. McGeoch et al., Journal of Molecular Biology 181, 1-13, 1985.

S.L. McKnight and R. Kingsbury, Science 217, 316- 324., 1982.

16. L.J.N. Ross et al., Journal of General Virology 1789-1804, 1989.

17. L.J.N. Ross et al., Journal of General Virology 72, 949-954, 1991.

18. J. Sambrook et al., Molecular Cloning A Laboratory Manual Second Edition, Cold Spring Harbor Press, 1989.

19. M. Zijil et al., Journal of Virology 62, 2191- 2195, 1988.

20. Maniatis et al., Intervirology 16, 201-217,' 1981.

21. S.L. Mansour et al., Proc. Natl. Acad. Sci. USA 82, 1359-1363, 1985.

22. C. Thummel et al., Cell 33, 455-464, 1983.

23. D. Scolnick, Cell 24, 135-143, 1981.

24.* C. Thunimel et al., Cell 23, 825-8361, 1981.

Y. Haj-Ahtned and F.L. Graham, J. of Virology 57, 267- 274, 1986.

26. M. Mackett et al., Proc. Natl. Acad. Sdi. USA 79, 7415-7419, 1982.

27. D. Panicali and E. Paoletti, Proc. Natl. Acad.

Sci. USA 79, 4927-4931, 1982.

147 28. E. Paoletti et al. Proc. Natl. acad. Sci. USA 81, 193-197, 1984.

29. G.L. Smith et al., Nature 302, 490-495, 1983.

J.H. Gillespie et al., J. Clin. Microbiology 23, 283-288, 1986.

31. D. Panicali et al., Proc. Natl. Acad. Sci. USA 80, 5364-5368, 1983.

32. G.L. Smith et al., Pro Natl. Acad. Sci. USA 7155-7159, 1983.

15 33. G.L. Smith et al., Science 224, 397-399, 1984.

34. M. Mackett et al., Science 227, 433-435, 1985.

E.S. Moccarski et al., Cell 22, 243-255, 1980.

L.E. Post and B. Roizman, Cell 25, 227-232, 198-1.

41. K.L. Poffenberger et al., Proc. Natl. Acad. Sdi.

USA 80, 2690-2694, 1981.

42. M.G. Gibson and P.G. Spear, Journal of Virology 48, 396-404, 1983.

43. Lee et al., Proc. Natl. Acad. Sci. USA 79, 6612-6616, 1982.

44. M. Shih e t al. Proc. Na tl Acad. Sci. USA 81, 5867-5870, 1984.

45. R. Desrosiers et al. Ninth Annual Herpesvirus meeting, Seattle, Abstract #280, 1984.

46. M. Arsenakis and B. Roizman, in "The High 148 Society for Microbiology, Washington 1985 (Proceedings of the First Annual Southwest Foundation for Biomedical Research International Symposium, Houston, Texas, 8-10 November 1984).

47. L.E. Post et al., Tenth International Herpesvirus Workshop, Ann Arbor, August 1985.

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49. A.M. Griffin, Journal of General Virology 72, 393-398, 1991.

S 15 50. D.R. Thomsen et al., Gene 16, 207-217, 1981.

51. Carpenter, D.E. and Misra, V. Journal of General Virology 72 3077-3084 (1991).

20 52. Kibenge, Jackwood, Mercado, C.C., Journal of General Virology 71 569-577 (1990).

S53. Fukuchi et al., J. Virologu 51 102-109, 1984.

54. Fukuchi et al., J. Virologu 53 994-997, 1985.

Ross, et al., Virus Genes 7 33-51, 1993.

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57. Ross, et al., J. General Virology 64:2785-2790, 1983.

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149 G.L. Smith, Journal of General Virology 74, 1725- 1740 (1993).

61. B. Scgumacher, et al., Virology 203, 144-148 (1994) 150 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: SYNTRO CORPORATION (ii) TITLE OF INVENTION: Recombinant Herpesvirus of Turkeys And Uses Thereof (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: John P. White STREET: 1185 Avenue of the Americas CITY: New York STATE: New York COUNTRY: USA S(F) ZIP: 10036 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk 0. COMPUTER: IBM PC compatible S* OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 09-AUG-1995

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: White, John P REGISTRATION NUMBER: 28,678 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212)278-0400 TELEFAX: (212)391-0526 TELEX: 422523 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3350 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 129..2522 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGATACGATC GGTCTGACCC GGGGGAGTCA CCCGGGGACA GCCGTCAAGG CCTTGTTCCA GGATAGAACT CCTCCTTCTA CAACGCTATC ATTGATGGTC AGTAGAGATC AGACAAACGA 120 151 ATA CGG AGC CTT CTG ATG CCA ACA ACC GGA CCG GCG TCC ATT CCG GAG Gly Pro Ala Ser Ile Pro Giu 000.

0.* a 0..

Ile

ACA

Thr

ACT

Thr

CCT

Pro

TAC

Tyr

TAC

Tyr

ACA

Thr

ACC

Thr

TTG

Leu

GTA

Val

GGG

Giy 175

AAA

Lys

ATA

Ile

GGG

Giy

CTC

Leu

GTA

Val 255 Arg

CCC

Pro

GTG

Val

GGC

Gly

AAG

Lys

AAC

Asn

CTT

Leu

TTC

Phe

ATG

Met

GGG

Giy 160

TAT

Tyr

ATG

Met

ACT

Thr

GTA

Val

AGC

Ser 240

CTG

Leu Ser

TGG

Trp,

GGG

Gly

TCA

Ser

TTC

Phe

TAC

Tyr

CCT

Pro

CAA

Gin

TCT

Ser 145

GAA

Giu

GTO

Val

GTA

Val

GCA

Aila

ACA

Thr 225

OTT

Val

GC

Gly Leu

AGA

Arg

GAC

Asp

ATT

Ile

GAT

Asp

TGC

Cys

GGT

Gly

GGA

Gly 130

GCA

Al a

GGG

Gly

AGO

Arg

GCC

Al a

GCC

Ala 210

ATC

Ilie

GGG

Gly

GCC

Ala

AGC

Ser

ACA

Thr

GTG

Val

CGG

Arg

AG

Arg

GGC

Gly 115

AGC

Ser

ACA

Thr

GTC

Val

CTT

Leu

ACA

Thr 195

OAT

Asp

ACA

Thr

GGA

Gly

ACC

Thr Met 20

ACA

Thr

GG

Gly

GGT

Gly

ATG

Met

CTA

Leu 100

GTT

Val

CTG

Leu

GCC

Ala

ACC

Thr

GGT

Gly 180

TGT

Cys

GAT

Asp

CTG

Leu

GAG

Glu

ATC

Ile 260 Pro

CTC

Leu

TCA

Ser

GCT

Ala

CTC

Leu 85

GTG

Val

TAT

Tyr

AGT

Ser

AAC

Asn

GTC

Val 165

GAC

Asp

GAC

Asp

TAC

Tyr

TTC

Phe

CTC

Leu 245

TAC

Tyr Thr

TCA

Ser

GGG

Gly

CAC

His 70

CTG

Leu

AGT

Ser

GCA

Ala

GAA

Glu

ATC

Ile 150

CTC

Leu

CCC

Pro

AGC

Ser

CAA

Gin

TCA

Ser 230

GTG

Val1

CTC

Leu Thr

GT

Gly

CTA

Leu 55

TAC

Tyr

ACT

Thr

CGG

Arg

CTA

Leu

CTG

Leu 135

AAC

Asn

AGC

Ser

ATT

Ile

AGT

Ser

TTC

Phe 215

.GCC

Ala

TTT

Phe

ATA

Ile CAG AGA Gin Arg 40 ATT GTC Ile Val ACA CTG Thr Leu 0CC CAG Ala Gin AGT CTC Ser Leu 105 AAC GGC Asn Oly 120 ACA OAT Thr Asp GAC AAA Asp Lys TTA CCC Leu Pro CCC GCA* Pro Ala 185 GAC AGO, Asp Arg 200 TCA TCA Ser Ser AAC ATT Asn Ile CGA ACA Arg Thr GOC TTT Giy Phe 265

CTG

Leu

TTT

Phe

CAG

Gin

AAC

Asn

ACA

Thr

ACC

Thr

GTT

Val

ATT

Ile

ACA

Thr 170

ATA

Ile

CCC

Pro

CAG

Gin

GAT

Asp

AGC

Ser 250

OAT

Asp

ACC

Thr

TTC

Phe

AGC

Ser

CTA

Leu

GTG

Val

ATA

Ile

AGC

Ser 000 Gly 155

TCA

Ser

.GGG

Gly

AGA

Arg

TAC

Tyr 0CC Ala 235

GTC

Vai

GG

Gly

TAC

Tyr

CCT

Pro

AAT

Asn

CCG

Pro

AGO

Arg

AAC

Asn

TAC

Tyr 140

AAC

Asn

TAT

Tyr

CTT

Leu

GTC

Val

CAA

Gin 220

ATC

Ile

CAC

His

ACA

Thr

AAT

Asn

GGA

Gly 000 Gly 0CC Ala TrCA Ser

GCC

Ala 125

AAT

Asn

GTC

Val

GAT

Asp

GAC

Asp

TAC

Tyr 205

CCA

Pro

ACA

Thr

GGC

Gly

ACG

Thr TTo Leu

TTC

Phe

AAC

Asn

AGT

Ser

AGC

Ser 110

GTG

Val

GG

Gly

CTA

.Leu

CTT

Leu

CCA

Pro 190

ACC,

Thr

GOT

Oly

AGC

Ser

CTT

Leu

GTA

Val 270 218 266 314 362 410 458 506 554 602 650 698 746 794 842 ATC ACC AGG OCT GTG 0CC OCA AAC ACT 000 CTG ACG ACC GGC ACC GAC Ile Thr Arg Ala Val Ala Ala Asn Thr 275 Gly 280 Leu Thr Thr Gly Thr Asp 152 AAC CTT ATG CCA TTC AAT CTT GTG ATT CCA Asn Leu Met Pro Phe Asn Leu Val Ile Pro ACA AAC GAG ATA ACC CAG Thr Asn Giu Ile Thr Gin 290 0 *rose 0* 0 se.

so** 0 .eve ease 800

CCA

Pro

CAG

Gin

ACG

Thr 335

GTG

Val

GTG.

Val

GTT

Val

TTG

Leu

ACA

Thr 415

CTC

Leu

ATC

Ile

CCA

Pro

CTG

Leu

TCA

Ser 495

GCC

Al a

CAG

Gin

ATC

Ilie

GCA

Ala 320

ATC

Ile

GCC

Ala

AGC

Ser

ACA

Thr

ATA

Ile 400

AGG

Arg

AAC

Asn

CGG

Arg

CCT

Pro

CTG

Leu 480

GGA

Gly

GCC

Al a

AAT

Asn

ACA

Thr 305

GG

Giy

CAT

His

TAC

Tyr

AAC

Asn

GAA

Giu 385

CTG

Leu

GAG

Giu

TCT

Ser

GCC

Al a

GCC

Al a 465

GGC

Gly

AAA

Lys

GAC

Asp

CCC

Pro

TCC

Ser

GAT

Asp

GGT

Giy

GAA

Giu

TTC

Phe 370 TAC4 Tyr4

AGT

Ser

TAC

TPyr

CCC

Pro

ATA

Ile 450

GCT

Al a

GAT

Asp

GCA

Al a

AAG

Lys

GTA

Val 530 kLTC Ilie

CAG

31n

GGC

Gly kGA Arg 355

GAG

Glu

GGC

Gly

GAG

Glu

ACT

Thr

CTG

Leu 435

AGG

Arg

CCC

Pro

GAG

Giu

AGA

Arg

GGG

Gly sis

GTC

Val

AAA

Lys

ATG

Met

AAC

Asn 340

GTG

Val

CTG

Leu

CGA

Arg

AGG

Arg

GAC

Asp 420

AAG

Lys

AGG

Arg

CTA

Leu

GCA

Al a

GCT

Al a 500

TAC

Tyr

GAC

Asp

CTG

Leu4

TTA

Leu 325

TAT

Tyr

GCA

Ala

ATC

Ile

TTT

Phe

GAC

Asp 405

TT

Phe

ATT

Ile

ATA

Ile

GCC

Ala

CAG

Gin 485

GCC

Al a

GAG

Glu

GGG

Giy

GAG

flu 310

TGG

rrp

CCA

Pro

ACA

l'hr

CCA

Pro

GAC

Asp 390

CGT

Arg

CGT

Arg

GCA

Ala

GCT

Ala

CAT

His 470

GCT

Ala

TCA

Ser

GTA

Val

AT'I

Ile 295 ATA4 Ile TCG4 Ser

GGG'

Gly

GGA.

Giy

AAT

Asn 375

CCA

Pro cTr Leu

GAA

Giu

GGA

Gly

GTG

Val 455

GCA

Ala

GCT

Ala

*GGC

Gly

GTC

Val1

*CTT

Leu 535

GTG

Val1

GCA

Ala

GCC

Ala

TCC

Ser 360

CCT

Pro

GGA

Gly

GGC

Giy

TAC

Tyr

GCA

Ala 440

CCG-

Pro

ATT

Ile

TCA

Ser

CGC

Arg

GCG

Al a 520

GCT

Ala

ACC

Thr

AGA

Arg

CTC

Leu 345

GTC

Val GAA4 Giu

GCC

Ala

ATC

Ile

TTC

Phe 425

TTC

Phe

GTG

Val

GGG

Gly

GGA

Gly

ATA

Ile 505

AAT

Asn

TCA

Ser rcc 3er 3GG 'iY 330

CGT

krg

GTT

Ial

CTA

Leu

P.TG

Me t

AAG

[Ls 410

ATG

Met

GGC

Gly

GTC

Val

GAA

Giu

ACT

Thr 490

AGG

Arg

CTA

Leu

CCI

Pro 300 AAA AGT Lys Ser 315 AGC CTA Ser Leu CCC GTC Pro Val ACO GTC Thr Val GCA AAG Ala Lys 380 AAC TAC Asn Tyr 395 ACC GTC Thr Val GAG GTG Giu Val TTC AAA Phe Lys TCC ACA Ser Thr 460 GGT GTA Giy Vai 475 GCT CGA Ala Arg CAG CTG Gin Leu TTC GAG Phe Gin GGG GTA Gly Val 540

GGT

Giy

GCA

Ala

ACG

Thr

GCT

Ala 365

AAC

Asn

ACA

Thr

TGG

Trp

GCC

Al a

GAC

Asp 445

TTG

Leu

GAC

Asp

GCC

Al a

ACT

Thr

GTG

Val 525

CTC

GGT

Gly

GTG

Val

CTA

Leu 350

CG

Gly

CTG

Leu

AAA

Lys

CCA

Pro

GAG

Asp 430

ATA

Ile

TTC

Phe

TAC

Tyr

GCG

Al a

CTC

Leu 510

CCC

Pro

CC

1034 1082 1130 1178 1226 1274 1322 1370 1418 1466 1514 1562 1610 1658 1706 1754 1802 Leu Arg GGT GCA GAG Gly Ala His 545 AAC CTC GAC TGC Asn Leu Asp Cys TTA AGA GAG GGT Leu Arg Glu Giy

GCC

Al a 555 ACG GTA TTC Thr Leu Phe 153 GTT ATT ACG ACA GTG GAA GAC GCC ATG ACA CCC AAA Val Ile Thr Thr Val Glu Asp Ala Met Thr Pro Lys CCT GTG Pro Val GCA TTG Ala Leu 560 565 a.

AAC AGC Asn Ser 575 CCT CCA Pro Pro GTC TAT Val Tyr GAC TAC Asp Tyr CTG TCC Leu Ser 640 GCC ATA Ala Ile 655 GCT ATG Ala Met TTT AGA Phe Arg GCT GGT Ala Gly TTC ATC Phe Ile 720 TAC CTC Tyr Leu 735 CTT GCC Leu Ala CGT CAG Arg Gin TGC ACT

AAA

Lys

TCT

Ser

GGA

Gly

ACC

Thr 625

AAA

Lys

GCT

Ala

ACG

Thr

AGC

Ser

CCC

Pro 705

AAA

Lys

AAC

Asn

ATG

Met

AGC

Ser

CAG

ATG

Met

CAAS

Gin

TAT

Tyr 610

GTT

Val

GAT

Asp

TAC

Tyr

GGA

Gly

ACC

Thr 690

GGA

Gly

CGT

Arg

CTA

Leu

GCT

Al a

AAT

Asn 770

TGT

Phe

AGA

Arg 595

GCT

Ala

GTC

Val

CCC

Pro

ATG

Met

GCC

Al a 675

AAG

Lys

GCA

Ala

TTC

Phe

CCA

Pro

GCA

Al a 755

GGA

Gly

GTT

GCT GTC Ala Val 580 GGA TCC Gly Ser CCA GAT Pro Asp CCA ATA Pro Ile ATA CCT Ile- Pro 645 GAT GTG Asp Val 660 CTC AAT Leu Asn CTC, GCC Leu Ala TTC GAT Phe Asp CCT CAC Pro His 725 TAC CTT Tyr Leu 740 TCA GAG Ser Giu AGC AGC Ser Ser CAT GTG

ATT

Ile

TTC

Phe

GGG

Gly

GAT

Asp 630

CCT

Pro

TTT

Phe

GCT

Ala

ACT

Thr

GTA

Val 710 Asn

CCA

Pro

TTC

Phe

AGC

Ser

GCT

GAA

Glu

ATA

Ile

GTA

Val 615

GAT

Asp

ATT

Ile

CGA

Arg

TGT

Cys

GCA

Ala 695

AAC

Asn

CCA

Pro

CCC

Pro

AAG

Lys

CAA

Gin 775

GGA

GGC

Gly

CGA

Arg 600

CTT

Leu

GTC

Val

GTG

Val

CCC

Pro

GGC

Gly 680

CAC

His

ACC

Thr

CGC

Arg

AAT

Asn

AGA

Arg 760

CGT

Arg

AGA

570.

GTG CGA Val Arg 585 ACT CTC Thr Leu CCA CTG Pro Leu TGG GAC Trp Asp GGA AAC Gly Asn 650 AAA GTC Lys Val 665 GAG ATT Glu Ile CGA CTT Arg Leu GGG CCC Gly Pro GAC TGG Asp Trp, 730 OCA GGA Ala Gly 745 CCC CGA Pro Arg GGA CCC Gly Pro GAA TGG

GAA

GiU

TCT

Ser

GAG

Giu

GAC

Asp 635

AGT

Ser

CCA

Pro

GAG

Giu

GGC

Gly

AAC

Asn 715

GAC

Asp

CGC

Arg

ACT

Thr

ACT

Thr

GAT

GAC CTC Asp Leu GGA CAC Gly His 605 ACT GGG Thr Gly 620 AGC ATT Ser Ile GGA AAT Gly Asn ATC CAT Ile His AAA GTA Lys Val 685 CTT AAG Leu Lys 700 TGG GCA Trp Ala AGG' CTC 'Arg Leu CAG TAC Gin Tyr CGA GAG Arg Glu 765 ATT CCA Ile Pro 780 TGT GAC

CAA

Gin 590

AGA

Arg

AGA

Arg

ATG

Met

CTA

Leu

GTG

Vai 670

AGC

Ser

TTG

Leu

ACG

Thr

CCC.

Pro

CAC

His 750

TGC

Cys

ATC

Ile

TGA

1850 1898 1946 1994 2042 2090 2138 2186 2234 2282 233.0' 2378 2426 2474 2522 2582 2642 2702 2762 Cys Thr Gin Cys Val His Val Ala Giy Arg Glu Trp Asp Cys Asp 785 790 795 CATGGCCAAC TTCGCACTCA GCGACCCGAA CGCCCATCGG CGACCACAAG CAGGCAGCAA GTCGCAAAGG GCCAAGTACG GAGGCTCGGG GCCCCCACAC CAGAGGAAGC ACAGAGGGAA GAAGATGGAG ACCATGGGCA TCTACTTTGC AACACCAGAA ATGCGAAATT TTTTTGCAAA GGACAGCAGG CTACGGAGTG AA.AGACACAC GGATCTCAAA TGGGTAGCAC TCAATGGGCA 154

CCGAGGGCCA

AACGAGGACT

ATCCTAAGGG

T'TCATAGACG

CAGATGAAAG

CTACCAAAGC

CTGGATCAGG

CACCCGCGCA

GGTCCCCAAA

AAGTACCTTC

AGCCCCGGCC

ATCTAGACTA

CAGCTACGTC

AAGTTGCCAA

ATCTGCTCTT

CCAAGCCAAA

ACCGTCTCTG

GGTGTGGACA.

AAAAAAAAAk

TGAGGCGGAA

AGCTAAAGTA

CGTGCATGCA.

AGATCTACGG

AGTCTATGAA

GACTGCGATG

ACCCAATGCT

ATGAGGACCT

CAATTCGGCC

AGAACCAGCC

CGGGCAGAAC

GAGAAGAGCC

GGCTCCAGGA

ATCAACCATG

GAGATGAAGC

CCAACACAGA

TGAGTGAGGC

TTACAACATC

GGATCCCTCG

ACACGAGAAA

GGTTGGCATC

CAGGCAGAGC

GACGTGGCCC

ATCGCAATCC

GACCCCCTGG

TCCTGGGAGT

CCAAATTGGA

AGGGATCC

TACGGACCCA

AGAAGAACAA

ACCCCAAGCT

AAACCAAGAA

CAGGCGGGCT

TCGGCTGGGG

CTCCCGACAA

TCCGTTCGCG

2822 2882 2942 3002 3062 3122 3182 3242 3302 3350

S

INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 797 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

S

1 Ser. Leu Asn Leu Gin Asp Gin Thr Gin Gin Ile Val Pro 5 10 Leu Met Pro Thr Thr Gly Pro Ala 25 Ser Ile Pro Phe Ile Arg Glu~ Thr Pro Leu Thr Val Phe Pro Gly Trp Arg Ser Thr Leu Gly Asp Thr.Gly Ser Ser Gly Gin Arg Leu Thr Tyr Gl-y Ser Ile Gly Leu His Tyr Ile Val Phe Phe Val Gly Ala Thr Leu Gin Phe 70 Leu 75 Leu Gly Asfl Tyr Lys Asp Arg Met Leu Thr Ala Gin 90 Thr Pro Ala Ser Tyr Asn 95 Tyr Cys Arg Leu Val 100 Pro Gly Gly Val Tyr 115 Gin Giv Ser Leu Ser Ser Arg Ser Leu 105 Val Arg Ser 110 Val Thr Phe Ala Leu Glu Leu 135 Asn Gly Tkhr 120 Thr Asp Val Ile Asn Ala 125 Ser Tyr Asn Gly Leu Met 140 130 Ser Ala 145 Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val Gly 150 155 160 Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr 165 170 175 Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys Met 155 0 0.0.0.

Val Ala Thr 195 Ala Ala Asp 210 Thr Ilie Thr 225 Val Gly Gly Gly Ala Thr Arg Ala Vai 275 Met Pro Phe 290 Thr Ser Ile 305 Gly Asp Gin His Giy Gly Tyr Glu Arg 355 Asn Phe Glu 370 Giu Tyr Gly 385 Leu Ser Glu Glu Tyr Thr Ser Pro Leu 435 Ala Ilie Arg 450 Ala Ala Pro 465 Gly Asp Glu Lys Ala Arg Asp Lys Gly 515 180 Cys Asp Leu Giu Ile 260 Ala Asn Lys Met Asn 340 Val Leu Arg Arg Asp 420 Lys Arg Leu Ala Al a 500 Tyr Asp Tyr Phe Leu 245 Tyr Ala Leu Leu Leu 325 Tyr Ala Ile Phe Asp 405 Phe Ile Ile Al a Gin 485 Al a Ser Gin Ser 230 Vai Leu Asn Val Giu 310 Trp Pro Thr Pro Asp 390 Arg Arg Ala Ala His 470 Ala Ser Se'- Phe 215 Ala Phe Ile Thr Ile 295 Ile Ser Giy Gly Asn 375 Pro Leu Glu Gly Val 455 Ala Al a Gly Asp 200 Ser Asn Arg Gly Gly 280 Pro Val Ala Ala Ser 360 Pro Gly Gly Tyr Ala 440 Pro Ile Ser Arg 185 Arg Ser Ile Thr Phe 265 Leu Thr Thr Arg Leu 345 Val Glu -Ala Ile Phe 425 Phe Val Gly Gly Ile 505 Pro Arg Gin Tyr Asp Ala 235 Ser Val 250 Asp Gly Thr Thr Asn Giu Ser Lys 315 Gly Ser 330 Arg Pro Val Thr Leu -Ala Met Asn 395 Lys :,Thr 410 Met Glu Gly Phe Val Ser Giu Gly 475 Thr Ala 490 Arg Gin Val Gin 220 Ile His Thr Gly Ile 300 Ser Leu Val Val Lys 380 Tyr Val Val Lys Thr 460 Val Arg Leu 190 [yr Thr Ile Thr 205 Pro Gly Gly Vai rhr Ser Leu Ser 240 31y Leu Val Leu 255 rhr Val Ile Thr 270 rhr Asp Asn Leu 285 rhr Gin Pro Ile G1y Gly Gin Ala .320 kla Val Thr Ile 335 rhr Leu Val Ala 350 Al1a Gly Val Ser 365 Fsn-Leu Val Thr Thr Lys Leu Ile 400 Trp Pro Thr Arg 415 Ala Asp Leu An 430 Asp Ile Ile Arg 445 Leu Phe Pro Pro Asp Tyr Leu Leu 480 Ala Ala Ser Giy 495 Thr Leu Ala Ala 510 Val Pro Gin Asn 525 Giu Val Val Ala Asn Leu Phe Gin 520 Pro Val Val Asp Gly Ile Leu Ala Ser Pro Giy Val Leu Arg Giy Ala 156 530 535 540 His Asn Leu Asp Cys Val Leu Arg Glu Gly Ala Thr Leu Phe Pro Val 545 550 555 560 Val Ile Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu Asn Ser 565 570 575 Lys Met Phe Ala Val Ile Glu Gly Val Arg Glu Asp Leu Gin Pro Pro 580 585 590 Ser Gin Arg Gly Ser Phe Ile Arg Thr Leu Ser Gly His Arg Val Tyr 595 600 605 Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg Asp Tyr 610 615 620 i Thr Val Val Pro Ile Asp Asp Val Trp Asp Asp Ser Ile Met Leu Ser 625 630 635 640 Lys Asp Pro Ile Pro Pro Ile Val Gly Asn Ser Gly Asn Leu Ala Ile 645 650 655 Ala Tyr Met Asp Vai Phe Arg Pro Lys Val Pro Ile His Val Ala Met 660 665 670 Thr Gly Ala Leu Asn Ala Cys Gly Glu Ile Glu Lys Val Ser Phe Arg 675 680 685 Ser Thr Lys Leu Ala Thr Ala His Arg Leu Gly Leu Lys Leu Ala Gly 690 695 700 Pro Gly Ala Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe Ile 705 710 715 720 Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr Leu 725 730 735 Asn Leu Pro Tyr Leu Pro Pro Asn Ala Gly Arg Gln Tyr His Leu Ala 740 745 750 Met Ala Ala Ser Glu Phe Lys Arg Pro Arg Thr Arg Glu Cys Arg Gin 755 760 765 Ser Asn Gly Ser Ser Ser Gin Arg Gly Pro Thr Ile Pro Ile Cys Thr 770 775 780 Gin Cys Val His Val Ala Gly Arg Glu Trp Asp Cys Asp 785 790 795 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 5426 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS 157 LOCATION: 73. .1182 OTHB3 INFORMATION: /product= (ix) FEATURE: NAME/KEY: CDS LOCATION: 1306. .2574 OTHER INFORMATION: /product= (ix) FEATURE: NAME/KEY: CDS LOCATION: 2790. .4259 OTHER INFORMATION: /product= (ix) FEATURE: NAME/KEY: CDS LOCATION: 4701. .5339 OTHER INFORMATION: /product= "HVT UL42" "HVT UL43" "1HV gA"f "IlVT *fl.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGATCCGAGC TTCTACTATA CAACGCGGAC GATAATTTTG TCCACCCCAT CGGTGTTCGA GAAAGGGTTT TT ATG ATG GCA GGA ATA ACT GTC GCA TGT GAC CAC ACT Met Met Ala Gly Ile Thr Val Ala Cys Asp His Thr GCA GGA GAG Ala Gly Giu GCT CAT ACA CCC Ala His Thr Pro

GAG

Giu 20 GAT ATG CAA AAG Asp Met Gin Lys ACT ATA TCG GCA Thr Ile Ser Ala

AAP.

Lys TGG AGG ATT Trp Arg Ile 108 156 204 252 ATA TTG Ile Leu 30 GCA GOG GAA AAA Ala Gly Giu Lys TTC ATG Phe Met 35 TCG, TTG AAA TCG Ser Leu Lys Ser

ATC

Ile GTC AGT TGT GTG .Val Ser Cys Val AAC CCC CTT CTC Asn Pro Leu Leu ACG -TTT Thr, Phe 55 GGC GCA GAT.

Gly -Ala Asp

GGG.

Gly CTC ATT GTA CAA Leu Ile Vai Gin ACT GTC TGC GGA Thr Vai Cys Gly CGC ATT TTT GTT Arg Ile Phe Val CCA ATC Pro Ile GAC CGT GAT Asp Arg Asp TTT CTA GCA Phe Leu Ala

TCC

Ser TrC AGC GAA TAT Phe Ser Giu Tyr

GAA

Giu 85 TGG CAT GGG .CCA Trp His Gly Pro ACT GCG ATG Thr Ala Met GCA TTC AAA Ala Phe Lys TTA ACT GAT TCC Leu Thr Asp Ser

AGA

Arg 100 CGC ACT CTT TTA Arg Thr Leu Leu

GAT

Asp 105 TGT GAA AAG AGA AGG GCA ATT GAC GTC TCC TTT ACC Cys Giu Lys Arg Arg Ala Ile Asp Vai Ser Phe Thr 110 115 120 TTC GCG GGA GAG Phe Ala Gly Giu

CCT

Pro 125 CCA TGT AGG CAT Pro Cys Arg His ATC CAA GCC GTC Ile Gin Ala Val

ACA

Thr 135 TAC ATO ACC GAC Tyr Met Thr Asp

GGT

Gly 140 GGT TCA GTA TCG Gly Ser Val Ser

AAT

Asn 145 ACA ATC ATT AAA Thr Ile Ile Lys

TAT

Tyr 150 GAG CTC TOG AAT Giu Leu Trp Asn GCG TCT Ala Ser 155 ACA ATT TTC CCC CAA AAA ACT CCC GAT OTT ACC TTT TCT CTA AAC AAA 588 Thr Ile Phe Pro Gin Lys Thr Pro Asp Val Thr Phe Ser Leu Asn Lys 160 165 170 1.58 CAA CAA TTG Gin Gin Leu 175 AAC AAA ATA TTG Asn Lys Ile Leu

GCC

Ala 180 GTC GCT TCA AAA Val Ala Ser Lys

CTG

Leu 185 CAA CAC GAA Gin His Giu GAA CTT Glu Leu 190 GTA TTC TCT TTA Val Phe Ser Leu

AAA

Lys 195 CCT GAA GGA GGG Pro Glu Gly Gly

TTC

Phe 200 TAC GTA GGA ACG Tyr Val Gly Thr

GTT

Val 205 TGT ACT GTT ATA Cys Thr Val Ile

AGT

Ser 210 TTC GAA GTA GAT Phe Giu Val Asp

GGG

Gly 215 ACT GCC ATG ACT Thr Ala Met Thr

CAG

Gin 220 TAT CCT TAC AAC Tyr Pro Tyr Asn

CCT

Pro 225 CCA ACC TCG GCT Pro Thr Ser Ala CTA GCT CTC GTA Leu Ala Leu Val GTA GCA Val Ala 235 TGC AGA AAG Cys Arg Lys AGT GGT AAA Ser Gly Lys 255

AAG

Lys 240 AAG GCG AAT AAA Lys Ala Asn Lys

AAC

Asn 245 ACT ATT TTA ACG Thr Ile Leu Thr GCC TAT GGA Ala Tyr Gly 250 GCA 'rrT AGA Ala Phe Arg CCC TTT TGT GTT Pro Phe Cys Vai

GCA

Ala 260 TTG GAA GAT ACT.

Leu Giu Asp Thr

AGT

Ser 265 AAT ATC Asn Ilie 270 GTC AAT AAA ATC Val Asn Lys Ile

AAG

Lys 275 GCG GGT ACG TCG Ala Gly Thr Ser

GGA

Gly 280 GTT GAT CTG GGG Val Asp Leu Gly

TTT

Phe 285 TAT ACA ACT TGC Tyr Thr Thr Cys CCG CCG ATG CTA Pro Pro Met Leu

TGT

Cys 295 ATT CGC CCA CAC Ile Arg Pro His

GCA

Al a 300 636 684 732 780 828 876 924 972 1020 1068 1116 1164 1219 1279 1332 1380 1428

S

TTT GGA AGT CCT Phe Gly Ser Pro

ACC

Thr 305 GCA.TTC CTG TT Ala Phe Leu Phe AAC ACA GAC TGT Asn Thr Asp Cys ATG ACA Met Thr .315 ATA TAT GAA Ile Tyr Giu AAA CGC ATC Lys Arg Ile 335

CTG

Leu 320 GAA GAA GTA AGC Glu Giu Val Sex

GCC

Al a 325 GTT GAT GGT Val Asp Gly AAC GAA TAT TTC Asn Giu TPyr Phe

CCA

Pro 340 ACA GTA TC G-CAG Thr Val1 Ser Gin GCA ATC CGA GCA Ala Ile Arg Ala 330 GCT ACT TCC AAG Ala Thr Ser Lys 345 AGG AAA ACC ACC Arg Lys Thr Thr AAG AGA Lys Arg 350 AGA GCG Arg Ala 365 AAA CAG TCG CCG Lys Gin Ser Pro

CCC

Pro 355 CCT ATC GAA AGA Pro Ile Giu Arg

GAA

Giu 360 GAT ACC CAA Asp Thr Gin TAAAATGCCA GACAAACCCG GCATCCTGGT TAGAGGGCAG 370 GTGGGCTGGG CCAACCTTCA CGGGCGTCCG ACAGATCGGT GACACTCATA CGTTAACTAA ACGCCGGCAG CTTTGCAGAA. GAAAAT ATG CCT TCC GGA GCC AGC TCG AGT CCT Met Pro Ser Giy Ala Sex Ser Ser Pro 1 CCA CCA GCT TAT ACA TCT GCA GCT CCG CTT GAG ACT TAT AAC AGC TGG Pro Pro Ala Tyr Thr Ser Ala Ala Pro Leu Giu Thr Tyr Asn Ser Trp 15 20 CTA AGT GCC TTT TCA TGC GCA TAT CCC CAA TGC ACT GCG GGA AGA GGA Leu Sex Ala Phe Ser Cys Aia Tyr Pro Gin Cys Thr Ala Giy Arg Gly 30 35 159 GGC AAG AAG TGT ATA COG TGT ATA GTG ATC AGT GTA

CAT

His CGA CAA, Arg Gin

AAT

Asn Giy LYS LYS CyS- TGT TCC Cys Ser GTG GCA Val Ala ACG GTA Thr Val ACG ATA Thr Ilie TTT ATA Phe Ilie GAG GCG Glu Ala ATC GAA Ile Giu 155 ACA TCC Thr Ser 170 AAG. GGC Lys Oly ATA CCA Ile Pro ACA ATA Thr Ile TCC ACT Ser Thr 235 TCT CGT Ser Arg 250 CTT TAT Leu Tyr TCG TCA Ser Ser

TTA

Leu

TTA

Leu

TGT

Cys

AGA

Arg

TTT

Phe

CTT

Leu 140

GTA

Vai

CAC

His

GG

Gly

AAC

Asn

TTG

Leu 220

TGC

Cys

CTC

Leu

GAA

Giu

ATA

Ile

GTG

Val

ATT

Ile

GTA

Val

CTT

Leu

GCT

Ala 125

GCC

Ala

ACA

Thr

AAC

Asn

ATT

Ile

ATA

Ile 205

GCT

Ala

TAT

Tyr

GGA

Gly

GAA

Giu

TTT

Phe 285

TOC.

Cys

CCG

Pro

ATT

Ile

TCG

Ser 110

ATA.

Ile

ATC

Ile

TCT

Ser

TAT

Tyr

TTT

Phe 190

CAC

His

ATA

Ile

TAT

Tyr

CAT

His

GAC

Asp 270

CTA

Leu

ATC

Ile

CTT

Leu

GCC

Ala 95

GAA

Glu

ATC

Ile

AGT

Ser

TTG

Leu

GTC

Val 175

CAT

His

CCA

Pro

AAT

Asn

CGC

Arg

GGT

Gly 255

GTA

Val

TGT

Cys

OCT

Ala

ATC

Ile 80

GGA

Gly

ACG

Thr

GCT

Ala

AAT

Asn

GCG

Ala 160

TOC

Cys

GCT

Ala

ATC

Ile

ATC

Ile

AAT

Asn 240

TAC

Tyr

TAT

Tyr

TAT

Tyr

GCA

Ala 65

GAT

Asp

TTC

Phe

CTT

Leu

TCC

Ser

ACT

Thr 145

TGT

Cys

ATT

Ile

TAC

Tyr

CCT.

Pro

OCT

Ala 225

TGC

Cys

ACA

Thr

AGT

Ser

GCC

Ala Ilie Arg Cys CAT TTA GCT His Leu Ala CAA AAC AGA Gin Asn Arg ATC GCT ACG Ile Ala Thr 100 ATG CTA GTG Met Leu Val 115 GTT GCG GAA Val Ala Glu 130 ACT TAC AAA Thr Tyr Lys TTT GTT'ATG Phe Val Met TCA ACG GCA Ser Thr Ala .180 CAC GGA ACA His Oly Thr 195 CTC GCG G Leu Ala Gly 210 AGA GAT GCA Axg Asp Ala CGC GAG AGG Arg Oiu Arg ATC CCT TCT Ile Pro Ser 260 TTT GAC GCA Phe Asp Ala 275 ATG GGO CTT Met Oly.Leu 290

GTT

Val

OCT

Ala

TTT

Phe

GGC

Gly

ACA

Thr

ACT

Thr

CTC

Leu 165

G

Gly

TTA'

Leu

TTT

Phe

AGC

Ser

ACT

Thr 245

CCC

Pro

OCT

Al a

ACA

Thr

ACC

Thr

TAC

Tyr

OCT

Ala

AAG

Lys

CTG

Leu

OCA

Ala 150

GGG

Gly

GAC

Asp

CTC

Leu

CTT

Leu

OCT

Ala 230

ATA

Ile

GGT

Gly

AAA

Lys

ACA

Thr

GTG

Val

GGA

Oly

GCA

Ala

CCG

Pro

ATC

Ile 135

TTG

Leu

OCA

Ala

TTO

Leu

GOT

Gly

OCA

Ala 215

ACA

Thr

CTT

Leu

GCC

Aia

GCC

Gly

CCG

Pro 295 TCG GGA Ser Gly AAC TGT Asn Cl's CGA CTT Arg Leu 105 GCG CAG Ala Gin 120 AAT AAC Asn Asn CGA, ATA Arg Ile ATA ATT Ile Ile ACT TG Thr Trp 185 ATA ACA Ile Thi 200 GTC TAT Val Tyr TTA TTA Leu Leu COC CCT Arg Pro OAT ATG Asp Met 265 CAT TAT His Tyr 280 CTG ATT Leu Ile Ile Val Ile Ser Val 1476 1524 1572 1620 1668 1716 1764 1812 1860 1908 1956 2004 2052 2100 2148 2196 2244

ATT

Ile GCC CTC CAT AAA TAT ATO Ala Leu His Lys Tyr Met 300

C

Ala 305 GGC ATT AAA AAT TCG TCA GAT TG Gly Ile Lys Asn Ser Ser Asp Trp 310 160 ACT GCT ACA TTA CAA GCC ATG TAC GGG CTT GTC TTG GGA TCG CTA TCG 2292 Thr Ala Tkir Leu Gin Gly Met Tyr Gly Leu Val Leu Gly Ser Leu Ser 315 320 325 TCA CTA TCT ATT CCA TCC AGC AAC AAC GAT GCC CTA ATT CGT CCC ATT 2340 Ser Leu Cys Ile Pro Ser Ser Asn Asn Asp Ala Leu Ile Arg Pro Ile 330 335 340 345 CAA ATT TTC ATA TTG ATA ATC GGT GCA CTG GCC ATT GCA TTG GCT GGA 2388 Gin Ile Leu Ile Leu Ile Ile Gly Ala Leu Ala Ile Ala Leu Ala Gly 350 355 360 TGT GGT CAA ATT ATA GGG CCT ACA TTA TTT CC GCG AGT TCG GCT GCG 2436 Cys Cly Gin Ile Ile Gly Pro Thr Leu Phe Ala Ala Ser Ser Ala Ala 365 370 375 ATG TCA TGT TTT ACA TCT ATC AAT ATT CGC GCT ACT AAT AAG GGT GTC 2484 Met Ser Cys Phe Thr Cys Ile Asn Ile Arg Ala Thr Asn Lys Gly Val *380 385 390 AAC AAA TTG GCA GCA GCC ACT CTC GTG AAA TCT GTA CTG GGC TTC ATT 2532 Asn Lys Leu Ala Ala Ala Ser Val Val Lys Ser Vai Leu Gly Phe Ile 395 -400 405 ATT TCC CCC ATG CTT ACT TGC GTG CTA TTA CCA CTA TCG TGATAGATCG 2581 Ile Ser Cly Met Leu Thr Cys Val Leu Leu Pro Leu Ser 410 415 420 TCGGTCTGCG CATCGCCCAT GCTGGCGGAA CGCTCTTTCG AACCGTGAAT AAAACTTTGT 2641 ATCTACTAAA CAATAACTTT GTGTTTTATT GAGCGGTCGA AAACAATGAG GAGCTGCAAT 2701 TTAAAGCTAA CCCCATACGC CGCGCGGGTA AACACCATTT TATACCATAT TACGCATCTA 2761 TCCAAACTTC TTCCAGAACC GCAAGTAT ATG GTT TCC AAC ATG. CCCTT -CTA 2813 Met Val Ser Asn Met Arg Val Leu 1 CCC GTA CTG CCC CTC ACG CGA TOG CTG GGC ATA TTT CTA CTT CTC TCT 2861 Arg Val Leu Arg Leu Thr Cly Trp Val Cly le.Phe Leu Val Leu Ser .10 i5 TTA CAG CAA ACC TCT TGT CCC GCA TTG CCC CAT AAC GTC GAT'ACC CAT 2909 Leu Gin Cmn Thr Ser Cys Ala Cly Leu Pro His Ash Vai Asp Thr His 30 35 CAT ATC CTA ACT TTC AAC CCT TCT CCC ATT TCG CCC GAT GGC CTT CCT 2957 His Ile Leu Thr Phe Asn Pro Ser Pro Ile Ser Ala Asp Gly Val Pro 50 TTG TCA GAG GTG CCC AAT TCG CCT ACG ACC CAA TTA TCT ACA ACT GTC 3005 Leu Ser Glu Val Pro Asn Ser Pro Thr Thr Clu Leu Ser Thr Thr Val 65 GCC ACC AAG ACA GCT CTA CCC ACG ACT GAA AGC ACT ACT TCC TCC GAA 3053 Ala Thr Lvs Thr Ala Val Pro Thr Thr Ciu Ser Thr Ser Ser Ser Ciu' 80 GCG CAC CCC AAC TCT TCT CAC AAA ATA CCT GAT ATA ATC TGC GAC CCA 3101 Ala His Arg Asn Ser Ser His Lys Ile Pro Asp Ile Ile Cys Asp Arg 95 -100 CAA GAA GTA TTC GTA TTC CTT AAC AAT ACA CGA AGA ATT TTG TGT GAC 3149 Glu Giu Val Phe Val Phe Leu Asn Asn Thr Gly Arg Ile Leu Cys Asp 105 110 115 120 CTT ATA GTC GAC Leu Ilie Val Asp

CCC

Pro 125 CCT TCA GAC GAT Pro Ser Asp Asp 161

GAA

Glu 130 TGG TCC AAC TTC Trp Ser Asn Phe GCT CTT Ala Leu 135 GAC GTC ACG Asp Val Thr

TTC

Phe 140 AAT CCA ATC GAA Asn Pro Ile Glu

TAC

Tyr 145 CAC GCC AAC GAA His Ala Asn Glu AAG AAT GTA Lys Asn Val 150 GAG GTT GCC CGA GTG GCC GGT CTA TAC GGA GTA CCG GGG TCG GAT TAT Giu Val Ala Arg Val Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr 155 160 165 GCA TAC Ala Tyr 170 CCT AGG AAA TCG Pro Arg Lys Ser

GAA

Giu 175 TTA ATA TCC TCC Leu Ile Ser Ser

ATT

Ile 180 CGA CGG GAT CCC Arg Arg Asp Pro a. a. a

CAG

Gin 185 GGT TCT TTC TGG Gly Ser Phe Trp

ACT

Thr 190 AGT CCT ACA CCC Ser Pro Thr Pro

CGT

Arg 195 GGA AAT AAA TAT Giy Asn Lys Tyr ATA TGG ATT AAT Ile Trp Ile Asn

AAA

Lys 205 ACA ATG CAC ACC Thr Met His Thr

ATG

Met 210 GGC GTG GAA GTT Gly Val Giu Val AGA AAT Arg Asn 215 GTC GAC TAC Val Asp Tyr TTT AAT CGC Phe Asn Arg 235

AAA

Lys 220 GAC AAC GGC TAC Asp Asn Gly Tyr

TTT

Phe 225 CAA GTG ATA CTG Gin Val Ile Leu CGT GAT AGA Arg Asp Arg 230 GTG TGC CAA Val Cys Gin CCA TTG GTA GAA Pro Leu Val Glu

AAA

Lys 240 CAT ATT TAC ATG His Ile Tyr Met

CGT

Arg 245' a CGA CCC Arg Pro 250 GCA TCC GTG GAT Ala Ser Vai Asp

GTA

Val 255 TTG GCC CCT Leu Ala Pro CCA GTT Pro Val .260 CTC AGC GGA GAA Leu Ser Gly Giu 3197 3245 3293 3341 3389 3437 3485 3533 3581 3629 3677 3725 3773 3821 3869 3917 3965

AAC

Asn 265 TAC AAA GCA TCT Tyr Lys Ala Ser

TGC

Cys 270 ATC GTT AGA CAT Ilie Vai Arg His

T

Phe 275 TAT CCC CCG GGA Tyr-Pro Pro Gly

TCT

Ser 280 GTC TAC GTA TCT TGG AGA CGT AAC GGA AAC Val Tyr Val Ser Trp Arg Arg Asn Giy Asn 285 290 ATT GCC ACA'*CCC .le Ala.-Thr Pro CGC AAG Arg Lys 295 GAC CGT GAC Asp Arg Asp CTA GTA TCC Leu Val Ser 315

GGG

Gly 300 AGT TTT TGG TGG Ser Phe Trp Trp

TTC

Phe 305 GAA TCT GGC CGC Giu Ser Gly Arg GGG GCC ACA Gly Ala Thr 310 TCT CCT CCA Ser Pro Pro ACA ATA ACC CTC Thr Ile Thr Leu

GGA

Gly 320 AAC TCT GGA CTC Asn Ser Giy Leu

GAA

Giu 325 AAG GTT Lys Vai 330 TCC TGC TTG GTA Ser Cys Leu Vai

GCG

Ala 335 TGG AGG CAA GGC Trp Arg Gin Gly

GAT

Asp 340 ATG ATA AGC ACA Met Ile Ser Thr

TCG

Ser 345 AAT GCT ACA GCT Asn Ala Thr Ala

GTA

Val 350 CCG ACG GTA TAT Pro Thr Val Tyr

TAT

Tyr 355 CAC CCC CGT ATC His Pro Arg Ile CTG GCA TTT AAA~ Leu Ala Phe Lys

GAT

Asp 365 GGG TAT GCA ATA Gly Tyr Ala Ile

TGT

Cys 370 ACT ATA GAA TGT Thr Ile Giu Cys GTT CCC Vai Pro 375 TCT GGG ATT Ser Giy Ile

ACT

Thr 380 GTG AGG TGG TTA Val Axg Trp Leu

GTT

Val 385 CAT GAT GAA CCC His Asp Giu Pro CAG CCT AAC Gin Pro Asn 390

ACA

Thr

TAT

Tyr

ACG

Thr 425

TTT

Phe

CCG

Pro

GGT

Gly

AAT

Asn 162 ACT TAT GAT ACT GTG GTT ACA GGT CTC TGC AGG Thr Tyr Asp Thr Val Val Thr Gly Leu Cys Arg 395 400 AGA AAT CTC GCC AGT CGG ATT CCA GTC CAG GAC Arg Asn Leu Ala Ser Arg Ile Pro Val Gin Asp 410 415 420 AAG TAT ACG TGC AGA CTA ATT GGA TAT CCG TTC Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe 430 435 CAA AAT TCC GAA TAT TAT GAT GCA ACG CCG TCG, Gin Asn Ser Glu Tyr Tyr Asp Ala Thr Pro Ser 445 450 ATG ATT GTA ACA ATT ACG GCC GTT CTA GGA CTG Met Ile Val Thr Ile Thr Ala Val Leu Gly Leu 460 465 ATT GGT ATC ATT ATC ACA GCC CTA TGC TTT TAC Ile Gly Ile Ile Ilie Thr Ala Leu Cys Phe Tyr 475 480 TAAGATTAAC CATCGTATGT GATATAAAAA TTATTAAGTG ACC ATC GAT Thr Ile Asp 405 AAC TGG GCG Asn Trp, Ala GAC GTG GAT Asp Val Asp GCA AGA GGA Ala Arg Gly 455 GCC TTG TTT Ala Leu Phe 470 CTA CCG GGG Leu Pro Gly 485

TTATAACCGA

CGT

Arg

AAA

Lys

AGA

Arg 440

ATG

Met

TTA

Leu

CGG

Arg S.

B

490

*SSS

S

a *5 S

S

TCGCATTCTT

TTGTTTCGTA

CACGCAGCGG

CGGGACGTAC

A.AGTCTCCGA

GATACGGATA

CAATCACAA

CTGTTTCGAT

GATGACTCAT

CCAAAP.TGCC

ATCATGTGGC

ATATCAAGCT

CCGTACAATC

ATCGCTGGGG

TCACAATAAA

GTTCAGTCCG

CATTATGTTA

GCACGTTAAI

CACGGCCAAA

GCTGAGTAGA

TATATCATAT

GAA

Giu

GAT

Asp

TGG

Trp

TTC

Phe

TAC

Tyr

GGT

Gly

GAT

Asp

AGC

Ser

CGA

Arg

GTT

Val.

ATG

Met

TAT

Tyr

GAT

Asp

GGC

Gly

TCG

Ser

CTC

Leu

GCC

Ala

AGT

Ser

CGC

Arg

ACG

Thr

ATC

Ilie

GTA

Val

ATG

Met

TGC

Cys

GAT

Asp

GAA

Giu

TGT

Cys

GCG

Ala

GAA

Giu

ATG

Met

CTC

Leu

ACA

Thr

TGT

Cys

GCA

Ala 60

TCG

Ser

CGC

Arg

GTT

Val

GAT

Asp

GGG

Gly 45

GTA

Val

GGA

Giy

GTG

Val

GTG

Val

AGA

Arg 30

TGT

Cys

TTG

Leu

ACA

Thr

GCC

Ala TAAAATGGTA TTGTAATCAG CACCATCGCA CGTGATGTCA AAAATACGTA TTTTTGGTAT TTTTTACTCC AAACGCGGTA TTTAAAACAT CGTATACGGT GCCGCTACAT TAAAAATCGC ACGTCGGTAA TAATCTTACG CATCGAATGT TTTCCTATAT AGTTACTCAG TAGTGATACA AAGA ATG ATG TCG CCC ACC CCT.

Met Met Ser Pro Thr Pro 1 GTT CGT GGA CGT CTC CGA ATG ATG Val Arg Gly Axg Leu Arg Met Met i5 GAG CAA CGA CAT CCA CGT ACG ACT Giu Gin Arg His Pro Arg Thr Thr ACG ATA GGA ATG GTA TTT ACC ATA Thr Ile Gly Met Val Phe Thr Ile TTG GGA TCA CTA TTC ACT GTT TCA Leu Gly Ser Leu Phe Thr Val Ser 65 TGT CCC GAT GAA TGG ATT GGT TTG Cys Pro Asp Giu Trp Ile Gly Leu so GGG AAA AAT GCA ACT CAT CTT GAG Gly Lys Asn Ala Thr Asp Leu Giu 95 100 4013 4061 4109 4157 4205 4253 4306 4366 4426 4486 4546 4606 4666 4718 4766 4814 4862 4910 4958 5006 S

*S.SSS

S

163 GCG TTG GAT ACA TGT GCT CGG CAT AAC AGC AAA Ala Leu Asp Thr'qys Ala Arg His Asn Ser Lys 105 110 AAC GCC AAA GTT CTG GTT GAA GCT ATC GCC CCA Asn Ala Lys Val Leu Val Giu Ala Ile Ala Pro 120 125 GCA GCA TAT GGG GAA GTC TTC CGG TTA AGG GAC Ala Ala Tyr Gly Giu Val Phe Azg Leu Arg Asp 135 140 145 ATA CGA CCT ACC ATG OGA GGA CCC GTG TCG GCA Ile Arg Pro Thr Met Gly Gly Pro Val Ser Ala 155 160, TOT ACC GTT ATA TOT CAG CGA CCC AGO CCT CTA Cys Thr Val Ile Cys Gin Arg Pro Arg Pro Leu 170 175 ATC ATT AGA OAT GCC CGC GTG TAT CTT CAT TTA Ile Ile Arg Asp Ala Arg Val Tyr Leu His Leu 185 190 TAT GAA GTC TAC GCC TCT GTC CTC TCT AAT GCG Tyr Glu Val Tyr Ala Ser Val Leu Ser Asn Ala 200 205 CCTCTAACGG TTACTGTGTT TATTATCCAA TCACACCATA GATCTTTATT TCATATAATG INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 369 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 Met Met Ala Giy Ile Thr Val Ala Cys Asp His 1 5 10 His Thr Pro Olu Asp Met Gin Lys Lys Ti-p Arg 25 Glu Lys Phe Met Thr Ile Ser Ala Ser Leu Lys 40 Val Lys Asn Pro Leu Leu Thr Phe Gly Ala Asp 55 Gly Thr Val Cys Gly Gin Arg Ile Phe Val Pro 70 75 Phe Ser Glu Tyr Glu Trp His Gly Pro Thr Ala 90 CTT ATT GAC TTC GCA Leu Ile Asp Phe Ala 115 TTC GOT OTO CCA AAT Phe Oly Val Pro Asn 130 AGC AAA ACC ACG TOT Ser Lys Thr-Thr Cys 150 GAC TOT CCT OTA ACA Asp Cys Pro Val Thr 165 AGT ACC ATG TCT TCC Ser Thr Met Ser Ser 180 GAA CGA COC GAT TAT Glu Arg Arg Asp Tyr 195 ATG AGT-AAA TAAAAACGCA Met Ser Lys 210 GACATTATTA CAATAATATG 5054 5102 5150 5198 5246 5294 5346 5406 5426 Thr Ile Ser Gly Ile Met Lys Giu Ala Ile Ile Leu Asp Phe Cys Pro Gly Leu Val Ile Axg Leu Giu 110 Pro Giu Ala Ser Val Asp Ala Lys Cys Ala Gly Cys Gin Ser Leu Arg Arg Asp Ala Ser Ile Arg 100 Asp Arg Val Thr Ser Leu Thr Asp 105 Phe Al a Al a Phe Gly 164 115 120 125 His Leu Ile Gin Ala Val Thr Tyr Met Thr Asp Gly Gly Ser Val. Ser 130 135 140 Asn Thr Ile Ile Lys Tyr Giu Leu Trp Asn Ala Ser Thr Ile Phe Pro 145 150 155 160 Gin Lys Thr Pro Asp Val Thr Phe Ser Leu Asn Lys Gin Gin Leu Asn 165 170- 175 Lys Ilie Leu Ala Val Ala Ser Lys Leu Gin His Glu Glu Leu Val. Phe 180 185 190 Ser Leu Lys Pro Giu Giy Gly Phe Tyr Val Gly Thr Val Cys Thr Val.

195 200 205 le Ser Phe Giu Val Asp Gly Thr Ala Met Thz Gin Tyr Pro Tyr Asn 210 215 220 Pro Pro Thr Ser Ala Thr Leu Ala Leu Val Val Ala Cys Arg Lys Lys 225 230 235 240 *Lys Ala Asn. Lys Asn Thr Ile Leu Thr Ala Tyr Gly Ser Gly Lys Pro 245 250 255 Phe Cys Val Ala Leu Giu Asp Thr Ser Ala Phe Arg Asn Ile Val. Asn 260 265 270 Lys Ile Lys Ala Gly Thr Ser Gly Val Asp Leu Gly Phe Tyr Thr Thr 275 280 285 Cys Asp Pro Pro Met Leu Cys Ile Arg Pro His Ala Phe Gly Ser Pro 290 295 300 Thr Ala Phe Leu Phe Cys Asn Thr Asp Cyr. Met..Thr Ile Tyr Glu Leu 305 310 315 320 Giu Giu Val Ser Ala Val Asp Gly Ala Ile Arg Ala Lys Arg Ile Asn *325 330 335 Giu Tyr Phe Pro Thr Vai Ser Gin Ala Thr Ser, Lys Lys Arg Lys Gin 340 345 350 Ser Pro Pro Pro Ile Glu Arg Giu Arg Lys Thr Thr Arg Ala Asp Thr 355 360 365 Gin INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 422 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Pro Ser Gly Ala Ser Ser Ser Pro Pro Pro Ala Tyr Thr Ser Ala 1 5 10 Ala Pro Leu Glu Thr Tyr Asn Ser Trp Leu Ser Ala Phe Ser Cys Ala 25 165 Tyr Pro Gin Cys Thr Ala Gly Arg 40 Gly His Arg Gin Asn Gly Lys Lys

C

C

C

S

dys Ala Asp Phe Leu Ser Thr 145 Cys Ile Tyr Pro Ala 225 Cys Thr Ser Ala Ala 305 Tyr Asn Gly Thr Ile Arg His Leu Gin Asn Ile Ala Met Leu 115 Vai Ala 130 Thr Tyr Phe Vai Ser Thr His Gly 195 Leu Ala 210 Arg Asp Arg Glu Ile Pro Phe Asp 275 Met Gly 290 Gly Ile Gly Leu Asn Asp Ala Leu 355 Leu Phe Cys Ala Arg Thr 100 Val Glu Lys Met Ala 180 Thr Gly Ala Arg Ser 260 Ala Leu Lys Val Ala 340 Ala I le Vai Ala Phe Gly Thr Thr Leu 165 Gly Leu Phe Ser Thr 245 Pro Ala Thr Asn Leu 325 Leu Ile Val Thr 70 Tyr Ala Lys Leu Ala 150 Gly Asp Leu Leu Ala 230 Ile ly Lys rhr Ser 310 Gly Ile kla Ile Ser 55 Val Ser Gly Asn Ala Arg Pro Ala 120 Ile Asn 135 Leu Arg Ala Ile Leu Thr Gly Ile 200 Ala Vai 215 Thr Leu Leu Arg Ala Asp Gly His 280 Pro Leu 295 Ser Asp Ser Leu Arg Pro Leu Ala 360 Val Gly Cys Leu 105 Gin Asn Ile Ile Trp 185 Thr Tyr Leu Pro Met 265 Tyr Ile rrp Ser lie 345 3iy Cys Val Thr 90 Thr Phe Glu Ile Thr 170 Lys Ile Thr.

Ser Ser 250 Leu Ser Ile Thr Ser 330 Gln Cys Ser Ala 75 Val Ile le Ala Glu 155 Ser Gly Pro Ile Thr 235 Arg Tyr Ser Ala Ala 315 Leu Ile Gly Leu Val Leu Ile Cls Val Arg Leu Phe Ala 125 Leu Ala 140 Val Thr His Asn Gly Ile Asn Ile 205 Leu Ala 220 Cys Tyr Leu Gly Glu'Glu Ile Phe 285 Leu His 300 Thr Leu Cys Ile Leu Ile Gin Ile 365 Cys Phe 380 Cys Pro Ile Ser 110 Ile 1le Ser Tyr Phe 190 His Ile Tyr His Asp 270 Leu Lys Gin Pro Leu 350 Ile Ile Leu Ala Glu Ile Ser Leu Val 175 His Pro Asn Arg Gly 255 Val Cys TyrI Gly I Ser 335 Ile Gly Ala Ile Gly Thr Ala Asn Ala 160 Cys Ala Ile Ile Asn 240 Tyr Tyr Tyr Met Met 320 Ser lie Pro Ala Ala Ser 370 Ser 375 Ala Ala Met Ser Thr Cs Ile 166 Asn Ile Arg Ala Thr Ann Lys Gly Val Ann Lys Leu Ala Ala Ala Ser 385 390 395 400 Val Val Lys Ser Val Leu Gly Phe Ile Ile Ser Gly Met Leu Thr Cys 405 410 415 Val Leu Leu Pro Leu Ser 420 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 489 amino acids TYPE: amino acid TOPOLOGY: linear 6S a 0@ 0 60 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Val Ser Asn Met Arg Val Leu Arg Val Leu Val Leu Pro Thr Thr Ile Asn Asp Tyr 145 Tyr Ile Thr Thr Phe 225 Gly Pro Ile 50 Thr Glu Pro Thr Giu 130 His Gly Ser Pro Met 210 Gln Ile His 35 Ser Glu Ser Asp Gly 115 Trp Ala Val Ser Arg 195 Gly Phe An Al a Leu Thr Ile 100 Arg Ser Asn Pro Ile 180 Gly Val Leu Val Asp Ser Ser Ile Ile Asn Glu Gly 165 Arg Asn Glu Val Asp Gly Thr 70 Ser Cys Leu Phe Lys 150 Ser Arg Lys Val Arg 230 Leu Thr Val 55 Thr Ser Asp Cys Al a 135 An Asp Asp Tyr Arg 215 Asp Ser His 40 Pro Val Glu Arg Asp 120 Leu Val Tyr Pro Phe 200 An Leu 25 His Leu Ala Ala Giu 105 Leu Asp Glu Al a Gin 185 Ile Val1 Gin Ile Ser Thr His 90 Giu Ile Val Val Tyr 17 0 Gly Trp Asp Gin Leu Giu Lys 75 Arg Val Val Thr Ala 155 Pro Ser Ile Tyr Arg 235 Arg Leu Thr Gly Thr Ser Cys Ala Thr Phe Ann Pro Val Pro Ann Ser Thr Ala Val Pro Ann Ser Ser His Phe Val Phe Leu -110 Asp Pro Pro Ser 125 Phe Asn Pro Ile 140 Arg Val Ala Gly Arg Lys Ser Glu 175 Phe'Trp Thr Ser 190 Asn Lys Thr Met 205 Lys Asp Asn Gly 220 Pro Leu Val Glu Trp Giy Ser Pro Thr 180 Lys Asn Asp Giu Leu 160 Leu Pro His Tyr Lys 240 Val Ile Leu Arg Phe Asn 167 *o His Al a Arg Gly Phe 305 Asn Arg Val Ile Val 385 Gly Pro Gly Ala Val 465 Leu Ile Pro His Asn 290 Glu Ser Gin Tyr Cys 370 His Leu Val Tyr Thr 450 Leu Tyr Pro Phe 275 Ile Ser Gly Gly Tyr ~355 Thr Asp Cys Gin Pro 435 Pro Giy Phe Met Val 260 Tyr Al a Gly Leu Asp 340 His Ilie GlU Arg Asp 420 Phe Ser Leu Tyr *Arg 245 Leu Pro Thr Arg Glu 325 Met Pro Glu Pro Thr 405 Asn Asp Ala Ala Leu 485 Val Ser Pro Pro Giy 310 Ser Ile Arg Cys Gin 390 Ile Trp Val Arg Leu 470 Pro Cys Gly Gly Arg 295 Ala Pro Ser Ile.

Val 375 Pro Asp Aia Asp.

Gly 455 Phe Gly Gin Glu Ser 280 Lys Thr Pro Thr Ser 360 Pro Asn Arg Lys Arg 440 Met Leu Arg Arg Asn 265 Val Asp Leu Lys Ser 345 Leu Ser Thr Tyr Thr 425 Phe Pro Gly Asn 250 Tyr Lys Ala Tyr Val Ser Arg Asp Gly 300 Val Ser Thr 315 Val Ser Cys 330 Asn Ala Thr Ala Phe Lys Gly Ile Thr 380 Thr Tyr Asp 395 Arg Asn Leu 410 Lys -Tyr Thr Gin Asn"Ser.

MetIle Val -460 Ile 'Gly 'Ile 475 Ser Trp 285 Ser Ile Leu Ala Asp 365 Val Thr Al a Cys; Glu 445 Thr Ile 255 Cys Ile Val 270 Arg Arg Asn Phe Trp Trp Thr Leu Gly 320 Val Ala Trp 335 Val Pro Thr 350 Gly Tyr*Ala Arg Trp-Leu Val Val Thr 400 Ser Arg Ile 415 Arg Leu Ile -430 Tyr. Tyr Asp Ile Thr Ala Ile Thr Ala 480 Pro Ala Ser Val Asp Val Leu INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 212 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Met Ser Pro Thr Pro Glu Asp Asp Arg Asp Leu Val Val Val Arg 1 5 10 Gly Arg Leu Arg Met Met Asp Ser Gly Thr Giu Thr Asp Arg Giu Gin 25 168 Ser Ile Arg His Pro Arg Thr Thr Trp Arg 40 Cys Cys Gly Cys Thr Ile Gly Met Val Phe Thr Ile Ser Asp Asn Lys Pro Asp 145 Ala Leu Leu Ala Leu Glu Ala Leu Phe 130 Ser Asp Ser Glu Met 210 Phe Thr Trp Ile Thr Asp 100 Ile Asp 115 Gly Val Lys Thr Cys Pro Thr Met 180 Arg Arg 195 Ser Lys Val Gly Leu Phe Pro Thr Val 165 Ser Ser 70 Leu Giu Ala Asn Cys 150 Thr Ser Phe 55 Tyr Gly Ala Asn Ala 135 Ile Cys Ile Val Met Tyr Leu Ala 120 Ala Arg Thr Ile Leu Ala Ser Asp 105 Lys Tyr Pro Val Arg 185 Val Met Cys 90 Thr Val Giy Thr Ile 170 Asp Ala Glu 75 Met Cys Leu Glu Met 155 Cys Ala Ala Ser Arg Ala Val Val 140 Giy Gin Arg Val Gly Val Arg Giu 125 Phe Gly Arg Val Leu Thr Ala His 110 Ala Arg Pro Pro Tyr 190 Leu Cys Gly Asn Ile Leu Val Arg 175 Leu Gly Pro Lys Ser Ala Arg Ser 160 Pro His Asp Tyr Tyr Glu Val Tyr Ala Ser Val Leu Ser Asn INFORMATION FOR SEQ ID NO:B: SEQUENCE CHARACTERISTICS: LENGTH: 1506 base pairs TYPE: nucleic acid STP.ANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .1506 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATG CTC ACG CCG CGT GTG TTA CGA GCT TTG GGG TGG ACT GGA CTC TTT Met Leu Thr Pro Arg Val Leu Arg Ala Leu Gly Trp Thr Gly Leu Phe 1 5 10 TTT TTG CTT TTA TCT CCG AGC AAC GTC CTA GGA GCC AGC CTT AGC CGG Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Giy Ala Ser Leu Ser Arg 25 GAT CTC GAA ACA CCC CCA TTT CTA TCC TTT GAT CCA TCC AAC ATT TCA Asp Leu Glu Thr Pro Pro Phe Leu Ser Phe Asp Pro Ser Asn Ile Ser 169 ATT AAC Ile Asn GGC GCG CCT TTA Gly Ala Pro Leu

ACT

Thr 55 GAG GTA CCT CAT Giu Val Pro His

GCA

Ala CCT TCC ACA GAA Pro Ser Thr Giu

AGT

Ser GTG TCA ACA AAT Val Ser Thr Asn

TCG

Ser 70 GAA AGT ACC AAT Giu Ser Thr Asn

GAA

Giu 75 CAT ACC ATA ACA His Thr Ile Thr

GA.

Giu ACG ACG GGC AAG Thr Thr Gly Lys

AAC

Asn GCA TAC ATC CAC Ala Tyr Ile His AAT GCG TCT ACG Asn Ala Ser Thr GAC AAG Asp Lys CAA AAT GCG Gin Asn Ala GAA GAP. GTT Giu Giu Val 115

AAC

Asn 100 GAC ACT CAT AAA Asp Thr His Lys

ACG

Thr 105 CCC AAT ATA CTC Pro Asn Ile Leu TGC GAT ACG Cys Asp Thr 110 GTT TGT ACT Val Cys Thr TTT GTT T'rC CTT Phe Val Phe Leu

AAC

Asn 120 GAA ACG GGA AGA Giu Thr Gly Arg

TTT

Phe 125 CTC AAA.

Leu Lys 130 GAT CTG Asp Leu 145 GTC GAC-CCC CCC Val Asp Pro Pro

TCG

Ser 135 GAT AGT GAP. TGG Asp Ser Glu Trp

TCC

Ser 140 AAC TTT GTT CTA Asn Phe Val Leu 192 240 288 336 384 432 480 528 576 624 672 ATC TTT AAC Ile Phe Asn CCA ATT GAP. TAC CAC Pro Ile Glu Tyr His 150 GCC AAC GAP. AAG AAT Ala Asn Giu Lys Asn 155

GTG

Val 160 GAP. GCG GCG CGT Giu Ala Ala Arg

ATC

Ile 165 GCT GGT CTC TAT Ala Gly Leu Tyr

GGA

Gly 170 GTC CCC GGA TCA Val Pro Gly Ser GAC TAT Asp Tyr 175 GCA TAC CCA Ala Tyr Pro CAG GGC ACA Gin Gly Thr 195

CGT

Arg 180 CAP. TCT GAP. TIP.

Gin Ser Glu Leu

ATT

Ile 185 TCT TCG ATT CGA Ser Ser Ile Arg CGA. GAT CCC Arg, Asp Pro 190 AAG TAC TTC Lys Tyr Phe TTT TGG ACG AGC Phe Trp, Thr Ser

CCA

Pro 200 TCA CCT CAT Ser Pro His GGA AAC Gly Asn 205 ATA TGG Ile Trp, 210 ATA AAC AAA ACA Ile Asn Lys Thr

ACC

Thr 215 AAT ACG ATG GGC Asn Thr Met Gly GTG GAA ATT AGA Val Giu Ile Arg 220 ATT ATG CGT GAC Ile Met Arg Asp

AAT

Asn

CAT

His 240

GTA

Val 225 GAT TAT GCT GAT Asp Tyr Ala Asp

AAT

Asn 230 GGC TAC ATG CAA Gly Tyr Met Gin

GTC

Val 235 TTT AAT CGG CCT Phe Asn Arg Pro

TTA

Leu 245 ATA GAT AAA CAT Ile Asp Lys His

ATT

Ile 250 TAC ATA CGT GTG Tyr Ile Arg Val TGT CAA Cys Gin 255 CGA CCT GCA Arg Pro Ala AAT TAC AAG Asn Tyr Lys 275

TCA

Ser 260 GTG GAT GTA CTG Vai Asp Val Leu

GCC

Ala 265 CCT CCA GTC CTC Pro Pro Val Leu AGC GGA GAP.

Ser Gly Gi 270 CCT GGA TCT Pro Giy Scr 816 GCA TCT TGT ATC Ala Ser Cys Ile AGA CAC TTT TAT Arg His Phe Tyr

CCC

Pro 285 GTC TAT Val Tyr 290 GTA TCT TGG AGA Vai Ser Trp Arg

CAG

Gin 295 AAT GGA AAC ATT Asn Gly Asn Ile

GCA

Ala 300 ACT CCT COG AAA Thr Pro Arg Lys GAT CGC GAT GGA AGT TTT TOG TGG TTC GAP. TCT GGT AGA GGA GCT ACO 6 960 170 Glu Ser 315

C

C

Asp 305

TTG

Leu

AAA

Lys

ACG

Thr

CTG

Leu

TCT

Ser 385

ACA

Thr

CAT

His

ACA

Thr

TTT

Phe

CCC.

Pro 465

GGG

Gly

AAA

Lys Arg

GTT

Val

ATA

Ile

AAT

Asn

GCT

Ala 370

GAG

Glu

ACT

Thr

AGA

Arg

AAA

Lys

CAA

Gin 450

ATG

Met

ATG

Met

AAT

Asn Asp Gly TCT ACA Ser Thr TCT TGT Ser Cys 340 GCC ACA Ala Thr 355 TTT AAA Phe Lys ATT ACT Ile Thr TAT AAT Tyr Asn AAT CTC Asn Leu 420 TAT ACC Tyr Thr 435 GAT TCG Asp Ser GTT ATT Val Ile GGG ATA Gly Ile ATT CGA Ile Arg 500 Ser

ATA

Ile 325

CTG

Leu

GCT

Ala

CAT

Asp

GTA

Val

ACT

Thr 405

CTC

Leu

TGC

Cys

GAA

Glu

ACG

Thr

ATC

Ile 485

TTA

Leu Phe 310

ACA

Thr

GTT

Val

ATC

Ile

GGG

Gly

CGG

Arg 390

GTG

Val

AGC

Ser

AGA

Arg

TAT

Tyr

GTT

Val 470

ATG

Met

TAA

Trp

TTG

Leu

GCC

Ala

CCG

Pro

TAT

Tyr 375

TGG

Trp

GTT

Val

CGC

Arg

CTC

Leu

TAC

Tyr 455

ACG

Thr

ACT

Thr Trp

GGA

Gly

TGG

Trp

ACG

Thr 360

GCA

Ala

TTA

Leu

ACA

Thr

ATT

Ile

ATA

Ile 440

GAT

Asp

GCA

Ala

GCC

Ala Phe

AAT

Asn

AAG

Lys 345

GTA

Val

ATA

Ile

GTA

Val

GGT

Gly

CCA

Pro 425

GGC

Gly

GCA

Ala

GTT

Vai

CTA

Leu TCA GGA ATT Ser Gly Ile 330 CAG GGT GAT Gin Gly Asp TAT CAT CAT Tyr His His TGT ACT ATA Cys Thr Ile 380 CAT GAT GAA His Asp Glu 395 CTC TGC CGG Leu Cys Arg 410 GTA TGG GAC Vai Trp Asp TAC CCC TTC Tyr Pro Phe ACT'CCA TCT.

Thr Pro Ser 460 TTG GGA TTG Leu Gly:.Leu 475 TGT TTA TAC Cys Leu Tyr 490

GAT

Asp

ATG

Met

CCC

Pro 365

GAA

Glu

GCG

Ala

ACC

Thr

AAT

Asn

GAT

Asp 445

GCA

'Ala

GCT

Ala

AAC

Asn

TTC

Phe

ATC

Ile 350

CGT

Arg

TGT

Cys

CAG

Gin

ATC

Ile

TGG

Trp 430

GAA

Glu

AGA

Arg

GTA

Val

TCC

Ser

CCC

Pro 335

AGC

Ser

TTA

Leu

GTC

Vai

CCT

Pro

GAT

Asp 415

ACG

Thr

CAT

Asp

GGA

Gly

ATT

Ile

ACA

Thr 495

CCC

Pro

ACG

Thr

TCC

Ser

CCC

Pro

AAC

Asn 400

CGC

Arg

AAA

Lys

AAA

Lys

ACA

Thr

TTA

Leu 480

CGA

Arg 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1506 Gly Arg Giy Ala Thr INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 501 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Leu Thr Pro Arg Val Leu Arg Ala Leu Gly Trp Thr Gly Leu Phe 1 5 10 Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Giy Ala Ser Leu Ser Arg 171 Asp Ile Ser Leu Asn Val Giu Gly Ser Thr Al a Thr Pro Pro Asn Thr Thr Gly Lys Asn a a Gin Giu Leu Asp 145 Giu Ala Gin Ile Vai 225 Phe Axg An Val Asp 305 Leu Lys Thr I2 Asr Giu Lys 130 Leu Ala Gly Trp 210 Asp Asn Pro Tyr T~yr 290 krg Iai Ile ~sn Ala Val 115 Val Ile Ala Pro Thr 195 Ile Tyr Arg Al a Lys 275 Val Asp Ser Ser Ala 355 Asn 100 Phe Asp Phe Arg Arg 180 Phe Asn Al a Pro Ser 260 Al a Ser

G

1 y rhr :Cys 340 rhr Asp Val Pro Asn Ile 165 Gin Trp, Lys Asp Leu 245 Val Ser Trp Ser Ile 325 Leu Ala *1 2

I

P

3

T

Pro Phe Leu 40 Leu Thr Giu 55 Ser Giu Ser 70 Ala Tyr Ile Thr His Lys Phe Leu Asn 120 Pro Ser Asp 135 Pro Ile Giu LS50 Uia Gly Leu ~er Giu Leu hr Ser Pro 200 7hr Thr Asn 215 isfn Gly Tyr .30 le Asp Lys hsp Val Leu Ile Val 280 rg Gin Asn 295 he Trp Trp 10 hr Leu Gly al Ala TrpI le Pro Thr 1 360 Se~ Val Tl His Thr 105 Giu Ser Tyr Tyr Ile 185 Ser Thr M1et Hlis klia 265 krg ?he k*sn ys ~45 Tal rPhe Pro *An IAsn 90 *Pro *Thr Glu His Giy 170 Ser Pro met Gin: Ile 250 Pro His Asn Giu Ser 330 Gin Tyr Asp Pro S His Glu 75 An An Gly Trp Ala 155 Val Ser His Gly Val 235 Tyr Pro Phe Ile Ser 315 3 1

Y

~iis Al.

64 Hi~ Alg Ile Arg Ser 140 Asn Pro Ile Giy Val 220 Ile Ile Val Tyr Ala 300 Gly Ile Asp His aPro 3Thr Ser Leu Phe 125 *Asn Gly Arg Asn 205 Giu -Met Arg Leu Pro 1 285 Thr I Arg C Asp E Met I Pro P, 365 S e Thi Cys 110 Val Phe Lys Ser Arg 190 Lys Ile J'al' ;er ro ro 'ly ~he lie so0 Lrg r- Thr SThr Asp Asp Cys Vai Asn Asp 175 Asp Tyr Arg Asp Cysi 255 Gly Giy Arg Ala IJ Pro 1 335 Ser 'I Leu E ;er Asn Ile Ser Giu Glu Lys Thr Thr Leu Val 160 Tyr Pro Phe An His 240 Gln 3lu 3er .4'S rhr ro 'hr ~er Leu Ala Phe Lys Asp Giy Tyr Ala Ile Cys Thr Ile Giu Cys Val Pro 172 370 375 380 Ser Giu Ile Thr Val Arg Trp Leu Vai His Asp Glu Ala Gin Pro Asn 385 390 395 400 Thr Thr Tyr Asn Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg -405 410 415 His Az-g Asn Leu Leu Ser Arg Ile Pro Val Trp Asp Asn Trp Thr Lys 420 425 430 Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Giu Asp Lys 435 440 445 Phe Gin Asp Ser Giu Tyr Tyr Asp Al a Thr Pro Ser Ala Arg Gly Thr 450 455 460 Pro Met Vai Ilie Thr Val Thr Ala Val Leu Gly Leu Ala Vai Ile Leu 465 470 475 480 Gly Met Gly Ile Ile Met Thr Ala Leu Cys Leu Tyr Asn Ser Thr Arg **485 490 495 Lys Asn Ile Arg Leu 500 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1734 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CMS LOCATION: 1._1734 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GAC CGC GCC GTT AGC CAA GTT GCG TTA GAG AAT GAT GAA AGA GAG 48 Met Asp Arg Ala Val Ser Gin Val Ala Leu Giu Asn Asp Giu Arg Giu 1 5 10 GCA AA.A AAT ACA TGG CGC TTG ATA TTC CGG ATT GCA ATC TTA TTC TTA 96 Ala Lys. Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu 25 ACA GTA GTG ACC TTG GCT ATA TCT GTA GCC TCC CTT TTA TAT AGC ATG- 144 Thr Vai Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser Met 40 COG GCT AGC ACA CCT AGC CAT CTT GTA GGC ATA CCG ACT AGG ATT TCC 192 Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser 55 AGG GCA GAA GAA AAC ATT ACA TCT ACA CTT GOT TCC AAT CAA OAT GTA 240 Arg Ala Giu Giu Lys Ile Thr Ser Thr Leu Giy Ser Asn Gin Asp Val 70 75 so 173

GTA

Val GAT AGG ATA TAT AAG CAA GTG GCC CTT GAG TOT Asp Arg Ile Tyr Lys Gin Vai Ala Leu Glu Ser CCA TTG GCA TTG Pro Leu Ala Leu TTA AAT Leu Asn CAG ATT Gin Ile GAC OCA Asp Pro 130 GCT AGT Ala Ser 145 AAT TTT Asn Phe TCA TTT Ser Phe TTG TCT Leu Ser GGT GTG Gly Val 210 CGT TCC Arg Ser 225 AGT GCA Ser Ala ACA GAG Thr Giu GGG AGG Giy Arg ACA ACA Thr Thr 290 GGA TCT Gly Ser 305 AAA CCC Lys Pro TAO AAG Tyr Lys

ACT

Thr

AAT

Asn 115

GAT

Asp

GAT

Asp

ATC

Ile

GAO

Asp

GGA

Gly i195

OTO

Leu

ATO

Ile

ACT

Thr

GAA

Giu

TTA

Leu 275

TTA

Leu

TTT

Phe

AAT

Asn

CGA

Arg

GAG

Glu 100

GGA

Gly

TAT

Tyr

GTC

Val

CG

Pro

ATG

Met 180

TGC

Cys

CGG

Arg

AAC

Asn

CCC

Pro

GAA

Oiu 260

GGG

Gly

TTC

Phe

ATT

Ile

ACA

Thr

TAO

Tyr 340 ACC ACA Thr Thr GOT OCA Ala Ala ATA 000 Ile Gly ACA TOA Thr Ser 150 000 COT Ala Pro 165 AGT GOT Ser Ala AGA GAT Arg Asp ACA TOT Thr Ser OTG GAO Leu Asp 230 OTG GGT Leu Giy 245 GAT TAT Asp Tyr TTC GAO Phe Asp GGG GAO Gly Asp GAO AGO Asp Ser 310 CCC AGT Pro Ser 325 AAT GAO Asn Asp Ile

AAC

Asn

GGG

Gly 135

TTO

Phe

ACT

Thr

ACC

Thr

CAC

His

GOA

Ala 215

GAO

Asp

TGT

Cys

AAC

Asn

GGO

Gly

TGG

Trp 295 000 Arg

GAC

Asp

ACA

Thr

ATG

Met

AAO

Asn 120

ATA

Ile

TAT

Tyr

ACA

Thr

CAT

His

TCA

Ser 200

ACA

Thr

ACC

Thr

GAT

Asp

TCA

Ser

CAA

Gin 280

GTG

Val

GTG

Val

ACT

Thr

TGO

cys

AAO

Asn 105

AGO

Ser

GGO

Gly

COO

Pro

GGA

Gly

TAO

Tyr 185

CAC

His

GG

Gly

CAA

Gin

ATG

Met

GOT

Ala 265

TAT

Tyr

GC

Ala

TOG

Trp

GTA

Val

OCA

Pro 345 9C GOA ATA Ala Ile 000 TG Gly Trp AAA GAA Lys Giu TOT GOA Ser Ala 155 TCA GGT Ser Gly 170 TGO TAO Cys Tyr TOA CAT Ser His AGG GTA Arg Val AAT OGGO Asn Arg 235 OTG TOO.

Leu Cy6 250 OTO COT Val Pro CAC GAA His Glu AAC TAO Asn Tyr TTO TCA Phe Ser 315 CAG GAA Gin Glu 330 OAT GAG Asp Glu ACA TOT Thr Ser 000 GOA Oly Ala 125 OTO ATT Leu Ile 140 TTT CAA Phe Gin TOO ACT Cys Thr ACC OAT Thr His CAG TAT Gin Tyr 205 TTO TTT Phe Phe 220 AAG TCT Lys Ser TOG AAA Ser Lys AOG CG Tbx Arg AAG GAO Lys Asp 285 OCA OGA Pro Gly 300 OTO TAO Val Tyr GO AAA Gly Lys CAA GAO Gin Asp

OTO

Leu 110

OCT

Pro

GTA

Val

GAA

Glu

OGA

Arg

AAT

Asn 190

TTA

Leu

TOT

Ser

TGO

Cs 000 Ala

ATG

Met 270

OTA

Leu

OTA

Val

OGA

Gly

TAT

Tyr

TAO

Tyr 350 TOT TAT Ser Tyr ATT OAT Ile His OAT GAT Asp Asp OAT OTG His Leu 160 ATA 000 Ile Pro 175 OTA ATA Val Ile OOA OTT Ala Leu ACT OTO Thr Leu AGT GTG Ser Val 240 ACO GAG Thr 0Th 255 GTA OAT Val His OAT OTO Asp Val 000 GOT Gly Gly 000 TTA Gly Leu 320 GTG ATA Val Ile 335 CAG ATT Gin Ile 336 384 432 480 528 576 624 672 720 768 816 864 912 960 1008 1056 174

A

S

A

A

CGA

Arg

ATA

Ile

GAC

Asp 385

GAA

Glu

GGG

Gly

AAC

Asn

CGG

Arg

TGT

Cys 465

AAC

Asn

GCA

Ala

CGC

Arg

TCA

Ser

ATT

Ile 545

TTA

Leu

GGC

Gly

ATG

Met

CAG

Gin 370

CCG

Pro

GGC

Gly

TCA

Ser

AAA

Lys

CCA

Pro 450

GTT

Val

CAC

His

AGA

Arg

ATA

Ile

ACT

Thr 530

GCT

Ala

CTA

Leu

TAG

GCC AAG TCT Ala Lys Ser 355 CAG GCT ATC Gin Ala Ile GTA CTG ACT Val Le Thr AGA ATT CTC Arg Ile Leu 405 TCA TAC TTC Ser Tyr Phe 420 ACA GCC ACT Thr Ala Thr 435 GGT AGT ATC Gly Ser Ile ACT GGA GTC Thr Giy Val ACC TTG CGA Thr Leu Arg 485 CTT AAC CCT Leu Asn Pro 500 ACT CGA GTG Thr Arg Val 515 TGT TTT AAA Cys Phe Lys GAA ATA TCT Glu Ile Ser GTT GAG ATC Val Glu Ile 565

TCG

Ser

TTA

Leu

GTA

Val 390

ACA

Thr

TCT

Ser

CTT

Leu

CCT

Pro

TAT

Tyr 470

GGG

Gly

GCG

Ala

AGT

Ser

GTG

Val

AAT

Asn 550

TAT

Tyr

TCT

Ser 375

CCG

Pro

GTA

Val

CCC

Pro

CAT

His

TGC

Cys 455

ACA

Thr

GTA

Val

TCT

Ser

TCA

Ser

GTC

Vai 535

ACT

Thr

AAG

Lys 360

ATC

Ile

CCC

Pro

GGG

Gly

GCG

Ala

AGT

Ser 440

CAG

Gin

GAT

Asp

TTC

Phe

GCA

Ala

AGC

Ser 520

AAG

Lys

CTC

Leu

CCT

Pro

AAA

Lys

AAC

Asn

ACA

Thr

TTA

Leu 425

CCT

Pro

GCT

Ala

CCA

Pro

GGG

Gly

GTA

Val 505

AGC

Ser

ACC

Thr

TTC

Phe

CGG

Arg

TCA

Ser

GTC

Val 395

CAT

His

TAT

Tyr

ACA

Thr

GCA

Ala

CCC

Pro 475

ATG

Met

-GAT.

Asp

AAA

Lys

AAG

Lys

GAA

Glu 555

TT

Phe

ACA

Thr 380

ACA

Thr

TTC

Phe

CCT

Pro

TTC

Phe

AGA

Arg 460

CTA

Leu

CTT

Leu

AGC

Ser

GCA

Ala

ACC

Thr 540

TTC

Phe

GGT

Gly 365

TCC

Ser

CTC

Leu

TTG

Leu

ATG

Met

AAT

Asn 445

TGC

Cys

ATC

Ile

GAT

Asp

ACA

Thr

GCA

Ala 525

TAT

Tyr

AGA

Arg

AAA

Lys

GGC

Gly

GGG

Gly

CAG

Gin 415

GTC

Val

TTC

Phe

AAC

Asn

TAT

Tyr

GAA

Glu 495

CGC

Arg

ACA

Thr.

CTC

Leu

GTC

Val

CGC

Arg

GAA

Glu

GCC

Ala 400

CGA

Arg

AGC

Ser

ACT

Thr

TCA

Ser

AGA

Arg 480

CAA

Gin

AGT

Ser

ACA

Thr

AGC

Ser

CCG

Pro 560 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1734 CTC AAA GAT GAC GGG GTT AGA GAA GCC AGG TCT Leu Lys Asp Asp Cly Val Arg Glu Ala 570 Arg Ser 575 INFORMATION FOR SEQ ID NO:11: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 577 amino acids TYPE: amino acid TOPOLOGY: linear 175 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:l11: Giu Asn Met 1 Asp Arg Ala Val Ser Gin Val Ala Leu Asp Glu Arg Glu Ala Lys Asn Thi

S.

S S

S

S

Thr Gly Arg Val Leu Gin Asp Ali 145 Asn Ser Leu Gly Arg 225 Ser Thr Gly Thr Gly 305 Lys Val Ala Ala Asp Asn Ile Pro 130 Ser Phe Phe Ser V1al 210 Ser Al a flu Arg rhr 290 Ser Pro *Val *Ser Giu Arg Thr Asn 115 Asp Asp Ile Asp Gly 195 Leu Ile Thr Giu Leu 275 Leu Phe Asn Thr Thr Glu Ile Giu 100 Gly Tyr Vai Pro Met 180 Cys Arg Asn Pro Giu 260 Giy Phe Ile rhr Trp Leu Pro Lys Tyr Thr Ala Ile Thr Ala 165 Ser Arg Thr Leu Leu 245 Asp Phe Gly Asp Pro 325 Arg Ala Ser Ile 70 Lys Thr Ala Gly Ser 150 Pro Ala Asp Ser Asp 230 Gly Tyr Asp Asp Ser 310 Ser Leu Ile Asp 55 Thr Gin Ile Asn Giy 135 Phe Thr Thr His Ala 215 Asp cys Asn Gly Trp 295 Arg Asp Iif Sez 40 Let Ser Val Met Asn 120 Ile Tyr Thr His Ser 200 Thr Thr Asp Ser Gin 280 Val Val1 Thr Phe 25 Val IVal *Thr Ala Asn 105 *Ser Gly Pro Gly 185 His Gly.

Gin.

Met .Ala 265 Tyr Ala Trp Val Arg Ala Gly Leu Leu 90 Ala Gly Lys Ser Ser 170 Cys Set Arg Asn Leu 250 Val His Asn Phe Gln 330 Ile Ser Ile Gly 75 Giu Ile Trp Glu Ala 155 -Giy Tyr His Val Arg 235 Cys Pro Glu Tyr Ser 315 Glu Ala Leu Pro Ser Ser Thr Gly Leu 140 Phe Cys; Thr Gin' Phe 220 Lys Ser Thr Lys Pro 300 Val Gly Ile Leu Thr Asn Pro Ser Al1a 125 Ile Gin Thr His Tyr 205 Phe Ser Lys Arg Asp 285 Gly Tyr Lys Leu Tyr Arg Gin Leu Leu 110 Pro Val Giu Arg Asn 190 Leu Ser Cys Ala Met 270 Leu Val Gly Tyr Phe 5cr Ile Asp Al a Ser Ile Asp His Ile 175 Val Ala Thr Ser Thr 255 Val Asp Gly Gly Val 335 Leu Met Ser Val Leu Tyr His Asp Leu 160 Pro* Ile Leu Leu VJal 240 flu iis Jal fly 1,eu 320 Ile

S

S. S 55 176 Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Giu Gin Asp Tyr Gin Ile 340 345 350 Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly Lys Arg 355 360 365 Ile Gin Gin Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Giu 370 375 380 Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala 385. 390 395 400 Glu Gly Arg Ile Leu Thr Val Gly Thr 5cr His Phe Leu Tyr Gin Arg 405 410 415 Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser 420 425 430 Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr 435 440 445 00.00: *Arg Pro Gly Ser Ile Pro Cys Gin Ala Ser Ala Arg Cys Pro Asn Ser .450 455 460 Cys Val Thr Gly Val Tyr Thz Asp Pro Tyr Pro Leu Ile Phe Tyr Arg S 465 470 475 480 Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Gly Glu Gin 9485 490 495 Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp 5cr Thr Ser Arg Ser Soo 505 510 Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala Tyr Thr Thr 515 520 .525 Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr .Cys Leu Ser 530 535 540 Ile Ala Giu Ile Ser Asn Thr Leu Phe Gly:Glu Phe Arg Ile Val Pro- 545 550 555 560 *Leu Leu Vai Giu Ile Leu Lys Asp Asp Gly Val Arg Glu Ala Arg Ser 9565 570 575 Gly INFORMATION FOR SEQ ID NO:i2: SEQUENCE CHARACTERISTICS: LENGTH: 1662 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .1662 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 177 ATG GGC Met Gly TCC AGA CCT TCT ACC AAG AAC CCA GCA CCT ATG ATG Ser Arg Pro 5cr Thr Lys Asn 1 5 9.99.

9 p.

.9 9 0 9.

a a 99S*SS 9 S 9 **99

I

6 9 .9 5 ATC CGG Ile Arg GAT GGC Asp Giy GCA GTC Ala Val CTC CTC Leu Leu TTG GAT Leu Asp GAC TCT Asp Ser AGA CAG Arg Gin GTT GCA Val Ala 130 AAA CAA Lys Gin 145 ACC AAT Thr Asn GTG GCA Val Ala ACA GCT Thr Ala GAG CTC Giu Leu 210 ATC ACT Ile Thr 225 CTA GCT Leu Ala AAC AAT Asn Asn GTC GCG Val Ala AGG CCT Arg Pro AAC ATA Asn Ile CCG AAT Pro Asn GCA TAC Ala Tyr ATC CGT Ile Arg 100 GGG COC Gly Arg 115 ACT GCC Thr Ala AAT GCT Asn Ala GAG GCT Giu Ala OTT GG Val Gly 180 CAG GAA .Gin Glu 195 AAC CTG Asn Leu TCA CCT Ser Pro GGT GG Giy Gly CAA CTC Gin Leu 260

CTG

Leu

CTT

Leu

TAC

Tyr

CTG

Leu

AAC

Asn 85

AGG

Arg

CTT

Leu

GCA

Ala

GCC

Ala

GTG

Val 165

AAG

Lys

TTA

Leu

TAC

Tyr

GCC

Ala

AAT

Asn 245

AGC

Ser

GTA

Val

GCA

Ala

ACC

Thr

CCA

Pro 70

AGG

Arg

ATA

Ile

ATA

Ile

CAA

Gin

AAC

Asn 150

CAT

His

ATG

Met

GAC

Asp

CTA

Leu

TTA

Leu 230

ATG

Met

TCA

Ser

CTG

Leu

GCT

Ala

TCA

Ser 55

AAG

Lys

ACA

Thr-

CAA

Gin

GC

Gly

ATA

Ile 135

ATC

Ile

GAG

Giu

CAG

Gin

TGC

Cys

ACC

Thr 215

AAC

Asn

GAT

Asp

TTA

Leu

ACT

Ser

GCA

Ala 40

TCC

Ser

GAT

Asp

TTG

Leu

GAG

Giu

GCC

Ala 120

ACA

Thr

CTC

Leu

GTC,

Val

CAG

Gin

ATC

Ile 200

GAA

Giu

AAG

Lys

TAC

Tyr

ATC

Ile

TGC

Cys 25

GGA

Giy

CAG

Gin

AAG

Lys

ACC

Thr

TCT

Scr 105

ATT

Ile

GCG

Ala

CGA

Arg

ACT

Thr

TTC

Phe 185

AAA

Lys

TCG

Scr

CTG

Leu

TTA

Leu

GT

Gly 265 Pro

ATC

Ile

ATT

Ile

ACA

Thr

GAG

Giu

ACT

Thr 90

GTG

Val

ATT

Ile

GCC

Ala

CTT

Leu

GAC

Asp 170

GTT

Val

ATT

Ile

ACT

Thr

ACT

Thr

TTG

Leu 250

AGC

Ser CTG ACT Leu Thr

TOT

Cys

GTG

Val

GGA

Gly

GCA

Ala 75

TTG

Leu

ACT

Thr

GGC

Gly

GCA

Ala

AAA

Lays 155

GGA

Gly

AAT

Asn

GCA

Ala

ACA

Thr

ATT

Ile 235

ACT

Thr

GGC

Gly CCG GCA Pro Ala OTT ACA Val Thr TCA ATC Ser Ile TGT GCG Cys Ala CTC ACC Leu Thr ACA TCT Thr Ser GGT GTG Gly Val 125 GCT CTG Ala Leu 140 GAG AGC Giu Ser TTA TCG' Leu.:Ser GAC CAA Asp Gin CAG CAA Gin Gin 205 GTA TTC Val Phe 220 CAG GCA Gin Ala AAG TTA Lys Leu TTA ATC Leu Ile

AAC

Asn

GGA

Gly

ATA

Ile

AAA

Lys

CCC

Pro

GGA

Gly 110

GCT

Ala

ATA

Ile

ATT

Ile

'CAA

Gin

TTT

Phe 190

OTT

Val

GGA

Gly

CTT

Leu

GT

Gly

ACC

Thr 270 TCC ATT.

Scr Ile GAC AAA Asp Lys GTT AAG Val* Lys GCC CCC Ala Pro CTT GGT Leu Giy GGG, GGG Gly Cly CTT GGG Leu Gly CAA GCC Gin Ala GCC OCA Ala Ala 160 CTA. GCA Leu Ala 175 AAT AA.A Asn Lys GOT GTA Gly Val CCA CAA Pro Gin TAC AAT Tyr Asn 240 ATA GGG Ile Gly 255 GOT AAC Gly Asn 96 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816 Ala Pro Met Met 178 ~CA CAG ACT CAA CTC TTG GOT 0* .0 *000 0*0*0* 0 0 0* 0 0*

CCT

ProI

CTA

Leu

ACC

Thr 305

AAA(

Lys

TAC

Tyr

TTC

Phe

GCC

Ala

ACT.

Thr 385

TGT

Cys

TCT

Ser

ACT

Thr

TCA

Ser

ACT

Thr 465

TTA

Leu

AGC

Ser

GTT

Ele

XT

?ro rTA .jeu

;TG

Jai

TGT

Cys

CCT

Pro

TGT

cys 370

PATC

Ile

GTA

Vai

CTA

Leu

TTA

Leu

ATA

Ile 450

GAG

Glu

GAG

Giu

ACA

Thr TT1 CTA TAC GAC I Leu Tyr Asp E 275 TCA GTC GG Ser Val Gly TCC GTA AGC Ser Val Ser GTG ACA CGG Val Thr Arg 325 ATA GAA ACT Ile Giu Thr 340 ATG TCC CCT Met Ser Pro4 355- ATG TAC TC-A Met Tyr Ser AAA GGC TCA Lys Gly Ser AAC CCC CCG Asn Pro Pro 405 ATA GAT AAA Ile Asp Lys 420 AGO CTC AGT Arg'Leu Ser 435 CAA GAT TCT Gin Asp Ser CTT GGG MAT Leu Gly Asn GAA AGC AAC Glu Ser Asn 485 TCT OCT CTC Ser Ala Leu 500 GGT ATA CTT

ATAC

~er

LDAC

ksn kCA rhr 310 .3TC Jal 77AC ksp,

GGT

Gly

AAG

Lys

GTC

Val 390

GOT

Gly

CAA

Gin

GGG

Gly

CAA

Gin

GTC

Val 470

AGA

Arg AT1 Ile

AGC

Gin

CTA

Leu 295

ACC

Thr

GOT

Gly

TTA

Leu

ATT

Ile

ACC

Thr 375

ATC

Ile

ATC

Ile

TCA

Ser wA Gbli

GTI

Val 451

'AA(

Asi

~AN

Ly~

ACE

Th:

CT'

Thr 280

AAT

Asn

AGO

Arg

TCT

Ser

GAT

Asp

TAC

Tyr 360

GAA

Giu

OCT

Ala

*ATA

Sle

TGC

*Cys

TTC

Phe 440

SATA

Sle

AAC

1 Asr

SCT;

s Lei.

CTA'j r Ty~ G AT' u Iii 521 Gin Leu Leu Gly IleC 285 AAT ATG COT 0CC ACC Asn Met Arg Ala Thr 300 OGA TTT 0CC TCG OCA Gly Phe Ala Ser Ala 315 GTG ATA GAA GAA CTT Val Ile Giu Oiu Leu 330 TTA TAT TGT ACA AGA Leu Tyr Cys Thr Arg 345 TCC TGC TTG AGC GGC Ser Cys Leu Ser Oly 365 GOC GCA CTT ACT ACA> Gly Ala Leu Thr Thr 380 AAC TGC AAG ATO ACA.

Asn Cys Lys Met Thr 395 TCG CAA AAC TAT OGA Ser Gin Asn Tyr.Oly 410 AAT OTT TTA TCC !TTA Asn Val Leu Ser Leu 425 OAT GTA ACT TAT CGO *Asp Val Thr Tyr Gin.

445 ATA ACA GGC MAT CTT Ile Thr Gly Asn Leu 460 TCG ATC AGT MAT 0CC Ser Ile Ser Asn Ala 475 ~GAC MAA GTC MAT GTC Asp Lys Val Asn Vai 490 rATC OTT TTG ACT ATC Sle Vai Leu Thr Ile 505 r CTA OCA TGC TAC CTA e Leu Ala Cys Tyr Leu 0 525 [rAC TTG4 Cryr Leu4 -TT GTC 1,eu Val 3AC ACC k.sp Thr 335 PLTA GTA.

Ile Val 350 AAT ACA RAsn Thr CCA TAT Pro Tyr ACA TOT Thr Cys GMA GCC Giu Ala 415 GOC GG Gly Gly 430 AO MAT Lys -Asn OAT ATC AspIlie TTO AAT Leu Asn MAA CTG Lys Leu 495 ATA TCT Ile Ser 510 ATG TAC Giu

CCA

Pro 320

TCA-

Ser

ACG

Thr

TCG

Ser

ATG

Met

AGA

Arg 400

GTO

Val.

ATA

Ile

ATC

Ile

TCA

Ser

MAG

Lys 480

ACC

Thr

CTT

Leu

MAG

:AG GTA ACT dln Val Thr 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 Val Phe Oly Ilie Leu Ser Le' 515 Met Tyr Lys CAA MO Gln Lys 530 GCO CAA CAA MO Ala Gin Gin Lys TTA TTA TGG CTT Leu Leu Trp Leu

GG

Gly 540 MAT MAT ACC CTA Asn Asn Thr Leu 179 GAT CAG ATG AGA GCC ACT ACA AAA ATG TGA 1662 Asp Gin Met Arg Ala Thr Thr Lys Met 545 550 INFORMATION FOR SEQ ID NO:13: Wi SEQUENCE CHARACTERISTICS: LENGTH: 553 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13: Met Gly Ser Axg Pro Ser Thr Lys Asn Pro Ala Pro Met Met ILeu Thr 1 5 10 le Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile 25 As p Gly Arg Pro Leu Ala Ala Ala Gly Ile Val-Val Thr Gly Asp Lys 40 Ala Val Asn Ile Tyr Thr Ser Ser Gin Thr Gly Ser Ile Ile Val Lys 55 Leu Leu Pro Asn Leu Pro Lys Asp Lys Giu Ala Cys Ala Lys Ala Pro 65 70 75 Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly 90 Asp Ser Ile Arg Azg Ile Gin Giu Ear Vai Thr Thr Ser.-Gly Gly Gly 100 105 110 Arg Gin Gly Arg Leu Ile Giy Ala Ile Ile Gly Gly Val Ala Leu Gly 11S 120 125 Val Ala Thr Ala Aia Gin Ilie Thr.Ala AlA Ala Ala Leu Ile Gin Ala *130* 135 1.140 Lys Gin Asn Ala Ala Asn Ilie Leu Arg Leu Lys.Glu Ser Ile Ala Ala 145 150 155 160 Thr Asn Giu Ala Val His Giu Val Thr Asp Gly Leu Ser Gin Leu Ala 165 170 175 Val Ala Val Gly Lys Met Gin Gin Phe Val Asn Asp Gin Phe Asn Lys 185 190 Thr Ala Gin Giu Leu Asp Cys Ile Lys Ile Ala Gin Gin Vai Gly Val 195 200 205 Giu Leu Asn Leu Tyr Leu Thr Giu Ser Thr Thr Val Phe Gly Pro Gin 210 215 220 Ile Thr Ser Pro Aia Leu Asn Lys Leu Thr Ile Gin Ala Leu Tyr Asn 225 230 235 240 Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly.

245 250 255 Asn Asn Gin Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn 260 265 270 180 Pro Ile Leu Tyr Asp Ser Gin 275 Thr Gin 280 Leu Leu Gly Ile Gin Val Thr 285 Leu Thr 305 Lys Tyr Phe Al a Thr 385 Cys Ser Thr Ser Thr 465 Leu Ser Val1 Gin Asp 545 Pro Ser 290 Leu Ser Vai Val Cys Ile Pro Met 355 Cys Met 370 Ile Lys Val Asn Leu Ile Leu Arg 435 Ile Gin 450 Giu Leu Giu Giu Thr Ser Phe Giy 515 Lys Ala 530 Gin Met Vai Val Thr Glu 340 Ser Gly Pro Asp 420 Leu Asp Gly Ser Al a 500 Ile Gin Arg Gly Ser Arg 325 Thr Pro Ser Ser Pro 405 Lys Ser Ser Asn Asn 485 Leu Leu Gin Al a Asn Thr 310 Val Asp Gly Lys Val 390 Gly Gin Gly Gin Val 470 Arg Ile Ser Lys Thr 550 Leu Asn 295 Thr Arg Giy Ser Leu Asp Ile Tyr 360 Thr Glu 375 Ile Ala Ile Ile Ser Cys Giu Phe 440 Val Ile 455 Asn Asn Lys Leu Thr Tyr Leu Ile 520 Thr Leu 535 Thr Lys Asn Gly Val Leu 345 Ser Gly Asa Ser Asn 425 Asp Ile Ser Asp Ile 505 Leu Leu Met Met Phe Ile 330 Tyr Cys Ala Cys Gin 410 Val Vai Thr Ile.

Lys 490 Vai Ala Trp Arg Ala 315 Glu Cys Leu Leu Lys 395 Asn Leu Thr Gly Ser 475 Val.

Leu Cys Leu Aia Thr 300 Ser Ala Giu Leu Thr Arg Ser Gly 365 Thr Thr 380 Met Thr Tyr Gly Ser Leu Tyr Gin 445 Asn Leu- Asn Aia Asn Val Thr Ile Tyr Leu 525 Giy Asn 540 Tyr Leu Asp Ile 350 Asn Pro Thr Giu Gly 430 Lys Asp Leu Lys Ile Met Asn Leu Vai Thr 335 Val Thr Tyr Cys Ala 415 Gly Asa Ile Asa Leu 495 Ser Tyr Thr Giu Pro 320 Ser Thr Ser Met Arg 400 Val Ile Ile Ser Lys 480 Thr Leu Lys Leu INFORMATION FOR SEQ ID NO:14: Wi SEQUENCE CHARACTERISTICS: LENGTH: 3489 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 181 (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .3489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TTG GTA ACA CCT CTT TTA CTA GTG ACT CTT TTG TGT GTA Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val

ATG

Met 1 CTA TGT Leu Cys AGT GCT GCT Ser Ala Ala GCC TTT AGA Ala Phe Arg GTA GTT AAT Val Val Asn

TTG

Leu TAT GAC AGT AGT Tyr Asp Ser Ser TAC GTT TAC TAC Tyr Val Tyr Tyr CCA CCT AAT GGT Pro Pro Asn Gly

TG

Trp 40 CAT TTA CAC GGG His Leu His Gly

GGT

Gly

TCT

Ser TAC CAA AGT Tyr Gin Ser GCT TAT GCG Ala Tyr Ala TCA CCT GG Ser Pro Gly ATT TCT AGC Ile Ser Ser TCT AAT AAT GCA Ser Asn Asn Ala

TGT

Cys ATT GTT GGT ACT Ile Val Gly Thr

ATT

Ile 70 CAT GOT GGT CGT His Gly Gly Arg

GTT

Val 75 GTT AAT. GCT TCT Val Asn Ala Ser

TCT

Ser ATA GCT ATG ACG Ile Ala Met Thr

GCA

Ala CCG TCA TCA GGT Pro Ser Ser Gly

ATG

Met 90 GCT TOG TCT AGC Ala Trp Ser Ser AGT CAG Ser Gln' TTT TGT ACT Phe Cys Thr CAT TGT TAT His Cys Tyr 115 AAT TTT TTA Asn Phe Leu 130

GCA

Ala 100 CAC TGT AAC TTT His Cys Asn Phe

TCA

Ser 105 OAT ACT ACA GTG Asp Thr Thr Val TTT GTI' ACA Phe Val Thr 110 CTT 'CAA AAG Leu, Gin Lys, AAA TAT GAT GG Lys Tyr Asp Gly

TOT

Cys 120 CCT ATA ACT GC Pro Ile -Thr Gly

ATG.

Met 125 144 192 240 288 336 384 432 480 528 576 624 672 COT GTT TCT Arg Val Ser GCT ATG Ala Met 135 AAA AAT GGC Lys Asn Gly CAG CTT TTC TAT AAT Gin Leu-,Phe Tyr Asn

TTA

Leu 145 ACA OTT ACT GTA Thr Val Ser Val

OCT

Ala I50 AAO TAC CCT ACT Lys Tyr Pro Thr

TTT

Phe 155 AAA TCA TTT CAG Lys Ser Phe Gin

TGT

Cys 160 OTT AAT AAT TTA Val Asn Asn Leu

ACA

Thr 165 TCC GTA TAT TTA Ser Val Tyr Leu

AAT

Asn 170 GOT OAT CTT OTT Gly Asp Leu Val TAC ACC Tyr Thr 175 TCT AAT GAG Ser Asn. Glu GOT GGA CCT Gly Gly Pro 195

ACC

Thr 180 ACA OAT OTT ACA Thr Asp Val Thr

TCT

Ser 185 OCA GOT GTT TAT Ala Gly Val Tyr TTT AAA OCT Phe Lys Ala 190 0CC CTO OCT Ala Leu Ala ATA ACT TAT AAA~ Ile Thr Tyr Lys

OTT

Val 200 ATO AGA AAA GTT Met Arg Lys Val

AAA

Lys 205 TAT TTT Tyr Phe 210 CCT AGA Pro Arg 225 GTT AAT GOT ACT Val Asn Oly Thr GOC TTO TTA OCA Gly Leu Leu Ala 230

GCA

Ala 215 CAA GAT OTT ATT Gin Asp Val Ile

TTO;

Leu 220 TOT OAT GGA TCA Cys Asp Gly Ser TOC CAO TAT AAT ACT Cys Gin Tyr Asn Thr 235 GOC AAT TTT TCA Gly Asn Phe Ser

GAT

Asp 240 182 GGC TTT TAT CCT TTT ATT AAT AGT AGT TTA GTT AAG CAG AAG TTT ATT 768

S

S

S

.55555 Gly

GTC

Val

ACT

Thr

AAT

Asn

TTT

Phe 305

ATG

Met

AAT

Asri

CCT

Pro

TGT

Cys

TAT

Tyr 385

GTT

Val

GTT

Val

GAT

Asp

ACC

Thr

ATT

Ile 465

TAT

Tyr Phe

TAT

Tyr

TTT

Phe

ATT

Ile 290

AAT

Asn

TAT

Tyr

AAT

Asn

CTT

Leu

TGT

Cys 370

TCA

Ser

ACT

Thr

ATA

Ile

TAT

Tyr

GAC

Asp 450

TTA

Leu

GGT

Giy Tyr

CGT

Arg

CAT

His 275

CTA

Leu

TTT

Phe

GGA

Gly

GGC

Gly

CAA

Gin 355

TAT

Tyr

GGT

Gly

AAG

Lys

ACT

Thr

AAT

Asn 435

TCA

S er

GAT

Asp

CTT

Leu Pro

GAA

Giu 260

AAT

Asn

ACT

Thr

TCC

Ser

TCT

Ser

TTG

Leu 340

GGT

Gly

GCT

Aila

GAG

Giu

AGC

Ser

CGA

Arg 420

ATA

Ile

GCT

Aila

ACA

Thr

ACT

Thr Phe 245

AAT

Asn

GAG

Giu

TAC

Tyr

TTT

Phe

TAT

Tyr 325

TGG

Trp

GGT

Gly

TAT

Tyr

TTA

Leu

GGT

Giy 405

CAC

His

TAT

Tyr

GTT

Val

TCT

Ser

TAT

Tyr 485 Ile Asn AGT GTT Ser Val ACT GGC Thr Giy CAA ACA Gin Thr 295 CTG AGT Leu Ser 310 CAC CCA His Pro TTT AAT Phe Asn TGC AAG Cys Lys TCA TAT Ser Tyr 375 GAT CTT Asp Leu 390 GGC TCT Gly Ser AAT TAT Asn Tyr GGC AGA Gly Arg AGT TAT Ser Tyr 455 GGT TCC Gly Ser 470 TAT AAG Tyr Lys Ser Ser AAT ACT Asn Thr 265 GCC AAC Ala Asn 280 CAA ACA Gin Thr AGT TTT Ser Phe AGT TGT Ser Cys TCA CTT Ser Leu 345 CAA TCT Gin Ser 360 GGA GGT Giy Gly AAT TTT Asn Phe CGT ATA Arg Ile AAT AAT Asn Asn 425 ACT GGC Thr Gly 440 AAT TAT Asn Tyr ATA GAC Ile Asp GTT AAC Val Asn Leu 250

ACT

Thr

CCT

Pro

GCT

Ala

GTT

Val

AAT

Asn 330

TCA

Ser

GTC

Vai

CCT

Pro

GAA

Glu

CAA

Gin 410

ATT

Ile

CAA

Gin

CTA

Leu

ATC

Ile

CCT

Pro 490 TTT ACG Phe Thr AAT CCT Asn Pro CAG AGT Gin Ser 300 TAT AAG Tyr LYS 315 TTT AGA Phe Arg GTT TCA Val Ser TTT AGT Phe Ser .TCG CTG Ser Leu 380 TGT. GGA Cys Giy 395 ~ACA GCC.

Thr. Al a ACT TTA Thr Leu GGT TTT Giy Phe GCA GAC Ala Asp 460 TTT GTT Phe Val 475 TGC GAA Cys Giu

TTA

Leu

AGT

Ser 285

GGT

Giy

GAG

Giu

CTA

Leu

ATT

Ile

GGT

Giy 365

TGT

Cys

CTG

Leu

ACT

-Thr

AAT

Asn

ATT

Ile 445

GCA

Aila

GTA

Val

GAT

Asp

CAC

His 270

GGT

Giy

TAT

Tyr

TCT

Ser

GAA

Giu

GCT

Ala 350

AGA

Arg

AAA

Lys

TTA

Leu

GAA

'Giu.

ACT

Thr 430

ACT

Thr

GGT

Giy

CAA

Gin

GTC

Val

AAT

Asn

GTT

Val

TAT

Tyr

AAT

Asn

ACT

Thr 335

TAC

Tyr

GCA

Ala

GGT

Gly

GTT

Vai

CCG

Pro 415

TGT

Cys

AAT

Asn

TTG

Leu

GGT

Gly

AAC

Asn 495

TTC

Phe

CAG

Gin

AAT

Asn

TT

Phe 320

ATT

Ile

GGT

Gly

ACT

Thr

GTT

Vali

TAT

Tyr 400

CCA

Pro

GTT

Val

GTA

Vai

GCT

Aia

GAA

Giu 480

CAG

Gin 816 864 912 960 1008 1056 11.04 1152 1200 1248 1296 1344 1392 1440 1488 1536 Vai Lys Gin Lys Phe Ile 255 CAG TTT GTA GTT TCT GGT GGT Ser Gly Gly AAA TTA GTA GGT ATT CTT ACT TCA CGT Lys Leu Vai Giy Ilie Leu Thr Ser Arg Gin Phe Val Val 500 183 AAT GAG ACT GGT TCT CAG CTT CTT GAG AAC CAG TTT TAC ATT AAA ATC 1584 Asn Giu Thr Gly Ser Gin Leu Leu Giu Asn Gin Phe Tyr Ile Lys Ile 515 1520 525- ACT AAT GGA ACA CGT CGT TTT AGA-CGT TCT ATT ACT GAA AAT GTT GCA 1632 Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Giu Asn Val Ala 530 535 540 AAT TGC CCT TAT GTT AGT TAT GGT AAG TTT TGT ATA AAA CCT GAT GGT .1680 Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly 545 550 555 560 TCA ATT GCC ACA ATA GTA CCA AAA CAA TTG GAA CAG TTT GTG GCA CCT 1728 Ser Ile Ala Thr Ile Vai Pro Lys Gin Leu Giu-Gin-Phe Vai Ala Pro 565 570 575 TTA CTT AAT GTT ACT GAA AAT GTG CTC ATA CCT AAC AGT TTT AAT TTA 1776 Leu Leu Asn Val Thr Giu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu 580 565 590 a ACT GTT ACA GAT GAG TAC ATA CAA ACG CGT ATG GAT AAG GTC CAA ATT 1824 Thr Val Thr Asp Giu Tyr Ile Gin Thr Arg Met Asp Lys Val Gin Ile 595 600 605 AAT TGT CTG CAG TAT OTT TOT GGC AAT TCT CTG GAT TOT AGA GAT.TTG 1872 Asn Cys Leu Gin Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu 610 615 620 TTT CAA CAA TAT GGO CCT OTT TOT GAC AAC ATA TTG TCT GTA GTA AAT 1920 Phe Gin Gin Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn 625 630 635 640 AGT ATT GOT CAA AAA GAA OAT ATG GAA CTT TTG AAT TTC TAT TCT TCT 1968 Ser Ile Gly Gin Lys Giu Asp Met Giu Leu Leu Asn Phe Tyr Ser Ser 645 650 655 ACT AAA CCO GCT GGT TTT AAT ACA CCA TTT CTT AGT AAT GTT AGC ACT 2016 Thr Lys Pro Ala Gly Phe Asn Thx Pro Phe Leu Ser Asn Val Ser Thr 660 665 670 GGT GAG TTT AAT ATT TCT CTT CTG. TTA ACA- ACT -CCTAG.T AOT CCT AGA 2064.

ly Oiu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg.

:675 680 -685 AGG COT TCT TTT ATT GAA GAC CTT CTA TTT ACA AGC GTT GAA TCT GTT 2112 Arg Arg Ser Phe Ile Oiu Asp Leu Leu Phe Thr Ser Val Giu Ser Val 690 695 700 GGA TTA CCA ACA GAT GAC GCA TAC AAA AAT TGC.ACT OCA GGA CCT TTA. 2160 Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu 705 710 715 720 GGT TTT CTT AAG GAC CTT OCG TOT GCT COT GAA TAT AAT GOT TTO CTT 2208 Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Giu Tyr Asn Gly Leu Leu 725 730 735 GTG TTG CCT CCC ATT ATA ACA GCA GAA ATO CAA ACT TTG TAT ACT AGT 2256 Val Leu Pro Pro Ile Ile Thr Ala Giu Met Gin Thr Leu Tyr Thr Ser 740 745 750 TCT CTA GTA GCT TCT ATG GCT TTT GOT GGT ATT ACT GCA GCT GOT GCT 2304 -Ser Leu Vai Ala Ser Met Ala Phe Gly Oly Ile Thr Ala Ala Oly Ala.

755 760 765 ATA CCT TTT GCC ACA CAA CTO CAG OCT AGA ATT AAT CAC TTG GGT ATT 2352 Ile Pro Phe Ala Thr Gin Leu Gin Ala Arg Ile Asn His Leu Gly Ile 770 775 780 184 ACC CAG TCA CTT TTG TTG AAG AAT CAA GAA AAA ATT GCT GCT TCC TTT Thr Gin Ser Leu Leu Leu Lys Asn Gin Giu Lys Ile Ala Ala Ser Phe 785 AAT AAG GCC Asn Lys Ala GCA TTA CAA Ala Leu Gin ACT GAG ACT Thr Glu Thr 835 GTG ATT CAA Val Ile Gin 850 CAA GTG GAT Gin Val Asp 865 GCA TCT GCT Ala Ser Ala TTA GCT ACT Leu Ala Thr TAC TCC TTT Tyr Ser Phe 915 GCA CCT AAT Ala Pro Asn 930 TTT GTT AAT Phe Val Asn 945 OCT AGT CAG Ala Ser Gin CAA GTT AAT Gin Val Asn AGA GCT ATT Arg Ala Ile 995 AAT TAT GTA Asn Tyr Val 1010 GAT GAT TTT Asp Asp Phe 1025

ATT

Ile

CAA

Gin 820

ATG

Met

GAA

Glu

CGT

Arg

AAG

Lys

CAG

Gin 900

TGT

Cys

GGT

Gly

OTT

Val

TAT

Tyr

GGT

Gly 980

ACT

Thr

AGT

Ser

OAT

Asp

GGT

Gly 805

ATT

Ile

GCA

Ala

ATC

Ile

CTT

Leu

CAG

Gin 885

AAA

Lys

GGT

Gly

ATA

Ile

ACT

Thr

GCA

Ala 965

AGT

Ser

GCA

Ala

GTA

Val

TTT

Phe 790 CGT ATG CAG GAA Arg Met Gin Glu CAA GAT GTT OTT Gin Asp Val Val 825 TCA CTT AAT AAA Ser Leu Asn Lys 840 TAC CAG CAA CTT Tyr Gin Gin Leu 855 ATA ACT GOT AGA Ile Thr Oly Arg 870 GCG GAG CAT ATT Ala Giu His Ile ATT AAT GAG TGT Ile Asn Giu Cys 905 AAT OGA CGA CAT Asn Gly Axg His 920 GTG TTT ATA CAC Val Phe Ile His 935 OCA ATA GTG GGT Ala Ile Val Gly 950 ATA OTA CCC GCT Ile Val Pro Aia TAC TAC ATC ACA Tyr Tyr Ile Thr 985 GGA OAT ATA GTT Gly Asp Ile Vai 1000 AAT AAG ACC GTC Asn Lys Thr Val 1015 AAT GAC OAA TTG Asn Asp Giu Leu 1030

GGT

Gly 810

AAT

Asn

AAT

Asn

GAC

Asp

TTG

Leu

AGA

Arg 890

OTT

Vai

OTT

Val

TTT-

Phe

TTT

Phe

AAT

Asn 970

GCA

Ala

ACG

Thr

ATT

Ile

TCA

Ser 795 TTT AGA AGT ACA Phe Arg Ser Thr AAG CAG AOT OCT Lys Gin Ser Ala 830 TTT GOT OCT ATT Phe Oly Ala Ile 845 0CC ATA CAA OCA Ala Ile Gin Ala 860 TCA TCA CTT TCT Ser Ser Leu Ser 875 GTG TCA CAA CAG Val Ser Gin Gin AAO TCA CAG TCT Lys Ser Gin Ser 910 CTA ACC ATA CCO -Leu Thr Ile Pro 925 TCT TAT ACT CCA Ser Tyr Thr Pro 940 TGT OTA AAG CCA Cys Vai Lys Pro 955 OGT AGO GOT ATT Oly Arg Giy lle CGA OAT ATO TAT Arg Asp Met Tyr 990 CTT ACT TCT TGT Leu Thr Ser Cys 1005 ACT ACA TTC OTA Thr Thr Phe Vai 1020 AAA TOG TOG AAT Lys Trp Trp Asn 1035

TCT

Ser 815

ATT

Ile

TCT

Ser

AAT

Asn

OTT

Vai

CGT

Arg 895

ATT

Ile

CAA

Gin

OAT

Asp

GCT'

Ala

TTT

Phe 975

ATO

Met

CAA

Gin

GAC

Asp

GAC

Asp 800

CTA

Leu

CTT

Leu

TCT

Ser

OCT

Ala

TTA

Leu 880

GAG

Glu

AGO

Arg

AAT

Asn

AGT'

Ser

AAT

Asn 960

ATA

Ile

CCA

Pro

OCA

Ala

AAT

Asn

ACT

Thr 1040 2400 2448 2496 2544 2592 2640 2688 2736 2784 2832 2880 2928 2976 3024 3072 3120 9 AAG CAT GAG CTA CCA GAC TTT GAC AAA TTC AAT TAC ACA OTA CCT ATA Lys His Giu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile 3168 1045 1050 1055 185 CTT GAC ATT GAT AGT GAA ATT OAT CGT ATT CAA GGC GTT ATA CAG GGT Leu Asp Ile Asp Ser Giu Ile Asp Arg Ile Gin Gly Val Ile Gin Gly 1060 1065 1070 CTT AAT GAC TCT TTA ATA GAC CTT GAA AAA CTT TCA ATA CTC AAA ACT Leu Asn Asp Ser Leu Ile Asp Leu Giu Lys Leu Ser Ile Leu Lys Thr 1075 1080 1085 TAT ATT AAG TGG CCT TGG TAT GTG TGG TTA GCC ATA GCT TTT GCC ACT Tyr Ile Lys Trp, Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Ala Thr 1090 1095 1100 ATT ATC TTC ATC TTA ATA CTA GGA TOG GTT TTC TTC ATG ACT GGA TOT Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys 1105 1110 1115 1120 TGT GOT TGT TGT TGT GGA TGC TTT OGC ATT ATG CCT CTA ATG AGT AA.G Cys Giy Cys Cys Cys Giy Cys Phe Gly Ile Met Pro Leu Met Ser Lys 1125 1130 2135 TGT GOT AAG AAA TCT TCT TAT TAC ACG ACT TTT GAT AAC GAT GTG GTA Cys Giy Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val 1140 1145 1150 ACT GAA CAA AAC AGA CCT AAA AAG TCT GTT TAA Thr Giu Gin Asn Arg Pro Lys Lys Ser Val 1155 1160 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1162 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 3216 3264 3312 3360 3408 3456 3489 0 ~0 0* Met S er Ala Val Cys Ile Phe His Asn (xi) S Leu Val Ala Ala Phe Arg Val Asn Ilie Val Ala Met Cys Thr Cys Tyr 115 Phe Leu 130

SEQUENCE

Thr Pro 5 Leu Tyr Pro Pro Ilie Ser Gly Thr Thr Ala Ala His 100 Lys Tyr Arg Val DESCRIPTION: SEQ ID Leu Leu Leu Val Thr 10 Asp Ser Ser Ser Tyr 25 Asn Gly Trp His Leu 40 Ser Giu Ser Asn Asn 55 Ile His Gly Gly Arg 70 Pro Ser Ser Gly Met Cys Asn Phe Ser Asp 105 Asp Gly Cys Pro Ile 120 Ser Ala Met Lys Asn 135 NO: Leu- Leu' Val Tyr His Gly Ala Gly Val Val 75 Ala Trp Thr Thr Thr Gly Gly Gin 140 Cyo Tyr Oly Ser Asn Ser Val Met 125 Leu Val.,Leu..Cys 115 Tyr Gin Ser Ala Tyr Ala Ser Pro Gly Ala Ser Ser Ser Ser Gin Phe Val Thr 110 Leu Gin Lys- Phe Tyr Asn 186 Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gin Cys 145 150 155 160 Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr 165 170 175 Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 180 185 190 (fly Gly Pro Ile Thr Tyr Lys Val Met Arg Lys Val Lys Ala Leu Ala 195 200 205 Tyr Phe Val Asn Gly Thr Ala Gin Asp Val Ile Leu Cys Asp Gly Ser 210 215 220 Pro Arg Gly Leu Leu Ala Cys Gin Tyr Asn Thr Gly Asn Phe Ser Asp 225 230 235 240 Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gin Lys Phe Ile se*:245 250 255 Val Tyr Arg Giu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe 260 265 270 Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gin e :275 280 285 Asn Ile Leu Thr Tyr Gin Thr Gin Thr Aia Gin Ser Giy Tyr Tyr Asn 290 295 300 Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Giu Ser Asn Phe 305 310 315 320 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile 325 330 335 Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly 340 345 350 Pro Leu Gin Gly Giy Cys Lys Gin Ser Val Phe Ser Giy Arg Ala Thr 355 360 365 Cys Cys Tyr Ala Tyr Ser Tyr Giy Gly Pro Ser Leu Cys Lys Gly Val Tyr Ser Giy Giu Leu Asp Leu Asn Phe Glu Cys Giy Leu Leu Val Tyr 385 390 395 400 Val Thr Lys Ser Gly Giy Ser Arg Ile Gin Thr Ala Thr Giu Pro Pro 405 410 415 Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val 420 425 430 Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gin Gly Phe Ilie Thr Asn Val 435 440 445 Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala 450 455 460 Ile Leu Asp Thr Ser Giy Ser Ile Asp le Phe Val Val Gin Gly Giu 465 470 475 480 Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Giu Asp Val Asn Gin 485 490 495 187 Gin Phe Vai Val Ser Gly Gly Lys Leu 500 N\50

S

S. S S. 55

S

*SS*

a *5*9

S

*5*5 a

S

S

*SS@Ss 5

S.

Asn Thr Asn 545 S er Leu Thr Asn Phe 625 Ser Thr Gly Arg Gly 705 Gly Val Ser 1 -1e Thr 785 Asn Ala Giu Thr 515 Asn Giy 530 Cys Pro Ile Ala Leu Asn Val Thr 595 Cys Leu 610 Gin Gin Ile Giy Lys Pro Giu Phe 675 Arg Ser 690 Leu Pro Phe Leu Leu Pro Leu Vai 755 Pro Phe 770 Gin Ser Lys Ala Leu Gin Gly thr Tyr Thr Val Asp Gin Tyr Gin Ala 660 Asn Phe Thr Lys Pro 740 Ala Ala Leu Ile Gin 820 Ser Gin Leu Arg Arg Phe 535 Val Ser Tyr 550 Ile Vai Pro 565 Thr Glu Asn Giu Tyr Ile Tyr Val Cys 615 Gly Pro Val 630 Lys Giu Asp 645 Gly Phe Asn Ile Ser Leu Ile Giu Asp 695 Asp Asp Ala 710 Asp Leu Aia 725 Ile Ile Thr Ser Met Ala Thr Gin Leu 775 Leu Leu Lys 790 Gly Arg Met 805 Ile Gin Asp Leu 520 Arg Gly Lys Val Gin 600 Gly Cys Met Thr Leu 680 Leu Tyr Cys Al a Phe 760 Gin Asn Gin Val Giu Arg Lys Gin Leu 585 Thr Asn Asp Giu Pro 665 Leu Leu Lys Ala Giu 745 Gly Al a Gin Glu Val1 825 Val Gly Asn Gin Ser Ile Phe Cys 555 Leu Giu 570 Ile Pro Arg Met Ser Leu Asn Ile 635 Leu Leu 650 Phe Leu Thr Thr Phe--Thr- Asn Cys ~715 Arkg*-Giu 730 Met Gin Giy Ile Arg Ile Glu Lys 795 Gly Phe 810 Asn Lys Ile Phe Thr 540 Ile Gin Asn Asp Asp 620 Leu Asn Ser Pro Ser 700 Thr Tyr Thr Thr Asn 780 Ile Arg Gin Leu Thr 510 Tyr Ile 525 Glu Asn Lys Pro Phe Val Ser Phe 590 Lys Val 605 Cys Arg Ser Val Phe Tyr Asn Val 670 Ser Ser 685 Val Giu Ala Giy Asn Gly Leu Tyr 750 Ala Ala 765 His Leu Ala Ala Ser Thr Ser Ala 830 Ser Arg Lys Ile Val Ala Asp Gly 560 Ala Pro 575 Asn Leu Gin Ile Asp Leu Val Asn 640 Ser Ser 655 Ser Thr Pro Arg Ser Val Pro Leu 720 Leu Leu 735 Thr Ser Gly Ala Gly Ile Ser Phe 800 Ser Leu 815 Ile Leu Thr Giu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser 835 845 188 Vai Ile Gin Giu Ile Tyr Gin Gin .Leu Asp Ala Ile Gin Ala Asn Ala 850 855 860 Gin Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu 865 870 875 880 Ala Ser Ala Lys Gin Ala Giu His Ile Arg Val Ser Gin Gin Arg Giu 885 890 895 Leu Ala Thr Gin Lys Ile Asn Giu Cys Vai Lys Ser Gin Ser Ile Arg 900 905 910 Tyr Ser Phe Cys Gly Asn Gly Arg His Val -Leu Thr Ile Pro Gin Asn 915 920 925 Ala Pro Asn Giy Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser 930 935 940 Phe Vai Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn 945 950 955 960 Ala Ser Gin Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile 965 970 975 Gin Vai Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro **980 985 990 Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gin Ala 995 1000 1005 Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn 1010 1015 1020 Asp Asp Phe Asp Phe Asn Asp Giu Leu Ser Lys, Trp Trp Asn Asp Thr *1025 1030 1035 1.040 Lys His Giu Leu Pro Asp Phe Asp Lys Phe.Asn Tyr Thr Val Pro Ile 1045 1050 1055 Leu Asp Ile Asp Ser Giu Ile Asp Arg Ile Gin Gly Val Ile Gin Gly :1060 1065 1070 Leu Asn Asp Ser Leu Ile Asp Leu Giu Lys Leu'Ser Ile Leu Lys Thr 1075 1080 1085 Tyr Ile Lys Trp Pro Trp Tyr Vai Trp Leu Ala Ilie Ala Phe Ala Thr 1090 1095 1100 Ile Ile Phe Ilie Leu Ile Leu Giy Trp Val Phe Phe Met Thr Gly Cys 1105 1110 ill5 1120 Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys 1125 1130 1135 Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Vai 1140 1145 1150 Thr Giu Gin Asn Arg Pro Lys Lys Ser Vai 1155 INFORMATION FOR SEQ ID NO:i6: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1846 base pairs TYPE: nucleic acid STRANDEDNESS: double 189 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genanic) (iii) HYPOTHETICAL: NO (iv) ANT'I-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1._-1846 (xi) SEQUENCE DESCRIPTION:. SEQ ID NO:l6:

ATG

Met 1 TTG GTG AAG TCA CTG TTT CTA GTG Leu Val Lys Ser Leu Phe Leu Val

ACC

Thr 10 ATT TTG TTT GCA Ile Leu Phe Ala CTA TGT Leu Cys is -ego.: AGT GCT AAT Ser Ala Asn GCT TTT AGG Ala Phe Arg

TTA

Leu 20 TAT GAC AAC GAA TCT TTT GTG TAT TAC Tyr Asp Asn Glu Ser Phe Val Tyr Tyr TAC CAG AGT Tyr Gin Ser GCT TAT.GCA Ala Tyr Ala CCA GGA CAT GGT Pro Gly His Gly CAT TTA CAT GGA His Leu His Gly

GGT

Gly GTA GTT Val Val so AAT GTG TCT AGT Asn Val Ser Ser AAT AAT AAT GCA Asn Asn Asn Ala ACT GCC CCA AGT Thr Ala Pro Ser

TGC

Cys ACT GCT GGT GCT Thr Ala Gly Ala

ATT

Ile 70 GGC TAC AGT AAG Gly Tyr Ser Lys

AAT

Asn TTC AGT GCG GCC Phe Ser Ala Ala 240 99 GTA GCC ATG ACT Val Ala Met Thr TTT TGT ACA GCT Phe Cys Thr Ala 100 CAT TGT TTT AAG His Cys Phe Lys 115

GCA

Ala as CCA CTA AGT GGT Pro Leu Ser Gly TCA TGG TCT Ser Trp Ser GCC :TCA TCT Ala Ser Ser CAC TGT AAT TTT His Cys Asn Phe

ACT

Thr 105 TCT TAT ATA Ser Tyr Ile GTG-TTT-OTT ACA Vai Phe -Val Thr AGC GGA TCT Ser Gly Ser

AAT

Asn 120 AGT TGT CCT TTG Ser Cys Pro Leu

ACA

Thr 125 GOT CTT ATT Oly Leu Ile 288 336 384 432 480 528 CCA AGC Pro Ser 130 GOT TAT ATT CGT Gly Tyr Ile Arg

ATT

Ile 135 GCT OCT ATG AAA Ala Ala Met Lys

CAT

His 140 GGA AGT CGT ACG Oly Ser Arg Thr C CT Pro 145 GGT CAC TTA TTT Gly His Leu Phe

TAT

Tyr 150 AAC TTA ACA GTT Asn Leu Thr Val

TCT

Ser 155 GTG ACT AAA TAT Val Thr Lys Tyr

CCT

Pro 160 AAG TTT AGA TCG Lys Phe Arg Ser

CTA

Leu 165 CAA TGT GTT AAT Gin Cys Val Asn

AAT

Asn 170 CAT ACT TCT GTA His Th~r Ser Val TAT TTA Tyr Leu 175 A.AT GGT GAC Asn Gly Asp GCA GGT GTC Ala Gly Val 195

CTT

Leu 180 OTT TTC ACA TCT Val Phe Thr Ser

AAC

Asn 185 TAT ACT GAA GAT Tyr Thr Giu Asp OTT GTA GCT Val Val Ala 190 AAA GTT ATG Lys Val Met CAT TTT AAA AGT His Phe Lys Ser

GGT

Gly 200 GGA CCT ATA ACT Oly Pro Ile Thr AGA GAG Arg Giu 210 GTC ATT Val Ile 225 AAT ACT Asn Thr ATT GTT Ile Val ACT TTG Thr Leu CCT AAT Pro Asn 290 GCT CAG Ala Gin 305 GTT TAT Val Tyr AGT TTT Ser Phe OTT TCA Val Ser T'rT AAT Phe Asn 370 COT OCT Axg Ala 385 TOT GGT Cys Gly ACT GCA Thr Ala ACT TTA Thr Leu GGT TTT Gly Phe 450 GCG GAG Ala Giu 465

GITT

Val

CTA

Leu

GGC

Gly

AAG

Lys

ACA

Thr 275

ACA

Thr

AGT

Ser

AGG

Arg

AGA

Arg

TTA

Leu 355

GGT

Gly

TGT

Cys

TTG

Leu

ACA

Thr

GGT

Gly 435

ATT

Ile

GGA

Gly

AAA

Lys

TGT

Cys

AAT

Asn

GAT

Asp 260

TTA

Leu

GOT

Gly

GGT

Gly

OAA

Giu

CCT

Pro 340

ATA

Ile

AAA.

Lys

AAA

Lys

TTA

Leu

CAA

Gin 420

AAG

Lys

ACT

Thr

GGA

Gly 0CC Ala

GAT

Asp

TTT

Phe 245

AAG

Lys

ACT

Thr

GOT

Giy

TAT

Tyr

AGT

Ser 325

GAA

Giu

TAC

Tyr

GCA

Ala

GGT

Gly

GTT

Val 405

CCA

Pro

TOT

Cys

AAT

Asn

TTA

Leu TTG GCT Leu Ala 215 GAC ACA Asp Thr 230 TCA OAT Ser Asp TTT ATT Phe Ile AAT TTC Asn Phe OTT GAC Val Asp 295 TAT AAT Tyr Asn 310 AAT TAT Asn Tyr ACC CTT Thr Leu GOT CCC Gly Pro ACT TOT Thr Cys 375 GTC TAT Val Tyr 390 TAT OTT Tyr Val CCT GTA Pro Val OTT OAT Val Asp OTA ACT Val Thr 455 OCT ATT Ala Ile 470

TAT

Tyr

CCT

Pro

GC

Gly

GTT

Val

ACG

Thr 280

ACT

Ser

TTT

Phe

ATG

Met

AAT

Asn

ATT

Ile 360

TGT

Cys

AGA

Arg

ACT

Thr

TTA

Leu

TAT

Tyr 440

OAT

Asp

TTA

Leu

TTT

Phe

AGA

Arg

TTC

Phe

TAT

Tyr 265

TTT

Phe

TTT

Phe

AAT

Asn

TAT

Tyr

GOT

Gly 345

CAA

Gin

TAT

Tyr

GOT

Giy

AAG

Lys

ACC

Thr 425

AAT

Asn

TTA

Leu

OAT

Asp 190 GTC AAT GOT Val. Asn Gly 220

GT

Oly

TAT

Tyr 250

CGT

Arg

AGT

Ser

ATT

Ile

TTT

Phe

OGA

Gly 330

TTG

Leu

GT

Gly

GCT

Ala

GAG

Giu

AGC

Scr 410

CAA

Gin

OTT

Val

OCT

Ala

ACA

Thr TTG TTA Leu Leu 235 CCT TTT Pro Phe GAA AGT Giu Ser AAT GAA Asn Giu TTA TAC Leu Tyr 300 TCA TTT Ser Phe 315 TCT TAC Ser Tyr TOG TCT Trp Ser GGT TGT Gly Cys TAT TCA Tyr Ser 380 CTA ACA Leu Thr 395 OAT GGC Asp Gly AAT TTT Asn Phe TAT GGT Tyr Gly ACT TCC Thr Ser 460 TCT GOT Ser Gly 475 OCA TGC Ala Cys ACT AAT Thr Asn AGT GTC Ser Val, 270 AGT GOT Scr Gly 285 CAG ACA Gin Thr CTG AGT Leu Ser CAT CCG His Pro AAT TCC Asn Ser .350

AAG:CAA

Lys Gin 365 TAC GGA Tyr Gly CAG CAT Gin His TCC COT Ser Arg TAT AAT Tyr Asn 430 AGA ACT Arg Thr 445 CAT AAT His Asn GCC ATA Ala Ile CAA TAT Gin Tyr 240 ACT AGT Thr Ser 255

AAT'ACT

Asn Thr 0CC CCT Ala Pro C.AA ACA Gin Thr AGT TTT Ser Phe 320 GCT TOT Ala Cys 335 C7T TCT Leu Ser TCT OTA Ser Val OGGA CCT 'GJy Pro =I GAA Phe Giu 400 ATA CAA Ile Gin 415 AAC ATC Asa Ile OGA CAA GJ-y Gin TAC TTA Tyr Leu GAC ATC Asp Ile 480 ACT GCA CXT OAT Thr Ala His Asp 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 191 TTC GTT GTA CAA Phe Val Val Gin

TGT

Cys

GGT

Gly

CAG

Gin

OTT

Val 545

TGT

Cys

GAA

Glu

CCT

Pro

ATG

Met

GAA

Glu

ATT

Ile

TTT

Phe 530

AAT

Asfl

ATA

Ilie

CAG

Gin

AAC

Asn

GAT

Asp 610 GAT GTT Asp Vai 500 CTC ACT Leu Thr 515 TAC ATT Tyr Ile GAA AAT.

Glu Asn AAA CCT Lys Pro TTT GTG Phe Vai 580 AGT TTT Ser Phe 595 AAG GTC Lys Vai GGT GAA Gly Giu 485 AAC CAA Asn Gin TCA CGT Ser Arg AAG ATC Lys Ile GTT ACG Val Thr 550 GAT GGT Asp Gly 565 GCA CCT Ala Pro AAC TTA Asn Leu CAA ATT Gin Ile CAG TTT Gin Phe AAT OAA Asn Glu 520 ACT AAT Thr Asn 535 AAT TOC Asn Cys TCA OTT Ser -Vai TTA CTT Leu Leu ACT OTT Thr Vai 600 AGO A Arg 615 490 OTA OTT Vai Val 505 ACT GGT Thr Gly OGA ACA Oly Thr CCT TAT Pro Tyr TCT CCT Ser Pro 570 AAT OTT Asn Val 585 ACA OAT Thr Asp

TCT

Ser

TCT

Ser

CAT

His

OTT

Vai 555

ATA

Ile

ACT

Thr

GAG

Glu

GGT

Gly

CAG

Gin

CGT

Arg 540

AGT

Ser

GTA

Val

GAA

Glu

TAC

Tyr

GT

Oiy

CCT

Pro 525

TCT

Ser

TAT

Tyr

CCA

Pro

AAT

Asn

ATA

Ile 605

AAA

Lys 510 C'r' Leu

AGA

Arg

GGC

Gly

AAA

Lys

OTG

Vai 590

CAA

Gin 495 TTA OTA Leu Vai GAA AAC Olu Asn COT TCT Aig Ser AAG TTT Lys Phe 560 GAA CTT Glu Leu 575 CTC ATA Leu Ile ACG COT Thr Arg TAT GOC CCT AAC TAC TAT AAG OTT AAT CTA Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu 1488 1536 1584 1632 1680 1728 1776 1824 1846 INFORMATION FOR SEQ ID NO:17: Met S er Ala Val Cys Val Leu Ala Phe Val Th2 Ala~ SEQUENCE CHARACTERISTICS: LENGTH: 6i5 amino acids TYPE: amino acid TOPOLOGY: iinear ii) MOLECULE TYPE: protein xi) SEQUENCE DESCRIPTION: SEQ ID N Val Lys Ser Leu Phe Leu Val Thr I 10 Asn Leu Tyr Asp Asn Oiu Ser Phe V 25 Arg Pro Gly His Gly Trp His Leu H 40 Asn Val Ser Ser Glu Asn Asn Asn 55 Ala Gly Ala Ile Oly Tyr Ser Lys 70 Met Thr Ala Pro Leu Ser Oly Met S 90 0:17: ie Leu al: Tyr is Giy la Giy Lsn Phe 75 ~er Trp Phe Gly Thr Ser Ser Ala Tyr Ala Ala Ala Ala Leu Gin Tyr Pro Al a Ser Cys Ser Al a Ser Ser Ser 192 006.0: S Sr .0 *0*S .00.0.

0 5* Phe Cys T His Cys P Pro Ser C 130 Pro Gly 1 145 Lys Phe I Asn Gly 2 Ala Gly Arg Glu 210 Val Ile 225 Asn Thr I Ile Val Thr Leu Pro Asn 290 Ala Gin 305 Val Tyr Ser Phe Val Ser Phe Asn 370 Arg Ala 385 cys Gly Thr Ala hr ,he ly Iis rg ksp Jal L95 Jal Leu Gly Lys Thr 275 Thr Ser Arg Arg Leu 355 Gly Cys Leu Thr Ala 100 Lys Tyr Leu Ser Leu 180 His Lys Cys Asn Asp 260 Leu Gly Gly Glu Pro 340 Ile Lys LyE Let Glx 42( His Ser Ile Phe Leu 165 Val Phe Ala Asp Phe 245 Lys Thr Gly Tyr Ser 325 Glu Tyr Ala Gl i Val 401 Cys Asn P Gly Ser

J

Arg Ile I 135 Tyr Asn I 150 Gin Cys Phe Thr Lys Ser Leu Ala 215 Asp Thr 230 Ser Asp Phe Ile Asn Phe Val Asp 295 Tyr As 310 Asn Tyr Thr Leu Gly Pro Thr Cys 375 Val Tyr 390 Tyr Val he sn .20 Qa eu fal Ser Gly 200 ryr Pro Gly Val Thr 280 Sex Phe Met Asi Il 36( Cya Ar Th Thr S 105 Ser C Ala Dv Thr Asn Asn 185 Gly Phe Arg Phe Tyr 265 Phe Phe Asn :Tyr 1 Gly 345 B Gin 3 s Tyr g Gly r Lys er :ys ret 7al ksn L70 Tyr Pro Val Gly Tyr 250 Arg Ser Ile Phe Gly 330 Leu Gly Ala Gi.

Se 41(

'I

Pro Leu '1 Lys His 140 Ser Vai 155 His Thr Thr Glu Ile Thr Asn Gly 220 Leu Leu 235 Pro Phe Glu Ser Asn Glu *Leu Tyr 300 Ser Phe 315 Ser Tyr Trp Ser Gly Cys Tyr Ser 380 Leu Thr 395 c Asp Gly 3 hr ly rhr 3er ksp yr 205 Thr Ala Thr Ser Ser 285 Gin Leu His Asn Lys 365 Tyl Glr Se Gly I Ser I Lys J Val.

Val 190 Lys I Ala I Cys Asn Val 270 Gly.

Thr Ser Pro Ser 350 Gin Gly 1 His r Arg ~eu krg ryr ryr 175 lal 6ral His 3Gl rhr 255 Asn Ala Gin Ser Ala 335 Leu Ser Gly Phe Ile 415 Ile Thr Pro 160 Leu Ala Met Asp Tyr 240 Ser Thr Pro Thr Phe 320 Cys Ser Val Pro Glu 400 Gin 'yr Ile Val Phe Val Thr 110 0 Pro Pro Val Leu Thr Gin Asn Phe Tyr Asn Asn Ile Al C;430 Thr Leu Gly 435 Lys Cys Val Asp Tyr 440 Asn Val Tyr Gly Arg 445 Thr Gly Gin 193 Gly Phe 450 Ile Thr Asn Val Thr 455 Asp Leu Ala Thr Ser 460 Gly His Asn Tyr Leu Ala Giu Gly 465 Gly Leu Ala Ile Leu.

470 Asp Thr Ser 475 Ala Ile Asp Phe Cys Giv Val Val Gin Gly 485 Glu Asp Val Asn 500 Ile Leu Thr Ser Giu Gin Arg Tyr Gly Pro Asn 490 Gin Phe Val Vai 505 Asn Giu Thr Giy Tyr Tyr Lys Ser Giy Gly Vai Asn Leu 495 Lys Leu Val 510 Leu Glu Asn Arg Arg Ser Ser Gin 515 Gin Phe Tyr 520 Asn Pro 525 Ser Ile Lys Ile 530 Val Asn.

Thr 535 Asn Gly Thr His Arg 540 Ser woo.

0..

Giu Asn Val 545 Cys Thr 550 Gly Cys Pro Tyr Tyr Gly Lys Phe 560 Ile Lys Pro Asp 565 Ala Ser Val Ser Pro 570 Val Val Pro Lys Giu Gin Phe Pro Asn Ser 595 Met Asp Lys .610 Val1 580 Phe Pro Leu Leu Asn 585 Thr Thr Glu Asn" Vai 590 Gin Giu Leu 575 Leu Ile Thr Arg Asn Leu Thr Val 600 Asp Glu Tyr Ile 605 Val Gin Ile Arg 615 INFORMATION FOR SEQ.ID NO:1B: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2116 base pairs TYPE: nucleic acid STRANDEDNESS: doubie TOPOLOGY: iinear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iB: TATAATTATC TAGCAGACGC AGGTATGGCT ATTTTAGATA CATCTGGTTC TTTGTTGCAC AAGGTGAATA TGGCCTTACT-TATTATAAGG CTAACCCTTG AACCAGCAGT TTGTAGTTTC TGGTGGTAAA TTAGTAGGTA TTCTTACTTC ACTGGTTCTC AGCTTCTTGA GAACCAGTTT TACATTAAAA TCACTAATGG TCTAGACGTT CTATTACTGC AAATGTHACA AATYGCCCTT ATGTTAGCTA TGTCTAAAAC CTGATGGYTC AGYTTCTGYT ATAGCACCAC NNNNNNNNNN NNNNNNN NNN NiNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNN NNNNNNN N NNN1NNNN NNNNNNNNNN DJ'NNNNNN NNNNNNNNNN GTTTGTGGCA ATTCTCTGGA TTGTAGAAAG TTGYTTCAAC AATATGGGCC

CATAGACATC

CGAAGACGTC

ACGTAATGAG

AACACGTCGT

TGGCAAGTTT

NNNNNN

TGTTTGBGAC

120 180 240 300 360 420 480 540

B

B

B

AACATATTGT

TATTCTTCTA

GATTTYAATA

GAAGATCTTT

AAGTGCACTG

GGCTTGCTTG

TTAGTAGCTT

CAACTGCAGG

GAAAAAATTG

ACATCTCTAG

GAGACTATGG

TACCAGCAAC

TTGTCATCAC

CAGCGTGAGT

TCCTTTTGTG

GTGTTTATAC

TTTTGTAARG

TCTATACAAG

ATTACTGCAG

AAGACCGTCA

AAATGGTGGA

CCTATACTTG

GACTCTCTAA

TATGTGTGGT

TTTTTC.ATGA

AGCAAGTGTG

CAAWACAGAC

CTGTGGTAAA

CTAAACCATC

TTTCTCTTYT

TAT7TACAAG

CAGGACCTTT

YN14NNNNCCC

CTATGGCTTT

CTAGAATTAA

CTGCCTCCTT

CATTACAACA

CATCAC'rTAA

TTGACGCCAT

TTTCTGTTT

TAGCTACTCA

GTAATGGACG

ACTTTACTTA

CCGCTAATGC

TTAATGGTAG

GAGATATAGT

TTACYACATT

ATGATACTAA

ACATTGGTAG

TAGACCTTGA

TAGCCATAGC

CTGGTTGTTG

GTAAGAAATC

CYAAAA

TAGTGTTGGT

TGGCTTTAAT

GGTTGACACC

TGTTGAATCT

AGGCTTCCTT

TATTATAACA

TGGTGGGATT

TCACTTGGGT

TAATAAGGCC

AGTYCAMGAT

TAAAAATTTK

ACAAGCAAAT

AGCATCTGCT

GAAAATTAAT

ACACGTTCTA

TACTCCAGAG

TAGTCAGTAT

TCACTACATC

TACGCTTACT

HGTAGACAAT

GCATGAGCTA

TGAAATTGAT

AACACTATCA

TTTTGSCACT

TGGTTGTTGT

TTCTTATTAC

194

CAAA.AAGAAG

ACACCAGTTT

TCCAGTAGTA

GTTGGATTAC

AAGGACCTBG

GCAGAAATGC

ACTGCAGCTG

ATTACCCAGT

ATTGGCCATA

GTTGTTAATA

GGTGCTATTT

GCTCAAGTGG

AAGCAGGCGG

GAGTGTGTTA

ACTATACCGC

AGTTTTGKTA

GCAATAGTGC

ACTGCACGAG

TCTTGTCAAG

GATGATTTTG

CCAGACTTTG

CGTATTCAAG

ATACTCAAAA

ATTATCTTCA

TGTGGATGCT

ACGACTTTGG

ATATGGAACT

TTAGTAATCT

CTACTGGGCG

CAACAGATGA

CGTGTGCTCG

AAACCTTGTA

GTGCTATACC

CACTTTTGC.A

TGCAGGAAGG

AGCAGAGTGC

CTTCTGTGAT

ATCGTCTTAT

AGTATATTAG

AATCACAGTC

AAAATGCACC

ATGTTACTGC

CTGCTAATGG

ATATGTATAT

CAAATTATGT

ATTTTGATGA

ACGAATTCAA

GCGTTATACA

CTTATATTAA

TCCTAATATT

TTGGCATTAT

ATAATGATGT

TCtJAAATCTC

YAGCACTGGC

CTCTTTTATT

AGCTTATAAA

TGAATATAAT

TACTAGTTCT

TTTTGCCACA

GAA.AAATCAA

TTTTAGAAGT

TATTCTTACT

TCAAGATATC

AACTGGTAGA

AGTGTCACAA

TATTAGGTAC

TAATGGTATA

AATAGTGGGT

CAGAGGTATT

GCCAAGAGAT

AAGTGTAMMT

CGAATTGTCA

TTACACAGTA

GGGCCTTAAT

GTGGCCTTGG

AGGGTGGGTG

TCCTCTAATG

GGTAACTGAA

600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 *1800 1860 1920 1980 2040 2100 2116 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 705 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 195 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Tyr Ser Lys Gly Leu Ser Tyr Pro Xaa Xaa 145 Val Pro Giu Phe Ser 225 Giu Giu Leu Ile Met 305 Gin Ile Ala Lys s0 Leu Arg Gly Xaa Xaa 130 Xaa Cys Val Asp Asn 210 Leu Asp Al a Al a Thr 290 Al a Leu Asp Asn Leu Glu Arg Lys Xaa 115 Xaa Xaa Gly Xaa Met 195 Thr Leu Leu Tyr Cys 275 Al a Phe Gin Ile Pro Val Asn Ser Phe 100 Xaa Xaa Xaa Asn Asp 180 Giu Pro Val Leu Lys 260 Ala Giu Gly Ala Phe Cys Gly Gin Ile Cys Xaa Xaa Xaa Ser 165 Asn Leu Val Asp Phe 245 Lys Arg Met Gly Arg Val Giu Ile Phe 70 Thr Leu Xaa Xaa Xaa 150 Leu Ile Leu Phe Thr 230 Thr Cys Giu Gin Ile 310 Ile Ala Asp Leu 55 Tyr Ala Lys Xaa Xaa 135 Xaa Asp Leu Asn Ser 215 Ser Ser Thr Thr 295 Thr Asn Gin Val 40 Thr Ile Asn Pro Xaa 120 Xaa Xaa Cys Ser Leu 200 Asn Ser Val Ala Asn 280 Leu Ala His Gly 25 Asn Ser Lys Val Asp 105 Xaa Xaa Xaa Arg Val 185 Tyr Leu Ser Glu Giy 265 Gly Tyr Ala Leu Glu Gin Arg Ile Thr 90 Gly Xaa Xaa Xaa Lys 170 Val Ser Ser Thr Ser 250 Pro Leu Thr Gly Gly Tyr Gin Asn Thr 75 Asn Ser Xaa Xaa Xaa 155 Leu Asn Ser Thr Thr 235 Val Leu Leu Ser Ala 315 Ile Gly Phe Glu Asn Xaa Xaa Xaa Xaa 140 Xaa Xaa Ser Thr .Gly 220 Gly Gly Gly Xaa Ser 300 Ilie Thr Leu Val Thr Gly Pro Ser Xaa 125 Xaa Xaa Gin Val Lys 205 Asp Arg Leu Phe Xaa 285 Leu Pro Gin Thr Val Gly Thr Tyr Xaa 110 Xaa Xaa Xaa Gin Gly 190 Pro Phe Ser Pro Leu 270 Xaa Val Phe Ser Tyr Tyr Ser Gly Ser Gin Arg Arg Val Ile Ala Xaa Xaa Xaa Xaa Xaa Xaa 160 Tyr Gly Gin Lys Ser Giy Asn Ile Phe Ile 240 Thr Asp 255 Lys Asp Pro Ile Ala Ser Ala Thr 320 Leu Leu Asn Tyr Leu Ala Asp Ala Gly Met Ala Ilie Leu Asp Thr Ser Gly 10 325 330 335 Gin Lys Asn Gin Glu Lys Ilie Ala Ala 345 Ser Phe Asn Lys Ala Ile Gly 350 196 His Met Gin Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gin Gin Val 355 360 365 Xaa Asp Vai Val Asn Lys Gin Ser Ala Ile Leu Thr Giu Thr Met Ala 370 375 380 Ser Leu Asn Lys Asn Xaa Gly Ala Ile Ser Ser Vai Ile Gin Asp Ile 385 390 395 400 Tyr Gin Gin Leu Asp Ala Ile Gin Ala Asn Ala Gin Vai Asp Arg Leu 405 410 415 Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gin 420 425 430 Ala Glu Tyr Ile Arg Val Ser Gin Gin Arg Giu Leu Ala Thr Gin Lys 435 440 445 :le Asn Giu Cys Val Lys Ser Gin Ser Ile Arg Tyr Ser Phe Cys Gly 450' 455 460 Asn Gly Arg His Vai Leu Thr Ile Pro Gin Asn Ala Pro Asn Gly Ile 465 470 475 480 Val Phe Ile His Phe Thr Tyr Thr Pro Glu Ser Phe Xaa' Asn Val Thr 485 490 495 Ala Ile Val Gly Phe Cys Lys Ala Ala Asn Ala Ser Gin Tyr Ala Ile 500 505 510 Val Pro Ala Asn Gly Arg Gly Ile Ser Ile Gin Val Asn Giy Ser His 515 520 525 Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Asp Ile Thr Ala Gl~y 530 535 540 Asp Ile Val Thr Leu Thr Ser Cys Gin Ala Asn Tyr Val Seir Val Xaa 545 550 555 560 Lys Thr Val Ile Thr Thr Xaa Val Asp Asn Asp Asp Phe Asp Phe Asp .:565 570 .575 Asp Giu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Giu Leu Pro Asp 580 585 590 Phe Asp Giu Phe Asn Tyr Thr Vai Pro Ile Leu Asp Ile Gly Ser Giu 595 600 605 Ile Asp Arg Ile Gin Gly Val Ile Gin Gly Leu Asn Asp Ser Leu Ile 610 615 620 Asp Leu Giu Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp 625 630 635 640 Tyr Val Trp Leu Ala Ile Ala Phe Xaa Thr Ile Ile Phe Ile LeaIle 645 650 655 Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Giy 660 665 670 Cys P-he Giy Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser 675 680 685 Tyr Tyr Thr Thr Leu Asp Asn Asp Val Val Thr Giu Gin Xaa Arg Pro 690 695 700 197 Lys 705 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GAATTCGAGC TCGCCCGGGG ATCCTCTAGA GTCGAC 36 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 13..57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CACAGCTCAA CA ATG AAG TGG GCA ACG TGG ATC GAT CCC GTC GTT TTA 48 Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu 1 5 CAA CGT CGT 57 Gin Arg Arg INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gin Arg Arg 1 5 10 198 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ACTCGGGCAG CGTTGGGTCC TGGGACTCTA GAGGATCGAT CCCCTATGGC GATCATC 57 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: 0. LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: S GCGCCCACGT GGCCTGGTAC AATTCGAGCT CGCCCGGGGA TCCTCTAGAG TCGACTCTAG AGGATCGATC CTCTAGAGTC GGCGGGACGA GCCCGCGAT 99 INFORMATION FOR SEQ ID

C.

SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TCCACAGGAC CTGCAGCGAC CCGCTTAACA GCGTCAACAG CGTGCCGCAG ATCGGGG 57 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear 199 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GTTGATCCCG GGAGATGGGG GAGGCTAACT GAAAC INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 103 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GCTCATGGTG GCCCCCGGGC GGTTCAACGA GGGCCAGTAC CGGCGCCTGG TGTCCGTCGA CCTGCAGGTC GACTCTAGAG GATCCCCGGG CGAGCTCGAA TTC 103 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GAATTCGAGC TCGCCCGGGG ATCCTCTAGA GTCGACGTCT GGGGCGCGGG GGTGGTGCTC TTCGAG 66 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 200 (ix) FEATURE: NAME/KEY: CDS LOCATION: 16..66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CTCCACAGCT CAACA ATG AAG TGG GCA ACG TGG ATC GAT CCC GTC GTT TTA 51 Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu 1 5 CAA CGT CGT GAC TGG 66 Gin Arg Arg Asp Trp INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30: Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gin Arg Arg Asp 1 5 10 Trp INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 132 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) "(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GAC GAC TCC TGG AGC CCG TCA GTA TCG GCG GAA ATC CAG CTG AGC GCC 48 Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Ile Gin Leu Ser Ala 1 5 10 GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT CAA AAA GAT CTA GAA 93 Gly Arg Tyr His Tyr Gin Leu Val Trp Cys Gin Lys Asp Leu Glu 25 TAAGCTAGAG GATCGATCCC CTATGGCGAT CATCAGGGC 132 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 31 amino acids 201 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Ile Gin Leu Ser Ala 1 5 10 Gly Arg Tyr His Tyr Gin Leu Val Trp Cys Gin Lys Asp Leu Glu 25 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: AACGAGGGCC AGTACCGGCG CCTGGTGTCC GTCGACTCTA GAGGATCCCC GGGCGAGCTC GAATTC 66 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID N:34: CAGGTCGAAG CTTGGGCGCT GCCTATGTAG TGAAATCTAT ACTGGGATTT ATCATAACTA GTTTA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICA: NO 202 (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID AATAATCTAT CACTTTGTCA TGGAGATGCC CAAGCTTCGA CGACTCCCTT GGCCATGATG AATGG INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: TATACCAGCT ACGGCGCTAG CATTCATGGT ATCCCGTGAT TGCTCGATGC TTTCCTTCTG AATTC S INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: AAGCTTGGCC TCGTCGTTAA TTAACCCAAT TCGAGCTCGC CCAGCTTGGG CTGCAGGTCG GGAAC INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 203 TGTTTCAGTT AGCCTCCCCC ATCTCCCGAC TCTAGAGGAT CTCGACATAG CGAATACATT TATGG INFORMATION FOR SEQ ID NO:39: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 130 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: AACGTATATA TTTTTCAC!GA CGTAGACCAC TATTGCCATG GACTCTAGAG GATCGGGTAC CGAGCTCGAA TTGGGAAGCT TGTCGACTTA ATTAAGCGGC CGCGTTTAAA CGGCCCTCGA. 120 GGCCAAGCTT 130 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs CB) TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO SEQUENCE DESCRIPTION: SEQ ID GTCGACGTCT GGGGCGCGGG GGTGGTGCTC TTCG.AGACGC TGCCTACCCC AAGACGATCG INFORMATION FOR SEQ ID NO:41: Wi SEQUENCE CHARACTERISTICS: CA) LENGTH: 60 base pairs TYPE: nucleic acid CC) STRANDEDNESS: double CD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: AGCTCAACAA TGAAGTGGGC AACGTGGATC GATCCCGTCG TTTTACAACG TCGTGACTGG INFORMATION FOR SEQ ID NO:42: 204 SEQUENCE CHARACTERISTICS: LENGTH: 120 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GAGCCCGTCA GTATCGGCGG AAATCCAGCT GAGCGCCGGT CGCTACCATT ACCAGTTGGT GTTGGTCTGG TGTCAAAAAG ATCCGGACCG CGCCGTTAGC CAAGTTGCGT TAGAGAATGA 120 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: ACACAGTCAC ACTCATGGGG GCCGAAGGCA GAATTCGTAA TCATGGTCAT AGCTGTTTCC INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: AAACCTGTCG TGCCAGCGAG CTCGGGATCC TCTAGAGGAT CCCCGGGCCC CGCCCCCTGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 205 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TCGTCCACAC GGAGCGCGGC TGCCGACACG GATCCCGGTT GGCGCCCTCC AGGTGCAGGA INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: AACCCCCCCC CCCCCCCCCC CCCCCCCCTG CAGGCATCGT GGTGTCACGC TCGTCGTTTG INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid S(C) STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TCGGATCCTC TAGAGTCGAC INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 2681 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 146..481 (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (602..1402) 206

C

CC CCC C

C

(ix) FEATURE: NAME/KEY: CDS LOCATION: 1599. .2135 (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (2308. .2634) (xi) SEQUENCE DESCRIPTION: SEQ ID 140:48: TTTATCGGAC CTTGGGTATT CAGGGGAACC CATCTGGTTG AAA TTGATCCTGG TTACCCCGAC CCAAI4TTTTA AGCCGGCTGG CC CCGCTTAAAA CTAGCCCCAA TATTGATGTG CAGATATAACAC AGACATGCTA CGGCGGTCAT CTCCCGAAGA CATCACCGAT TCC TATGTTATCG CGCATTCGTC GTACCATGCG CACCGCAGGA AT AGATCCAATG AATCGTATGT CTAATTACAC TCCAGGCGAA TGT ATATATTGAC GAACATGCTA GAAGGTGTCC TGATCACATA TGT TACACTTATG CCGATGTATG TGCACGGGCG ATATTTCTAT TGT GTAAACTACC ACAGGCTGTC CGGAAATCTA AGTTAATGAA TAP, CATTGCTTAG AATTGGACTA CTTTTAATYC TCTTTAATGT TCG TTTAATAAAC TTCAGCCTCT TCGCTTATTG TAGAAATTGA GTA AGCCGTCTTC GGAGAGTGTA CTC.GCCACGG TGGTTGGAAC ATC.

AATTTAAGC-A CGTCAGGTCT GTCGAGGACA AGAAATGGTT AAC TTATAAACGT TAAGCATTGT AAGCCCCCCG GCCGTCCGCA GC CGTGGGCTCC GGGACTATCA. CGGATGTCCA ATTCGCACAT GC CTCTCATTTC GAGAAATCTT CGGGGATCCA TCAGCAATGC GGG GTTTCAAATG AAGGTGCTCC AACACGGTCT TCAAAGCAAC CGG ACTGCAACTC CCCGCTGCAA TGATTGGTTA TAAACAGTAA TCT TTCGCCCGAC AATCCACGGC GCCCCCAAAG TTAAAAACCA TCC CTCTGTTAAA. AGAATATTGA CTGGCATTTT CCCGTTGACC GCC CACGATGTTG CACGGACGAC TTTGCAGTCA CCAGCCTTCC TTT AAATGTTTAT CGTAGGACCC ATATCCGTAA TA6AGGATGGG TCT CGCCTCGGCG TGGTAGTTCT CGAGGATACA TCCAAAGAGG TTG TCTTGTTAAA TGGAAAGTGC ATTTGCTTGT TCTTACAATC GCC CGCCTCGTTC ACACTTAAAC CACAAATAGT CTACAGGCTA TAT TCACATATGA CTAATATTCG GGGGTGTTAG TCACGTGTAG CCC ATGTTGGACG CGTCCTTATT CGCGGTGTAC TTGATACTAT GGC CATCCTCGTC ATCGTTAACA TCTCTACGGG TTCAGAATGT TTG TGCCCATCGT TGCAA6ATTAC AAGTCCGATC GCCATGACCG CGA

CCC...

C

C

TCATCC

GGTCCCT

GNNANCC

CTAACAA

AAATATA

ATGACAG

AATTTGT

ATT CAT

A~TAGAT

TATTAAA

TTCAMAA

ACTATGT

TAGTGTT

ACAATTT.

TATAATT

CTGTAGT

CATACCA

GTCTTCT

ATGTGTA

AGATATC

CCACCCC

GGCAGCA

AGTATTC

CCGAGTC

GGGAGCC

ATTGTGT

AGCGAGC

GCATGTC

.TAAGCCT

GACCCTGCAC

AGATAACCCC

GATCAATGGA

TGTGCCTGAT

GCTATATGAT

GTA7ATTGCG ATA7CACATG

TTTTTGKTA

GGTTAATACT

TAAAAACATC

TCATGTTCAA

CTACACGTCA

TCAATTATTC

ACTAGTATGC

TTTCTrAGGGT

CCCGATTCCC

GCAAACACAG

GGAAGTATAT

TTTGCGTCTT

CAAAGTACAG

CCCACCAACA

ACCCCATAGG

TCTCTACACT

TCGTTCACAG

AGACTGAAAC

GCATATAACG

ATGGGATATT

GTCGATCCTT

GTACCATGTG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 207 GCATTAGGGT GACATCTCGA TCATACATTA TAAGACCAAC GTGCGAGTCT TCCA.AAGACC

TGCACGCCTT

TTGAGACCAT

GATCTCCATT

TAGAGGGGGT

CTGCATTTTC

GCCGGTGGAG

ATAAAATAAT

AAATATTCAT

CTGTGAAAAT

CGTAGTTATC

GCGGTTATGG

TTTTAATCGC

TATGACCATC

GCCCCTAAGA

CNCTCGAAAT

CTTCTTCGGA TTGTCAACGG

TGTGCGTTTA

TTCTCACGCC

ATGTTTCC.AC

TGCTGCCGAA

TATACGGGAA

TGACCAGTGA

TACTTCTCCA

TACTACAGGA

GCAGACGTGC

TTTATGCGCA

GATCGATGTT

ACATCTCTCT

AATTGCAAAC

TACAAACTCG

ATGAACAATA

ACTATCCTGG

TGCCACTGTG

CAAACTTCAT

ACTAAATGTT

ACAATTTGTT

ATCCCAGGTC

TATATTTTTA

AACGCTTCGC

CAGAATCCAT

TGGGCAACTG

GGYTCATACC

TAACATNATT

CGCAATAGAC

GTTCTTCAGA

AAGCGGCATG

ACGCTGTAGA

ATGATAAGTT

CGCTATGCAA

CATAGAGGTC

TAATGTTAGT

ATTCTTTAGC

AGATGCAGGA

ATTTGAGTTA

GCATGTCCTA

CGTTATTTCA

CCGCTTGGGN

GNCGGGTTCC

CCCCGTACAT

ATCTATGCCC

CCATGGAAAG

CGATAATTAT

TTCTCCAGAT

AGAGATGCGT

TTTGGGCTAT

TTATTCAATG

GAGATGATGT

GTAACAATGT

CCGAAGTGCC

ATTGAACCAT

GATCTAAAAA

TAAGATATCA

ATATACAATC

T

ATATCTGGCG

GAGGGCTGCA

ACCATGAATA

TGTTGGATAT

GTGTACACGC

ATGTTATTAA

CATTGGTTGC

TATGACATTG

GCATAGTAGG

CAACAGTGCT

CCGATTTTTC

ATT7ACCCTY

TGTAGATTCC

CCATCTTGTC

1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2681 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 111 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: Met Cys Arg Tyr Asn Thr Xaa Xaa Arg Ser Met Giu 1 5 10 Arg Ser Ser Pro Glu Asp Ile Thr Asp Ser Leu Thr 25 Met Leu Ser Arg Ile Arg Arg Thr Met Arg Thr Ala 40 Ser Tyr Met Ile Asp Pro Met Asn Arg Met Ser Asn 55 Giu Cys Met Thr Gly Ile Leu Arg Tyr Ile Asp Glu 70 75 Cys Pro Asp His Ile Cys Asn Leu Tyr Ile Thr Cys Asp Met Leu. Arg Met Cys Leu Ile Gly Asn Lys Tyr Tyr Thr Pro Gly His Ala Arg Arg.

Thr Leu Met Pro 90 208 Met Tyr Val His Gly Arg Tyr Phe Tyr Cys Asri Ser Phe Phe Xaa 100 105 110 INFORMATION FOR SEQ ID i)SEQUENCE CHARACTERISTICS: LENGTH: 266 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) AN~TI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID Met His Phe Pro Phe Asn Lys Lys Cys Arg Giu Asn Thr Gln Pro Leu

*S

S

S

9* 5

S

Trp Ala Leu Gln Ser Leu Asp Val His 145 Arg Glu Cys Ile Cys 225 Met Arg Val 50 His Gin Thr Arg Phe 130 Leu Arg Leu Cys Ile 210 Leu Tyr Pro 35 Gly Arg Tyr Leu Leu 115 Ala Lys Phe Asp Cys 195 Glu Asn Pro Ile Gly Al a Ser Gly 100 Leu Gly Arg Leu Ile 180 Gly Thr Leu Arg Leu Trp Val Phe 85 Ala Phe Met Glu Glu 165 Arg Arg Leu Thr Giu Ile Lys Leu 70 Asn Pro Ile Pro Ser 150 Met Asp Pro Val Cys 230 Leu Thr Gly 55 Trp Arg Trp Thr Val 135 Gly Arg Ser Gly Asn 215 Arg Pro Asp 40 Arg Ile Giu Ile Asn 120 Ala Leu Asp Pro Gly 200 His His Arg 25 Met Leu Ser Asp Val 105 His Leu Gin Pro Gly 185 Leu Phe Ser Arg Gly Val Gly Ala 90 Gly .Cys Lys Pro Arg 170 Al a Gin Leu Asp Gly Pro Thr Gly 75 Asn Arg Ser Thr Ala 155 Lys His Cys Ser Val 235 Al a Thr Ala Gin Thr Asn Gly Val 140 Leu Ile Gly Leu Ser 220 Pro Tyr Ile Lys Arg His Ile Giu 125 Leu Leu Ile Ile Thr 205 Thr Thr Gly Asn Ser Giu Gly Leu 110 Leu Glu Met Cys Leu 190 Phe Asp Thr Val Ile Ser Asn Trp Pro Gin.

His Asp Met 175 Val Ile Leu Val.

Ala Leu Val Ala Phe Giu Ser Leu Pro 160 Cys Asn Arg Thr Al a 240 Ser Thr Leu Ser Giu Asp Gly Phe Giu His Asp Xaa Glu Tyr 245 250 Ser Ile 255 209 Ser Thr Ile Ser Glu Glu Ala Glu Val Tyr 260 265 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 178 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Met Ala Ala Ser Met Gly Tyr Ser Ser Ser Ser Ser Leu Thr Ser Leu 1 5 10 9 9

S

9. Arg Asn Ile Ser 65 Glu Asn His Glu Cys 145 Lys Cys Val Tyr Arg Lys Ser Lys Ala Gly 130 Trp Giu Ser Gin Lys Vai Asp Met Ala Thr 115 Val Ile Met Asn Ser Thr Leu Pro Ala 100 Ile Cys Ser Arg Val Asp Ser His Ile Cys Leu Phe Ala Val 165 Trp Arg Arg Ala 70 Ser His Asp His Phe 150 Tyr His His Ser 55 Phe Gly Gly Ala Cys 135 Ser Thr Val Asp 40 Tyr Phe Val Lys Vai 120 His Ala Arg Vai Arg Ile Phe Glu Glu 105 Asp Cys Ala Arg A-sp Asp Ile Gly Thr 90 Gly Asp Asp Glu Trp 170 Pro Lys Arg Leu 75 Ilie Cys Asn Asp Gin Ser Leu Pro Pro Ser Val Arg Tyr Lys 140 Thr Ile Pro Val Thr Thr Arg Ser Thr 125 Phe Ser Arg Val Cys Glu Ser Met Phe Asn Pro Leu Thr 175 Ala Gly Ser Ser Asn Ser Ile Asp Cys 160 Lys INFORMATION FOR SEQ ID N0:52: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 210 (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: Met Gly Leu Tyr Met Glu Pro Xaa Asn Xaa Val Ser Leu Gin Phe Leu 1 5 10 Arg Gly Gly Ile Tyr Met Ile Ser Xaa Pro Lys Arg Gly Met Xaa Gin 25 Arg Asp Val Met Val Ile Xaa Gly Lys Phe Phe Arg Ser Glu Ile Thr 40 Gin Leu Pro Lys His Arg Ser Arg Leu Lys Glu Lys Ser Asp Gly Ser 55 Ile Arg Thr Cys Met Asp Ser Val Arg Ile Asn His Asn Arg Ser Thr 70 75 Val Gly His Phe Gly Asn Ser Asn Ala Lys Arg Cys Thr Ser Ala Ile S. 85 90 Thr Thr Pro Thr Met His Ile Val Thr Pro Ala Ser 100 105 S INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Oligonucleotide Primer (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CTCGCTCGCC CATGATCATT AAGCAAGAAT TCCGTCG 37 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Oligonucleotide Primer (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CTGGTTCGGC CCATGATCAG ATGACAAACC TGCAAGATC 39 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 211 LENGTH: 57 base pairs TYPnB nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTCGGCGTGG TAGTTCTCGA GGCCTTAATT AAGGCCCTCG AGGATACATC CAAAGAG 57 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 63 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear i (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGGCGTGGTA GTTCTCGAGG CCTTAAGCGG CCGCTTAAGG CCCTCGAGGA TACATCCAAA GAG 63 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CGCAGGATCC GGGGCGTCAG AGGCGGGCGA GGTG 34 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 212 (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: GAGCGGATCC TGCAGGAGGA GACACAGAGC TG 32 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: TGTAGAGATC TGGCTAAGTG CGCGTGTTGC CTG 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TGTACAGATC TCACCATGGC TGTGCCTGCA AGC 33 TGTACAGATC TCACCATGGC TGTGCCTGCA AGC 33

Claims (23)

1. A recombinant herpesvirus of turkeys-Marek's disease virus chimera comprising a herpesvirus of turkeys unique long viral genome region and a Marek's disease virus unique short viral genome region.

2. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 1, wherein a foreign DNA sequence is inserted within a non-essential region of the herpesvirus of turkeys-Marek's disease virus chimera viral genome, and is capable of being expressed in a host cell.

3. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 2, wherein the foreign DNA sequence is inserted within an EcoR1 #9 fragment of the unique long region of the herpesvirus of turkeys-Marek's disease virus chimera viral genome.

4. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 2, wherein the foreign DNA sequence encodes a polypeptide.

The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 3, wherein the foreign DNA sequence encodes a polypeptide.

6. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 4, wherein the foreign DNA sequence encodes a cytokine or cytokine receptor.

7. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 6, wherein the cytokine if a chicken mylomonocytic growth factor (cMGF) or chicken interferon (cIFN).

8. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 4 or wherein the polypeptide is E. Coli beta-galactosidase. P:\OPER\MRO\2254914.CLM 21/1100 -214-

9. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 2 or 3, wherein the foreign DNA sequence encodes an antigenic polypeptide.

The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 9, wherein the antigenic peptide is from Marek's disease virus, Newcastle disease virus, Infectious laryngotracheitis virus, Infectious bronchitis virus or Infectious bursal disease virus.

11. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim wherein the antigenic peptide is Marek's disease virus glycoprotein A, Marek's disease virus glycoprotein B or Marek's disease virus glycoprotein D.

12. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim wherein the antigenic peptide is Newcastle disease virus fusion protein or Newcastle disease virus hemagglutinin-neuraminidase.

13. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim o wherein the antigenic peptide is infectious laryngotracheitis virus glycoprotein B, infectious laryngotracheitis virus glycoprotein I or infectious laryngotracheitis virus glycoprotein D.

14. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim wherein the antigenic peptide is infectious bronchitis virus spike protein or infectious bronchitis virus matrix protein.

The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim wherein the antigenic peptide is infectious bursal disease virus VP2, infectious bursal disease virus VP3 or infectious bursal disease virus VP4.

16. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 2, wherein the foreign DNA sequence is under control of an endogenous upstream herpesvirus promoter. P:\OPER\MRO\2254914.CLM -21/1/00 -215-

17. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 2, wherein the foreign DNA sequence is under control of a heterologous upstream promoter.

18. The recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 17, wherein the promoter is a HCMV immediate early promoter.

19. A vaccine comprising an effective immunizing amount of the recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 2 and a suitable carrier.

20. A multivalent vaccine comprising an effective immunizing amount of the recombinant herpesvirus of turkeys-Marek's disease virus chimera of claim 2 and a suitable carrier.

21. The recombinant herpesvirus of turkeys-Marek's disease virus chimera according to any one of claims 1 to 18 substantially as hereinbefore described with reference to the figures and/or examples.

22. The vaccine of claim 19 or the multivalent vaccine of claim 20 substantially as o hereinbefore described with reference to the figures and/or examples.

23. A composition as described herein. DATED this TWENTY-FIRST day of JANUARY, 2000 Syntro Corporation by DAVIES COLLISON CAVE Patent Attorneys for the applicant(s)