CA2626498C - Recombinant chimeric virus - Google Patents
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- CA2626498C CA2626498C CA2626498A CA2626498A CA2626498C CA 2626498 C CA2626498 C CA 2626498C CA 2626498 A CA2626498 A CA 2626498A CA 2626498 A CA2626498 A CA 2626498A CA 2626498 C CA2626498 C CA 2626498C
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Abstract
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 an 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. 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. Lastly, this invention provides homology vectors for producing a recombinant herpesvirus of turkeys, host cells, and vaccines and methods for immunization.
Description
Wo 96109291 PCr1US95/1ew RECOMBINANT CHIMERIC VIRUS
10 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.
BA XGROVND OF TBE INVKIITION
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:
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, e.g., which promoter sequence and termination sequence to use.
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 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;
10 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.
BA XGROVND OF TBE INVKIITION
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:
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, e.g., which promoter sequence and termination sequence to use.
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 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 applicable 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 host-specific 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 combination 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 5 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
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., Proc. Natl. Acad. Sci. USA, 1984); and the insertion of bovine growth hormone into herpes saimiri virus with an SV40 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 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 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 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.
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 applicable 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 host-specific 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 combination 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 5 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
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., Proc. Natl. Acad. Sci. USA, 1984); and the insertion of bovine growth hormone into herpes saimiri virus with an SV40 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 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 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 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.
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 passage and/or tedious regimes of administration have 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.
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 (S. Joshi, et al., J. of Virology, 1991), ribozymes (M. Wachsman, et al., J. of General Virology, 1989), suppressor tRNAs (R.A. Bhat, et al., Nucleic Acids Research, 1989), interferon-inducing double stranded RNA and numerous examples of protein therapeutics, from hormones, e.g., insulin, to lymphokines, e.g., interferons and interleukins, to naturals opiates. The discovery of these therapeutic agents and the elucidation of their structure and function does not make obvious the ability to use them in a viral vector delivery system.
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 EcoRl #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 IA-1C: Details of HVT Construction and Map 5 Data.
Figure 1A shows BamHI restriction fragment map of the HVT genome. Fragments are numbered in order of decreasing size; letters refer to small 10 fragments whose comparative size has not been determined.
Figure 1B shows BamHI #16 fragment of the HVT
genome showing location of (3-galactosidase gene insertion in S-HVT-001.
Figure 1C shows BamHI #19 fragment of the HVT
genome showing location of (3-galactosidase gene insertion.
Legend: B = BamHI ; X = XhoI ; H = Hindi I I ; P =
PstI; S = Sa1I; 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).
Figures 3A-3B: Details of S-SVT-003 Construction.
Figure 3A shows restriction map of HVT DNA in the region of the BamHI #16 fragment. This fragment is contained within large HindliI fragment. Figure 3A\also shows the XhoI site which was first changed to an EcoRI (R) 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).
.10 Figure 3B show the S-HVT-003 genome, including the location of the two inserted foreign genes, (3-gal and IBDV.
In Figure 3 : H = Hindlll; B = BamHI; X = XhoI;
R = EcoRI; Xb = XbaI; Hp = HpaI; S = Smal; UL =
unique long region; US = unique short region; IR
= internal repeat region; TR = terminal repeat region.
Figure 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.
FicTure 5:
Western blot indicating the differential expression of the 42kD (VP2) antigen in cellular lysates of S-HVT-003 infected cells (42kD present) ands 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.
Figures 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 (X) where applicants have made their insertion. Before the insertion, the XhoI was first changed to EcoRI (R) site by use of a "linker" and standard cloning procedures.
Figure 6B provides details of the construction of the P-gal gene and MDV gA gene inserted into the BamHI #16 fragment for use in homologous recombination. Beta-gal was under the control of the PRV gX gene promoter (gX) , 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, R-gal and MDV gA.
In Figure 6, H = Hindlll; B = BamHI; X = XhoI; R
= EcoRI; Xb = XbaI; UL = unique long region; US =
unique short region; IR = internal repeat region;
TR = terminal repeat region.
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 passage and/or tedious regimes of administration have 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.
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 (S. Joshi, et al., J. of Virology, 1991), ribozymes (M. Wachsman, et al., J. of General Virology, 1989), suppressor tRNAs (R.A. Bhat, et al., Nucleic Acids Research, 1989), interferon-inducing double stranded RNA and numerous examples of protein therapeutics, from hormones, e.g., insulin, to lymphokines, e.g., interferons and interleukins, to naturals opiates. The discovery of these therapeutic agents and the elucidation of their structure and function does not make obvious the ability to use them in a viral vector delivery system.
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 EcoRl #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 IA-1C: Details of HVT Construction and Map 5 Data.
Figure 1A shows BamHI restriction fragment map of the HVT genome. Fragments are numbered in order of decreasing size; letters refer to small 10 fragments whose comparative size has not been determined.
Figure 1B shows BamHI #16 fragment of the HVT
genome showing location of (3-galactosidase gene insertion in S-HVT-001.
Figure 1C shows BamHI #19 fragment of the HVT
genome showing location of (3-galactosidase gene insertion.
Legend: B = BamHI ; X = XhoI ; H = Hindi I I ; P =
PstI; S = Sa1I; 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).
Figures 3A-3B: Details of S-SVT-003 Construction.
Figure 3A shows restriction map of HVT DNA in the region of the BamHI #16 fragment. This fragment is contained within large HindliI fragment. Figure 3A\also shows the XhoI site which was first changed to an EcoRI (R) 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).
.10 Figure 3B show the S-HVT-003 genome, including the location of the two inserted foreign genes, (3-gal and IBDV.
In Figure 3 : H = Hindlll; B = BamHI; X = XhoI;
R = EcoRI; Xb = XbaI; Hp = HpaI; S = Smal; UL =
unique long region; US = unique short region; IR
= internal repeat region; TR = terminal repeat region.
Figure 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.
FicTure 5:
Western blot indicating the differential expression of the 42kD (VP2) antigen in cellular lysates of S-HVT-003 infected cells (42kD present) ands 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.
Figures 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 (X) where applicants have made their insertion. Before the insertion, the XhoI was first changed to EcoRI (R) site by use of a "linker" and standard cloning procedures.
Figure 6B provides details of the construction of the P-gal gene and MDV gA gene inserted into the BamHI #16 fragment for use in homologous recombination. Beta-gal was under the control of the PRV gX gene promoter (gX) , 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, R-gal and MDV gA.
In Figure 6, H = Hindlll; 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 (1acZ) 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, 30, 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 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 Z
gene (lacZ), Escherichia coli (E.Coli), polyadenylation signal (pA), and glycoprotein X
(gpX).
Ficrure 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
#6, P2 - BamHI #2, P3 - BamHI #13, and P4 - 4.0 kb BgIII to Stul sub-fragment of HVT genomic XbaI
fragment #5 (8.0 kb).
Detailed description of the P-galactosidase (1acZ) 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, 30, 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 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 Z
gene (lacZ), Escherichia coli (E.Coli), polyadenylation signal (pA), and glycoprotein X
(gpX).
Ficrure 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
#6, P2 - BamHI #2, P3 - BamHI #13, and P4 - 4.0 kb BgIII to Stul 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 junction A (SEQ ID NO: 34), junction B (SEQ ID NO:
35), 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, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction.
Figures 11A-11B:
Detailed description of the Hindlll 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 5 construction.
Figures 12A-12C:
Detailed description of the Sa1I fragment insert in Homology Vector 255-18.B16. Figure 12A shows 10 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: 40), junction B (SEQ ID NO: 41), junction C (SEQ ID NO:
42), junction D (SEQ ID NO: 43), junction E (SEQ
ID NO: 44), junction F(SEQ ID NO: 45), junction G
(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
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 #10 fragment of the HVT genome was converted into a Pacl 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 Pacl site to generate Plasmid 654-45.1 (SEQ ID NO. 55) and Figure 13B shows the XhoI site converted into a NotI site to generate Plamid 686-63.A1 (SEQ ID NO.
56).
Figure 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 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 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;
Nucl. 1599 to 2135; 179 amino acids) show significant sequence identity to the herpes 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 in the protein databases. Searches were performed on NCBI databases using Blast software.
Figure 15:
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 Pacl site in Plasmid 654-45.1 or a unique NotI site in Plasmid 686-63.A1.
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 junction A (SEQ ID NO: 34), junction B (SEQ ID NO:
35), 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, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction.
Figures 11A-11B:
Detailed description of the Hindlll 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 5 construction.
Figures 12A-12C:
Detailed description of the Sa1I fragment insert in Homology Vector 255-18.B16. Figure 12A shows 10 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: 40), junction B (SEQ ID NO: 41), junction C (SEQ ID NO:
42), junction D (SEQ ID NO: 43), junction E (SEQ
ID NO: 44), junction F(SEQ ID NO: 45), junction G
(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
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 #10 fragment of the HVT genome was converted into a Pacl 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 Pacl site to generate Plasmid 654-45.1 (SEQ ID NO. 55) and Figure 13B shows the XhoI site converted into a NotI site to generate Plamid 686-63.A1 (SEQ ID NO.
56).
Figure 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 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 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;
Nucl. 1599 to 2135; 179 amino acids) show significant sequence identity to the herpes 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 in the protein databases. Searches were performed on NCBI databases using Blast software.
Figure 15:
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 Pacl site in Plasmid 654-45.1 or a unique NotI site in Plasmid 686-63.A1.
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 not necessary for the viral infection or replication.
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 UL43 protein and a preferred insertion site within that open reading frame in a Xhol restriction endonuclease site.
Yet another insertion site is the HVT US2 gene, with a preferred insertion site within it being a Stul endonuclease site.
This invention provides a recombinant herpesvirus of turkeys comprising a herpesvirus of turkeys viral genome which contains a foreign DNA sequence inserted within the EcoRl #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 the herpesvirus of turkeys.
In one embodiment, the foreign DNA sequence is inserted within an Open Reading Frame A (ORFA) of the EcoRi #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 EcoRJ #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 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 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. 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), 5 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 EcoRl #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 20 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 Stul 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.
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 not necessary for the viral infection or replication.
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 UL43 protein and a preferred insertion site within that open reading frame in a Xhol restriction endonuclease site.
Yet another insertion site is the HVT US2 gene, with a preferred insertion site within it being a Stul endonuclease site.
This invention provides a recombinant herpesvirus of turkeys comprising a herpesvirus of turkeys viral genome which contains a foreign DNA sequence inserted within the EcoRl #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 the herpesvirus of turkeys.
In one embodiment, the foreign DNA sequence is inserted within an Open Reading Frame A (ORFA) of the EcoRi #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 EcoRJ #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 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 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. 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), 5 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 EcoRl #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 20 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 Stul 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 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.
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 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 such case, it is preferred that the antigenic polypeptide is Newcastle disease virus fusion (F) protein or Newcastle disease virus hemagglutinin-neuraminidase (HN), or a recombinant protein comprising E. coli B-galactosidase fused to Newcastle disease virus hemagglutinin-neuraminidase (HN). 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 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 virus fusion protein or Newcastle disease virus hemagglutinin-neuraminidase are inserted into a XhoI
site in EcoRl #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.
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 (F) protein.
Preferably, this recombinant herpesvirus of turkeys is designated S-HVT-048.
The invention further provides recombinant herpesvirus WO 96/05291 PCf/US95/10245 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 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 (F) protein and Newcastle disease virus hemagglutinin-neuraminidase (HN). Preferably, this recombinant herpesvirus of turkeys is designated S-HVT-050.
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 5 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
sequence encoding an antigenic polypeptide from 20 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 25 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 recombinant HVT contains foreign DNA sequences encoding MDV gB, MDV gA, MDV gD, ILTV gIand ILTV gD.
Preferably, this recombinant HVT is designated S-HVT-139 or S-HVT-140.
The invention further provides recombinant.herpesvirus 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 encoding 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 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.
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 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.
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 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 such case, it is preferred that the antigenic polypeptide is Newcastle disease virus fusion (F) protein or Newcastle disease virus hemagglutinin-neuraminidase (HN), or a recombinant protein comprising E. coli B-galactosidase fused to Newcastle disease virus hemagglutinin-neuraminidase (HN). 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 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 virus fusion protein or Newcastle disease virus hemagglutinin-neuraminidase are inserted into a XhoI
site in EcoRl #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.
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 (F) protein.
Preferably, this recombinant herpesvirus of turkeys is designated S-HVT-048.
The invention further provides recombinant herpesvirus WO 96/05291 PCf/US95/10245 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 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 (F) protein and Newcastle disease virus hemagglutinin-neuraminidase (HN). Preferably, this recombinant herpesvirus of turkeys is designated S-HVT-050.
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 5 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
sequence encoding an antigenic polypeptide from 20 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 25 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 recombinant HVT contains foreign DNA sequences encoding MDV gB, MDV gA, MDV gD, ILTV gIand ILTV gD.
Preferably, this recombinant HVT is designated S-HVT-139 or S-HVT-140.
The invention further provides recombinant.herpesvirus 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 encoding 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 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 EcoRl #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.
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 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 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
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.
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 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 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 .10 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, 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.
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 (F) 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 5 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 10 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 20 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 beta-galactosidase. In another embodiment the foreign DNA
sequence encodes a cytokine. In another embodiment the cytokine is chicken mylomonocytic growth factor (cMGF) or 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:
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 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 EcoRl #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.Al.
In one embodiment the polypeptide is antigenic in the animal into which the recombinant herpesvirus of.
turkeys is introducdd. 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 hemagglutinin-neuraminidase, infectious laryngotracheitis virus glycoprotein gB, infectious laryngotracheitis virus glycoprotein gD, infectious bronchitis virus spike protein, or infectious bronchitis virus matrix protein.
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 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 G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV
N),.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, .10 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
sequences present within the BamHI #16 fragment of the herpesvirus of turkeys genome. Preferably, the double-stranded 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 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 EcoR] #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. soli 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 effective immunizing amount of the recombinant 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.
This invention provides a vaccine useful for immunizing a bird against infectious laryngotracheitis 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 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 5 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.
10 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.
This invention provides a multivalent vaccine useful for immunizing a bird against Marek' s disease virus and infectious bursal disease virus which comprises an 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 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 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 105 to 107 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 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 5 minutes in a clinical centrifuge. Infected cells were resuspended in complete medium containing 20%
fetal bovine serum, 10% DMSO and stored frozen at -70 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 C in a humidified incubator with 5%
CO2 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 10 minutes at 5 C in a GS-3 rotor (Sorvall Instruments).
The resultant pellet was resuspended in cold PBS (20 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
5 Tris pH 7.5, 1 mM EDTA, and 1.5 mM MgCl2). NP40 (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 10 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 1%, respectively. One 15 hundred Al of proteinase-K (10 mg/ml; Boehringer Mannheim) was added per 4 ml of sample, mixed, and incubated at 45 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 5.2), and the nucleic acid precipitated at -70 C
for 30 minutes after the addition of 2.5 volumes of 25 cold absolute ethanol. DNA in the sample was pelleted by spinning for 20 minutes to 8000 rpm in an HB-4 rotor at 5 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), 30 and resuspended in 50 l/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 g/roller bottle of infected cells. All viral DNA was stored at approximately 10 C.
POLYMERASE.FILL-IN REACTION. DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KC1, 5 mM
WO 96/05291 PCT/US95n0245 MgC121 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 3SS-dATP (NEN). Reactions using both 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 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 * Trade-mark WO 96/05291 PC r/US95/10245 variation.
SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis et al.
(1982). DNA was blotted to nitrocellulose filters (S&S
BA85) in 20X SSC (1X ssc = 0.15M NaCl, 0.015M sodium citrate, pH 7.0), and prehybridized in hybridization solution consisting of 30% formamide, 1X Denhardt's solution (0.02% polyvinylpyrrolidone (PVP), 0.02%
bovine serum albumin (BSA), 0.02% Ficoll), 6X SSC, 50 mM NaH2PO41 pH 6.8, 200 g/ml salmon sperm DNA for 4-24 hours at 55 C. Labeled probe DNA was added that had been labeled by nick translation using a kit from Bethesda Research Laboratories (BRL) and one 31p_ labeled nucleotide. The probe DNA was separated from the unincorporated nucleotides by NACS column (BR7,) or on a Sephadex*G50 column (Pharmacia). After overnight hybridization at 55 C, the filter was washed once with 2X SSC at room temperature followed by two washes with O.1X SSC, 0.1% sodium dodecyl sulfate (SDS) for 30 minutes at 55 C. The filter was dried and autoradiographed.
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 5-10 plaque forming units per cell. When cytopathic effect was evident, but before total destruction, the * Trade-mark WO 96105291 P(T/US95110245 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 mis of CsCl solution (5.7 M CsCl, 25 mM sodium citrate pH 7.0) in Beckman SW41 centrifuge tube. The samples were centrifuged for 18 hrs at 20 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 Al glass distilled water, and 2.6 mis of guanidine-solution (7.5 M guanidine-HCL, 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 4 C at 10000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, recentrifuged at 13000 rpm, and the 25 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 C.
mRNA containing polyadenylate tails (poly-A) was * Trade-mark selected using oligo-dT cellulose (Pharmacia #27 5543-0). 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 LiCl, 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, 1mM EDTA pH 8.0, 0.1% sodium dodecyl sulfate) . This mRNA was reapplied to an oligo-dT 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 C for 18 hrs.
The RNA was resuspended in 50 Al distilled water.
Ten g poly-A RNA was denatured in 20 mM methyl mercury hydroxide for 6 min at 22 C. f3-mercaptoethanol was added to 75 mM and the sample was incubated for 5 min at 22 C. The reaction mixture for first strand cDNA
synthesis in 0.25 ml contained 1 g oligo-dT primer (P-L Bio-chemicals) or 1 g synthetic primer, 28 units placental ribonuclease inhibitor (Bethesda Research Labs #5518SA), 100 mM Tris pH 8.3, 140 mM KC1, 10mM
MgC121 0. 8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia) , 100 microcuries 32p-labeled dCTP (New England Nuclear #NEG-013H), and 180 units AMV reverse transcriptase (Molecular Genetics Resources #MG 101). The reaction was incubated at 42 C for 90 min, and then was terminated with 20mM EDTA pH 8Ø The sample was extracted with an equal volume of phenol /chloroform (1:1) and precipitated with 2 M ammonium acetate and 2 volumes of cold ethanol -20 C for 3 hrs. After precipitation and centrifugation, 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
NaCl). The leading edge of the eluted DNA fractions was pooled, and DNA was concentrated by lyophilization until the volume was about 100 l, then the DNA was precipitated with ammonium acetate plus ethanol as above.
The entire first strand sample was used for second 5 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.
.10 After second strand synthesis, the cDNA was phenol /chloroform extracted and precipitated. The DNA
was resuspended in 10 Al distilled water, treated with 1 g RNase A for 10 min at 22 C, and electrophoresed through a 1% agarose gel (Sigma Type II agarose) in 40 15 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 20 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
CaCl2, 80 moles dCTP, and 25 units terminal deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 Al. After 30 min at 37 C, the reaction was terminated with 10mM EDTA, and the sample 30 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 35 Research Labs #5355 SA/SB) in 200 Al of 0.01 M Tris pH
7.5, 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65 C for 2 min and then 57 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 l aliquots of cells. Transformed cells were plated on L-broth agar plates plus 10 g/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 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 (IX Fl0/199, 5% fetal calf serum, 2% glutamine, 1% non-essential amino acids, and 2% penicillin/streptomycin) containing 4 g/ml polybrene (stock 4 mg/ml in 1X HBSS). For cotransfections into CEF cells, 5 Ag of intact HVT DNA, and suspended in 1 ml of CEF media containing 30 g/ml polybrene (stock 4 mg/ml in 1X HBSS). The DNA-polybrene 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 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 1X HBSS for 4 * Trade-mark 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 C after the addition of 5 mis of CEF
growth media. The next day, the media was changed to remove any last traces of DMS.O 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 .10 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 -70 C.
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 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.,(19-88) 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.
Published restriction maps for the enzymes BamHI, HindIII, and XhoI permitted the use of subcloned fragments 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 the plates were frozen at -20 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 C. Clones which hybridized with the non-radioactive 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, * Trade-mark 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 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 pWE15 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 Stul site within the HVT US2 gene was established as a useful site for foreign DNA insertion utilizing the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUSES (see Example 6). The HVT US2 gene is located within the BamHI #1 fragment which contains five Stul sites. To facilitate the use of this site for insertion of foreign DNA by the Stul site within the US2 gene was converted to a unique Hindlll site. This was accomplished by partially digesting the BamHI #1 subclone with Stul, and then inserting a 10 kb fragment conferring kanomycin resistance (Neon) into the site using Hindlll linkers. The kanomycin resistance gene allowed positive selection of recombinant clones. The Neo` fragment was removed by digestion with Hindlll 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 10 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 Lipof ectin* (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% non-essential amino acids and 2% penicillin/Streptomysin (MEM+). Controls consisted of MEM+ with no DNA, with several ug of HVT DNA, or with 4 out of 5 of the 20 subclones. Separately, 30 Al 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 C in Flo/M199 (1:1) media containing 1% non-essential amino acids, 2%
penicillin/streptomycin, and 5% fetal calf serum (CEF+). Cells were transfected at a confluence of 90 -30 95%. For transfection, wells were aspirated and rinsed 3 times with MEM+, and then incubated 4 hours at 39 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 35 were then examined daily for the appearance of plaques.
Lipofectin with control HVT DNA resulted in the * Trade-mark 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.
BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. When the foreign gene encoded the enzyme Q-galactosidase, the plaques that contained the gene were visualized more easily. The chemical Bluogal7" (Bethesda Research Labs) was incorporated at the level of 200-300 g/ml into the agarose overlay during the plaque assay, and the plaques that expressed active /3-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 they replaced the /3-galactosidase gene; in this instance non-blue plaques were picked for purification of the recombinant virus.
SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT BVT
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 C. Once plaques have developed, the agarose overlay is removed from the dish, the monolayer rinsed lx with PBS, fixed with 100% methanol for 10 minutes at room temperature and the cells air dried.
WO %/05291 PCTIUS95110245 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 MgC12), and then incubated 10 minutes to overnight at room temperature with freshly prepared substrate solution (0.3 mg/ml Nitro Blue tetrazolium +
0.15 mg/ml 5-Bromo-4-Chloro-3-Indolyi 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 HVT STOCKS. When no suitable immunological reagent exists to detect the presence of 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 -.50-100 plaques/10 cm.dish, overlaid with nutrient agarose media and incubated for 4-5 days at 39 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 NaCl and 0.5M
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 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 g/ml) for one hour at 65 C. Radio-labeled probe DNA (alpha 32P-dCTP) is then added and the membranes incubated at 65 C
overnight (-12 hours). After hybridization the NC
membranes are washed two times (30 minutes each) with 2X SSC at 65 C, followed by two additional washes at 65 C with 0.5X SSC. The NC membranes are then dried and exposed to X-ray film (Kodak X-OMAT;kAR) at -70 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 beta-galactosidase (IacZ) gene was inserted into the HVT
EcoRI # 7 fragment at the unique Stul site. The marker 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 SaII 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 PvuI1 restriction fragment of plasmid pJF751 (Ferrari et al., 1985).
Fragment 3 is an approximately 754 base pair Ndel to * Trade-mark Sall 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 5 g/ml Concanavalin A and incubated at 39"for 48 hours.
Total RNA was isolated from the cells using guanidine isothionate lysis reagents and protocols from the Promega RNA isolation kit (Promega Corporation, Madison WI). 4 g of total RNA was used in each 1st strand reaction containing the appropriate antisense primers and ANN 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 * Trade-mark 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 10 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
20 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 25 such that the UL43 ORF is in the opposite transcriptional orientation to the pSP64 0-lacatamase gene.
HOMOLOGY VECTOR 172-63.1. The plasmid 172-63.1 was 30 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
35 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 Hindlll 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 inserted as a Sall 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 Sall 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 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 AvaII 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 Sinai to BamHI restriction sub-fragment of the HSV-1 BamHI restriction fragment N. Fragment 6 is an approximately 1812 base pair BamHI to PstI 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 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 constructed for the purpose of generating recombinant HVT. It contains an approximately 38,850 base pair region of genomic HVT DNA (see Figure 8). 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 8). 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.
SUBGENOMIC 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 8). 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 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 Pi and P2 (described in Figure 8). A bacterial strain containing this cosmid has been deposited 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. 75430.
SUBGENOMIC 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 8). 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 accordingq 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 8). A bacterial strain containing this cosmid has been deposited on March 3, 1993 .10 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. 75427.
HOMOLOGY VECTOR 435-47.1. The plasmid 435-47.1 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 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 Hindlll linker (CAAGCTTG) was then inserted into thetiunique Stul site of the EcoRI #7 fragment.
SUBGENOMIC CLONE 437-26.24. Plasmid 437-26.24 was constructed for the purpose of generating recombinant 5 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 10 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 20 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.
25 SUBGENOMIC 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 30 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, 35 1989), by joining two' restriction fragments from the following sources. The first fragment is an approximately 2970 base pair HindIII to BamHI
restriction fragment of pSP64 (Promega). The second fragment is the approximately 15,300 base pair BamHI to Stul 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 Hindlll 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 Hindlll site.
The MDV genes were inserted at the blunt ended Hindlll site as a blunt ended PstI to EcoRI fragment (see Figures 10A and 10B). The Hindlll and EcoRI sites were blunted by the Klenow fill in reaction. The PstI site was blunted by the T4 DNA polymerase reaction. Note 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 Sall 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 Hindlil 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 Bc1I 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 Safi restriction sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi et al., 1984). Note that the fragments are oriented such that Bc1I and NdeI sites are contiguous.
HOMOLOGY VECTOR 528-11.43. The plasmid 528-11.43 was constructed for the purpose of inserting the infectious laryngotracheitis (ILT) virus gB gene (A.M. Grifin, 1991) into HVT. The gB gene was inserted as an EcoRI
fragment into the homology vector 435-47.1 at the unique Hindlll site. The gB gene was inserted at the blunt ended HindIII site as a blunt ended EcoRI
fragment. The Hindill 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 Hindlil restriction enzyme site into which foreign DNA may be inserted.
When a plasmid containing a foreign DNA insert at the = CA 02626498 2008-04-10 Hindlll 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 fragment of pSP64 (Promega). The second fragment is an approximately 1368 base pair PvuI to Sall restriction fragment of pSP65 (Promega). The third fragment is the approximately 3400 base pair XhoI to XhoI fragment of 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 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 (Bi strain) . The last fragment is an approximately 784 base pair SmaI to Smal 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 Hindlll 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 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 is an approximately 1811 base pair AvaII to NaeI restriction fragment of the full length NDV HN
cDNA clone (B1 strain). The third fragment is an approximately 754 base pair NdeI to Sall 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 (B1 strain). 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).
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 Hindill site located between the MDV gA
and gB genes (see Junction B, Figure 10A). The HN gene 5 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 Sall to BamHI restriction sub-fragment of the PRV BamHI
15 fragment #10 (Lomniczi, et al., 1984) The second fragment is an approximately 1811 base pair AvaII to NaeI restriction fragment of the full length NDV HN
cDNA clone (B1 strain). The last fragment is an approximately 754 base pair NdeI to Sall restriction 20 sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984).
SUBGENOMIC CLONE 550-60.6. Plasmid 550-60.6 was constructed for the purpose of generating recombinant 25 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 30 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 35 approximately 4176 base pair EcoRV to BamHt 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 Hindlll and then resected with the Exolil 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 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, 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 Hindlll fragment into the homology vector 456-17.22 at the Hindlil 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 Hindlll 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 Hindlll 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 the synthetic DNA sequences indicated in Figures 11A
and 11B. Fragment 1 is an approximately 784 base pair SmaI to Sinai 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 Sall to NcoI sub-fragment of the MDV BglII 4.2 KB
genomic restriction fragment (Ross, et al., 1991).
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 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 Safi restriction sub-fragment of the PRV
BamHI restriction fragment #7 (Lomniczi, et al., 1984).
The fourth fragment is an approximately 1191 base pair Pstl to AvaII restriction sub-fragment of the HCMV
genomic XbaI E fragment (D.R. 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).
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 AvaII restriction sub-fragment of the HCMV genomic XbaI 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 AvaII and BclI sites are contiguous. The third fragment is an approximately 784 base pair Sinai 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 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.
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 1acZ 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 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 Bc1I restriction sub-fragment of the ILT KpnI genomic restriction fragment #8 (10.6 KB). 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). Note that the second and third fragments are oriented such that BclI
and NdeI 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
5 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 pWE15 (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 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 constructed for the purpose of generating recombinant HVT. It was isolated as an Ascl 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 .10 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 Xhol site (Nucleotide #1339-1344;=SEQ ID NO. 48) has been converted to a unique Pacl site using synthetic DNA
linkers. The Pacl site was used in insertion and expression of foreign genes in HVT. (See Figure 13A)..
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 686-63.A1. Plasmid 686-63.Al 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 14VT DNa. This region includes BamHI fragments 10 and 21, and approximately 1100 base pairs of fragment 6 and approxin~ately 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.
SUBGENOMIC 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 approximately 1100 base pair BamHI to AscI subfragment and an approximately 13,000 base pair AscI to NotI
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 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 #1. 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 Pacl site of plasmid 654-45.1. The IBDV VP2 gene uses the IBRV VP8 promoter and ILTV US3 polyadenylation signal. The cosmid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989). The first fragment is a 208 base pair Hindlil 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 primer used for reverse transcription and PCR was 5'-CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3' (SEQ ID NO.
53). The sense primer used for PCR was 5'-CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3' (SEQ ID NO.
54). The DNA fragment generated by PCR was cloned into the PCR-DirectT" vector (Clontech Laboratories, Inc., Pali Alto, CA). The IBDV VP2 fragment was subcloned next tot he VP8 promoter using BclI sites generated by the PCR primers. The DNA sequence at this junction adds amino acids methionine, aspartate and glutamine 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 Pacl 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 Hindlll 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 Pacl 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 Sail 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 NaeI restriction fragment of the full length NDV HN
cDNA clone (B1 strain). The third fragment is an approximately 754 base pair NdeI to SalI restriction subfragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984). The fourth fragment is an approximately, 1191 base pair PstI to Avail restriction subfragment 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 (Bi strain; SEQ ID NO: 12). The sixth fragment is an approximately 784 base pair SmaI to Sinai 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 721-38.1) contains the MDV gA, gD and gB genes inserted into a StuI site in the HVT US2 gene converted to a WO 96/05291 PC.T/US95110245 unique Hindlll 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 5 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 10 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 Stul 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 10 and 11) . The fourth fragment is an approximately 14,500 base pair Hindill 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 25 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 Stul site in the HVT US2 gene converted to a unique Hindill site within the BamHI #1 fragment of the 30 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 35 restriction digest with BamHI of S-HVT-106 and isolation of an approximately 46,300 base pari 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 6100 base pair BamHI fragment from cosmid vector pSY1626 derived from pHC79 (Bethesda Research Labs, Inc.) and pWE15 (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 Stul 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 (Lomniczi et al., 1984), the HCMV immediate early promoter (D.R. 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 11B), the approximately 450 base pair ILTV US3 polyadenylation site, and the MDV gB gene (Figures 10A
and 10B). The fourth fragment is an approximately 14,500 base pair Stul 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 HVT. Plasmid 729-37.1 contains the ILTV gD and gB genes inserted into the NotI site of plasmid 686-63.Al. 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 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 .10 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 contians the entire Smal B fragment and two Smal 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 base 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 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 SmaI 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.A8 contains the chicken myelomonocytic growth factor (cGMF) gene inserted into the Pacl site of plasmid 654-45.1. The cMGF gene uses the HCN V 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 Pacl site of HVT subgenomic clone 654-45.1. The first fragment is an approximately 1191 base pair PstI to Avail restriction subfragment of the HCMV genomic XbaI E fragment (D.R. 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 5'-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 784 base pair Smal to Smal 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.Al contains the chicken interferon gene inserted into the Pacl 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 Pacl 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: 60).
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 162 amino acids of the mature protein encoding chicken interferon. The third fragment is an approximately 784 base pair Smal to Smal restriction subfragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).
5 S-HVT-001 is a herpesvirus of turkeys (HVT) that contains the E. coli /3-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 10 a starting point to engineer the insertion of foreign genes into HVT. The BamHI restriction map of HVT is shown in Figure 1A. From this data, several different regions of HVT DNA into which insertions of foreign genes could be made were targeted. The foreign gene 15 chosen for insertion was the E. coli (3-galactosidase (lacZ) gene , which was used in PRV. The promoter was the PRV gpX promoter. The ZacZ gene was inserted into the unique long region of HVT, specifically into the XhoI 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 BluogalTM
(Bethesda Research Labs). Similarly, the lacZ gene has been inserted into. the Sail 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).
S-HVT-003 is a herpesvirus of turkeys (HVT) that contains the E. coli (3-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 3') (SEQ ID
NO: 2), two of which are antigens to provide protection against IBDV infections of chickens. Expression of the genes for both 0-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 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
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 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 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 V153. 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, 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 #10 fragment as an approximately 800 base pair Sail 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 BalI fragment followed by an approximately 40 base pair Ava I to Sma I fragment. The third segment of DNA (Segment 3, .10 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 Sinai 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 BamHI, #10 fragment as an approximately 330 base pair NaeI to AluI fragment. Additionally, segment four contains approximately 36 base pairs of HSV TK 5'untranslated leader sequence as a PstI to Bg1II fragment in which the PstI site has been joined to the AluI 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 5, 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 Bg1II
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 3') is under the control of the gpX promoter (segment 4), 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 Smal fragment (obtained from Bernard Roizman, Univ. of Chicago). The HpaI site of segment five was fused to the Smal 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 (5' to 3') the PRV promoter, the gpX TATA box, the gpX cap site, the first seven amino acids of gpX, the E. coli Q-galactosidase (1acZ) 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, 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 was engineered such that it was flanked by two EcoRI
restriction endonuclease sites. As a result, an approximately 9100 base pair fragment containing both 1acZ 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/1acZ
cassette. The following procedures were used to construct S-HVT-003 by homologous recombination. The IBDV/1acZ 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 1acZ (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 7). 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 5 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 10 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 1acZ gene and. the IBDV gene incorporated into its genome.
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.
25 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-30 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 35 dominant region of the IBDV 40 kd (VP2) capsid protein.
The filters were washed and treated with [1211] 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 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 immunologically indistinguishable from the authentic viral VP2 protein. In addition, the VP2 protein produced from S-HVT-003 comigrates with the 40 kd species 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.
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 received 0.5 ml of an inactivated IBDV vaccine inoculated 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 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.
DAY
Vaccine Group Bird No. 28 31 35 38 42 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 IBDV' 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 IBDV 262 <2 <2 <2 <2 1:4 1:4 263 <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
chicks were vaccinated with S-HVT-003 (20 with 0.2m1 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 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/1acZ cassette into the XhoI site of BamHI #16 does not result in detectable attenuation of virus.
Subsequent experiments examining the recovered virus .10 by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS
procedure confirmed the in vivo stability of S-HVT-003, by demonstrating /3-galactosidase expression in 100% of the viruses.
Harvest Date Day 4 Day 7 Sample Pi P2 Pi P2 T 2 2+ 2+
T 3 2+ 2+
T 4 + 4+
T 5 3+ 3+
15 T 6 2+ contaminated T 7 + 5+
T 8 + 5+
T 8 + 5+
T 9 + 5+
20 T10 + 5+
N = control, T = vaccinated CPE ranged from negative (-) to 5+
25 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 30 five control and fourteen vaccinated chicks were challenged with virulent IBDV by bi-lateral eyedrop (103=8EID50) . All birds were sacrificed 6-days post challenge and bursa to body weight ratios were calculated. A summary of the results is shown in 35 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 40 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 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.
ELISA VN
Sample Grout) HVT IBDV IBDV
C-0 (n=3) 0 0 <100 C-4 (n=2) 0 0 nd T-4 (n=5) 0 0 nd C-7 (n=2) 0 0 <100 T-7 (n=5) 0 0 <100 C-14 (n=5) 0 0 nd T-14 (n=14) 0 0 <100 C-21 (n=5) 0 0 nd T-21 (n=14) 1 0 <100 C-27 (n=5) 0 0 nd CC-27 (n=5) 0 5 nd 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.
Sample Group Body wt. Bursa wt. BBR
Control (n=5) 258.8 1.5088 0.0058 Challenge 209 0.6502 0.0031 Control (n=5) Challenge 215.5 0.5944 0.0027 Treated (n=10) 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 0.2m1 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 and incubated. Tests were developed with Tris-malcate buffer (TMB) + H202 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.
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 (9-galactosidase and IBDV antigens) that are recognized in the host by an immune response directed to these proteins.
WO %/05291 PCT/US95/10245 TABLE 5ti Serology: Herpes/IBDV ELISA titer Bleed Date 5 Bird# 11/3 11/10 11/14 11/24 12/1 12/8 12/15 12/22 Vaccinated and Challenged Control Challenged only Maximum titer level is 9 Example 3 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 /3-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 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 MDV gA gene. S-HVT-004 is a recombinant virus that contains both the /3-galactosidase gene and the MDV gA
gene incorporated into the genome.
Figure 6C shows the structure of S-HVT-004.
Example 4 NEWCASTLE DISEASE VIRUS
Newcastle disease virus (NDV) is closely related to PI-3 in overall structure. Hemagglutinin (HN) and fusion (F) 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.
INFECTIOUS BRONCHITIS VIRUS
Infectious bronchitis virus (IBV) is a virus of chickens closely related in overall structure to TGE.
Major neutralizing antigen of TGE was engineered for expression in PRV. 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 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.
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, 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 EcoRI-SaII 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-SaII 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 Stul site within HVT US2, previously utilized for the construction of S-HVT-012. The site was initially altered by insertion of a unique Hindlll 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
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 Stul site in HVT
US2 gene.
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-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 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 clinical signs of Marek's disease, then necropsied and observed for Marek's lesions. This study demonstrated the ability of HVT-045 and HVT-047 to provide 100% protection against challenge (Table 1). The commercial vaccine gave 96% protection, and 79% of the non-vaccinated chicks developed Marek's disease.
PROTECT SUSCEPTIBLE CHICKS AGAINST VIRULENT MAREK'S
DISEASE VIRUS
Marek's Protection Vaccine Group MD-5 Challenge RB1B Challenge S-HVT-046 20/20 Not Tested S-HVT-047 Not Tested 24/24 HVTa Not Tested 24/25 Controls 2/20 5/24 a Commercial Example 6 S-HVT-012 is a recombinant herpesvirus of turkeys that contains the E. coli (3-galactosidase (1acZ) gene inserted into the short unique region. The 1acZ 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 Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2382.
For insertion into the genome of HVT, the f3-galactosidase gene was introduced into the unique Stul site of the. cloned EcoRI fragment #7 of HVT, i.e., the fragment containing the Stul 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 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 IacZ
gene. S-HVT-012 is a recombinant virus that contains the 1acZ gene incorporated into the genome at the Stul site within the US2 gene of HVT.
S-HVT-012 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.
Examine 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, #7, and #9 (see figure i)) 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 Sall in EcoRI fragment #6 and StuI in EcoRI fragment V. A
0-galactosidase (1acZ) 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 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 1acZ 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 1000, clearly defining an appropriate site for the insertion of foreign DNA. The three homology vectors used to define these sites are described below.
Example 7A
Homology 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 also determined (see SEQ ID NO: 3). 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 (gC). The XhoI site interrupts an ORF
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: 5). 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.
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 Hindlll restriction site into which foreign DNA may be cloned. The Hindlil restriction site in plasmid results from the insertion of a Hindlll linker into the naturally occurring Stul site of EcoRI fragment V.
Hindlll 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 Stul indicated that this fragment contains open reading frames coding for US10, US2, and US3. The Stul site interrupts US2 at approximately amino acid 124, suggesting that the US2 gene is non-essential for HVT replication.
Example 7C
Homolocry 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 XhoI
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).
Example 8 S-HVT-014 is a recombinant herpesvirus of turkeys that contains the E. coli 3-galactosidase (lacZ) gene inserted into the long unique region. The IacZ 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 /3-galactosidase gene was introduced into the unique XhoI
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 XhoI site within the BarHI #10 fragment used for construction recombinant herpesvirues of turkeys described in Examples 16 through 19. Flanking regions of EcoRI fragment #9 were used for" homologous recombination. HVT DNA and plasmid DNA 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. S-HVT-014 is a recombinant virus that contains the IacZ 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.
Example 9 S-HVT-005 is a recombinant herpesvirus of turkeys that contains the E. coli f3-galactosidase (lacZ) gene inserted into the long unique region. The 1acZ 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 a galactosidase gene was introduced into an approximately 1300 base pair deletion of the XhoI #9 fragment-of HVT.
The deletion which lies between the unique M1uI and EcoRV sites removes the complete coding region of the HVT gA gene (see SEQ ID NO: 3). Flanking regions of XhoI fragment #9 were used for homologous recombination. HVT DNA and plasmid DNA 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, the DNA was analyzed for the presence of the 1acZ
gene. S-HVT-005 is a recombinant virus that contains the 1acZ 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.
Example 10 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 5), S-HVT-046 (Example 10A), S-HVT-047 (Example 10B), S-HVT-062 (Example 10C).
Example 10A S-HVT-046 S-HVT-046 is a recombinant herpesvirus of turkeys that 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 MDV antigens are more likely to elicit the proper antigenic response than the HVT equivalent antigen.
S-HVT-046 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 HindIll, 437-26.26 with BamHI and Hindlll, and 456-17.22 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.
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 with NotI, 437-26.24 with BamHI and HindIll, 437-26.26 with BamHI and Hindlll, 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.(gD) 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
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 HindlIl, 556-60.6 with BamHI and Hindlll, 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 pathogen free (SPF) chicks were vaccinated with either S-HVT-045, S-HVT-046, or S-HVT-047. Five days post-vaccination, vaccinated chicks, and non-vaccinated, control chicks were challenged with MDV. Following a 6-week 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 non-vaccinated 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-i 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 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.
VIRULENT MAREK'S VIRUS CHALLENGE
Study Vaccine Group Dosea Protectionb 1 S-HVT-045 2.2 X 103 24/24 (1000) 1 S-HVT-046 2.2 X 103 20/20 (1000) 1 S-HVT-047 2.2 X 103 24/24 (1000) 1 Controls 7/44 (16%) 1 HVT/SB-1 24/25 (96%) 2 S-HVT-062 7.5 X 102 32/32 (100%) 2 S-HVT-062 1.5 X 10' 22/22 (100%) 2 Controls 0/20 (0%) 2 HVT` 7.5 X 102 17/21 (81%) 2 HVT/SB-1 7.5 X 102 21/22 (95%) a PFU/0.2 ml.
No. protected/Total; Challenge 5 days post-vaccination.
` Commercial vaccine.
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 u4 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 255-18.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 1001i of the plaques were blue.
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 with NotI, 437-26.24 with BamHI and HindIll, 437-26.26 with BamHI and Hindlll, 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 under the control of the PRV gX 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-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 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 Hindlil, and 549-62.10 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.
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
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 Notl, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindlIl, 437-26.26 with BamHI and Hindlll, 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.
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
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 HindIll, 437-26.26 with BamHI and Hindlll, 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 USDA-licensed, conventional vaccine comprised of NDV
131/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-050) gave complete protection against a challenge that caused Newcastle disease in 100% of non-vaccinated control chicks. Recombinant virus S-HVT-049 gave 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-i 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 the ability of S-HVT-050 to provide protection greater than the commercial Marek's disease vaccines.
AGAINST VIRULENT NEWCASTLE AND MAREK'S DISEASE VIRUS
CHALLENGE
Protection (%) Vaccine Study Group Dosea NDVb MDVc 1 S-HVT-048 4.0 X 104 19/19 (100) 1 S-HVT-049 3.0 X 104 4/20 (20) 1 S-HVT-050 1.5 X 104 20/20 (100) 1 Controls 0/20 (0) 1 NDV B1/Bld 18/18 (100) 2 S-HVT-050 7.5 X 102 13/14 (93) 2 S-HVT-050 1.5 X 103 16/17 (94) 2 Controls 5/23 (22) 2 HVTd 20/26 (77) 2 HVT/SB_ld 10/12 (83) a PFU/0.2 ml.
b No. protected/Total; Challenge 3 weeks post-vaccination.
c No. protected/Total; Challenge 5 days post-vaccination.
d Commercial vaccine.
Example 12 Bivalent Vaccines Against Infectious Laryncgotracheitis 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 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 with NotI, 437-26.24 with BamHI and HindIll, 437-26.26 with BamHI and Hindill, 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.
= CA 02626498 2008-04-10 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 HindIll, 437-26.26 with BamHI and Hindlil, 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
genes are inserted in the unique short region in the same transcriptional orientation as the US2 gene. An E. coli 1acZ 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 plasmid 634-29.16 were co-transfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS
procedure into primary chick embryo fibroblast (CEF) 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 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.
VIRULENT' INFECTIOUS LARYNGOTRACHEITIS VIRUS CHALLENGE
Vaccine Group Dosea Protectionb S-HVT-051 28/30 (93%) 2.1 X 103 S-HVT-052 1.7 X 103 29/29 (100%) S-HVT-051 + 2.1 X 103 24/24 (100%) S-HVT-052 1.7 X 103 Controls 2/30 (7%) ILT 29/30 (97%) a PFU/0.2 ml.
b No.protected/Total; Challenge 2-3 weeks post-vaccination.
Commercial vaccine.
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.
S-HVT-096 is a recombinant herpesvirus of turkeys that contains the IBDV VP2 gene, under the control of the 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 HindlIl, 556-60.6 with BamHI, and 602-57.F1 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.
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.
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 HindlIl, 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.
Example 15 Vaccines utilizing HVT to express antigens from various pathogens.
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, Pasteurella spp, Haemophilus spp, Chlamydia spp, Mycoplasma spp, Campylobacter spp, Bordetella spp, Poultry nematodes, cestodes, trematodes, Poultry 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 Stul site converted into a Hindlil site in the HVT US2 gene. The 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-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 Pacl 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.
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 Pacl site in the EcoRl #9 (BamHI #10) fragment of the HVT genome. 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 13A and 15; SEQ ID NO: 48, 50). S-HVT-139 further contains the MDV gA, gD, and gB genes are inserted into the unique Stul site converted into a HindIll 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.SG6 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 S-HVT-140 is a recombinant herpesvirus of turkeys that ti contains the ILT virus gD and gI genes inserted into a unique XhoI site converted to a Pacl 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 Stul site converted into a Hindlll site in the HVT US2 gene. The ILTV gD and gI genes are expressed as overlapping transcripts from their own respective endogenous ILTV
promoters, and the MDV genes are also expressed from 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 PRV gX promoter. S-HVT-140 is useful as a vaccine in poultry against Infectious Laryngotracheitis, Marek's 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.
Example 17A HVT-126 S-HVT-126 is a recombinant herpesvirus of turkeys that contains the IBDV VP2 gene inserted into an XhoI site converted to a Pacl site in the EcoRl #9 (BamHI #10) 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
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
site converted to a Pacl site in the EcoRl #9 (BamHI
#10) 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) 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.
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 Pacl site in the EcoRl #9 (BamHI
#10) fragment of the HVT genome (Figures 13 A 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). 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 Hindlll 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 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 NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 722-60.E2 uncut.
= CA 02626498 2008-04-10 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 Pacl site in the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figures 13A and 15). S-HVT-128 further contains the MDV gA, gD, and gB
genes inserted into a unique Stul site converted into a Hindlll 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 with NotI, 1.72-07.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 the unique short region (see HVT-062) and the PRV gX
promoter-lacZ gene inserted into an XhoI 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 Pacl site in the EcoRl #9 (BamHI
#10) fragment within the unique long region of HVT.
(Figure 14; SEQ ID NOs: 48 and 50) The NDV HN gene is 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 BamHT.
Example 19 S-HVT-145 HVT/MDV recombinant virus vaccine S-HVT-145 is a recombinant virus vaccine containing MDV
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
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 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 Pacl site or NotI site in the EcoRl #9 (BamHI #10) fragment within the uniuqe long region of HVT/MDV recombinant virus (Figures 13 and 15).
A cosmid was constructed containing the entir MDV
unique short region. MDV genomic DNa contains several Smal sites in the uniuqe long and internal and terminal repeats of the virus, but no Sinai 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 and cloned into a blunt ended site of the cosmid vector pWE15. 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
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 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.
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 unique long/internal repeat junction and the unique long/terminal repeat junction (55, 56, and 57).
SB-1 strain is an MDV serotype 1 with attenuated pathogenicity. Vaccination with a combination of HVT
and SB-i 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 non-virulent 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-i 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.
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-i genomic DNA. To determine the appropriate sub-infectious dose, transfection of the SB-i 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-i which.
recombine withthe HVT unique short subgenomic clones.
Therefore, a virus resulting from recombination between overlapping homologous regions of the SB-1 and HVT
subgenomic fragments is highly favored. Alternatively, SB-i 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 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, Newcastle's disease virus and infectious bursal disease virus.
Example 20 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 (58), and chicken interferon (cIFN) is homologous to mammalian type 1 interferon (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 15.
Example 20A S-HVT-144 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 Pacl site in the EcoRl #9 fragment within the unique long region of HVT. The cMGF gene is in the opposite 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 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 A recombinant herpesvirus of turkeys contains the chicken interferon (cIFN) gene inserted into an XhoI
site converted to a Pacl 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 with NotI, 761-07.A1 with Asc I, 415-09.BA1 with BamHI.
Recombinant HVT expressing avian cytokines is combined with HVT expressing genes for avian disease antigens to enhance immune response. Additional cytokines that are 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 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 il, 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 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 Pacl 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 Stul site converted into a Hindlll 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.
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 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 Pacl site in the EcoRl #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 Stul site converted into a Hindlll 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 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, 672-07.C40 with NotI, 672-01.A40 with NotI, 761-07.A1 with Asc I, 722-60.E2 uncut.
Example 20E Recombinant HVT expressing Marek's disease virus genes and chicken 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 Pacl 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 Stul site converted into a Hindlll 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 an enhanced immune response in poultry against Marek's N
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.
Example 20F Recombinant HVT expressing Marek's disease virus genes, Newcastle disease virus genes and chicken myelomonocytic growth factor gene.
A recombinant herpesvirus of turkeys contains the chicken myelomonocytic growth factor (cGMF) gene inserted into an XhoI site converted to a Pacl site in the EcoRl #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 Stul site converted into a Hindlll 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
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 limited to humans, equine, bovine, porcine, canine and feline.
Recombinant HVT is useful as a vaccine against equine diseases when foreign antigens from diseases or disease 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 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
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, 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 growth 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 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 i3-galactosidase. cIFN added to the cell culture media reduced both the expression of 9-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, 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 evasion mechanism involves poxvirus soluble receptors for interleukins, interferon, or tumor necrosis factors which inactive the cytokines and allow viral replication (60). 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 (61). in a further embodiment, an interferon-negative CEF cell line is constructed by 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 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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11. Kawai and Nishizawa Mol. and Cell Bio. 4, 1172-1174, 1984.
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13. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1982.
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Laboratory Manual Second Edition, Cold Spring Harbor Press, 1989.
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82, 1359-1363, 1985.
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26. M. Mackett et al., Proc. Natl. Acad. Sci. USA 79, 7415-7419, 1982.
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Sci. USA 79, 4927-4931, 1982.
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81, 193-197, 1984.
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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 .10 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, 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.
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 (F) 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 5 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 10 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 20 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 beta-galactosidase. In another embodiment the foreign DNA
sequence encodes a cytokine. In another embodiment the cytokine is chicken mylomonocytic growth factor (cMGF) or 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:
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 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 EcoRl #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.Al.
In one embodiment the polypeptide is antigenic in the animal into which the recombinant herpesvirus of.
turkeys is introducdd. 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 hemagglutinin-neuraminidase, infectious laryngotracheitis virus glycoprotein gB, infectious laryngotracheitis virus glycoprotein gD, infectious bronchitis virus spike protein, or infectious bronchitis virus matrix protein.
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 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 G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV
N),.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, .10 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
sequences present within the BamHI #16 fragment of the herpesvirus of turkeys genome. Preferably, the double-stranded 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 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 EcoR] #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. soli 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 effective immunizing amount of the recombinant 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.
This invention provides a vaccine useful for immunizing a bird against infectious laryngotracheitis 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 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 5 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.
10 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.
This invention provides a multivalent vaccine useful for immunizing a bird against Marek' s disease virus and infectious bursal disease virus which comprises an 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 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 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 105 to 107 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 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 5 minutes in a clinical centrifuge. Infected cells were resuspended in complete medium containing 20%
fetal bovine serum, 10% DMSO and stored frozen at -70 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 C in a humidified incubator with 5%
CO2 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 10 minutes at 5 C in a GS-3 rotor (Sorvall Instruments).
The resultant pellet was resuspended in cold PBS (20 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
5 Tris pH 7.5, 1 mM EDTA, and 1.5 mM MgCl2). NP40 (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 10 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 1%, respectively. One 15 hundred Al of proteinase-K (10 mg/ml; Boehringer Mannheim) was added per 4 ml of sample, mixed, and incubated at 45 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 5.2), and the nucleic acid precipitated at -70 C
for 30 minutes after the addition of 2.5 volumes of 25 cold absolute ethanol. DNA in the sample was pelleted by spinning for 20 minutes to 8000 rpm in an HB-4 rotor at 5 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), 30 and resuspended in 50 l/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 g/roller bottle of infected cells. All viral DNA was stored at approximately 10 C.
POLYMERASE.FILL-IN REACTION. DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KC1, 5 mM
WO 96/05291 PCT/US95n0245 MgC121 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 3SS-dATP (NEN). Reactions using both 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 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 * Trade-mark WO 96/05291 PC r/US95/10245 variation.
SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis et al.
(1982). DNA was blotted to nitrocellulose filters (S&S
BA85) in 20X SSC (1X ssc = 0.15M NaCl, 0.015M sodium citrate, pH 7.0), and prehybridized in hybridization solution consisting of 30% formamide, 1X Denhardt's solution (0.02% polyvinylpyrrolidone (PVP), 0.02%
bovine serum albumin (BSA), 0.02% Ficoll), 6X SSC, 50 mM NaH2PO41 pH 6.8, 200 g/ml salmon sperm DNA for 4-24 hours at 55 C. Labeled probe DNA was added that had been labeled by nick translation using a kit from Bethesda Research Laboratories (BRL) and one 31p_ labeled nucleotide. The probe DNA was separated from the unincorporated nucleotides by NACS column (BR7,) or on a Sephadex*G50 column (Pharmacia). After overnight hybridization at 55 C, the filter was washed once with 2X SSC at room temperature followed by two washes with O.1X SSC, 0.1% sodium dodecyl sulfate (SDS) for 30 minutes at 55 C. The filter was dried and autoradiographed.
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 5-10 plaque forming units per cell. When cytopathic effect was evident, but before total destruction, the * Trade-mark WO 96105291 P(T/US95110245 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 mis of CsCl solution (5.7 M CsCl, 25 mM sodium citrate pH 7.0) in Beckman SW41 centrifuge tube. The samples were centrifuged for 18 hrs at 20 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 Al glass distilled water, and 2.6 mis of guanidine-solution (7.5 M guanidine-HCL, 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 4 C at 10000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, recentrifuged at 13000 rpm, and the 25 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 C.
mRNA containing polyadenylate tails (poly-A) was * Trade-mark selected using oligo-dT cellulose (Pharmacia #27 5543-0). 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 LiCl, 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, 1mM EDTA pH 8.0, 0.1% sodium dodecyl sulfate) . This mRNA was reapplied to an oligo-dT 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 C for 18 hrs.
The RNA was resuspended in 50 Al distilled water.
Ten g poly-A RNA was denatured in 20 mM methyl mercury hydroxide for 6 min at 22 C. f3-mercaptoethanol was added to 75 mM and the sample was incubated for 5 min at 22 C. The reaction mixture for first strand cDNA
synthesis in 0.25 ml contained 1 g oligo-dT primer (P-L Bio-chemicals) or 1 g synthetic primer, 28 units placental ribonuclease inhibitor (Bethesda Research Labs #5518SA), 100 mM Tris pH 8.3, 140 mM KC1, 10mM
MgC121 0. 8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia) , 100 microcuries 32p-labeled dCTP (New England Nuclear #NEG-013H), and 180 units AMV reverse transcriptase (Molecular Genetics Resources #MG 101). The reaction was incubated at 42 C for 90 min, and then was terminated with 20mM EDTA pH 8Ø The sample was extracted with an equal volume of phenol /chloroform (1:1) and precipitated with 2 M ammonium acetate and 2 volumes of cold ethanol -20 C for 3 hrs. After precipitation and centrifugation, 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
NaCl). The leading edge of the eluted DNA fractions was pooled, and DNA was concentrated by lyophilization until the volume was about 100 l, then the DNA was precipitated with ammonium acetate plus ethanol as above.
The entire first strand sample was used for second 5 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.
.10 After second strand synthesis, the cDNA was phenol /chloroform extracted and precipitated. The DNA
was resuspended in 10 Al distilled water, treated with 1 g RNase A for 10 min at 22 C, and electrophoresed through a 1% agarose gel (Sigma Type II agarose) in 40 15 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 20 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
CaCl2, 80 moles dCTP, and 25 units terminal deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 Al. After 30 min at 37 C, the reaction was terminated with 10mM EDTA, and the sample 30 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 35 Research Labs #5355 SA/SB) in 200 Al of 0.01 M Tris pH
7.5, 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65 C for 2 min and then 57 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 l aliquots of cells. Transformed cells were plated on L-broth agar plates plus 10 g/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 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 (IX Fl0/199, 5% fetal calf serum, 2% glutamine, 1% non-essential amino acids, and 2% penicillin/streptomycin) containing 4 g/ml polybrene (stock 4 mg/ml in 1X HBSS). For cotransfections into CEF cells, 5 Ag of intact HVT DNA, and suspended in 1 ml of CEF media containing 30 g/ml polybrene (stock 4 mg/ml in 1X HBSS). The DNA-polybrene 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 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 1X HBSS for 4 * Trade-mark 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 C after the addition of 5 mis of CEF
growth media. The next day, the media was changed to remove any last traces of DMS.O 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 .10 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 -70 C.
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 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.,(19-88) 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.
Published restriction maps for the enzymes BamHI, HindIII, and XhoI permitted the use of subcloned fragments 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 the plates were frozen at -20 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 C. Clones which hybridized with the non-radioactive 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, * Trade-mark 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 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 pWE15 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 Stul site within the HVT US2 gene was established as a useful site for foreign DNA insertion utilizing the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUSES (see Example 6). The HVT US2 gene is located within the BamHI #1 fragment which contains five Stul sites. To facilitate the use of this site for insertion of foreign DNA by the Stul site within the US2 gene was converted to a unique Hindlll site. This was accomplished by partially digesting the BamHI #1 subclone with Stul, and then inserting a 10 kb fragment conferring kanomycin resistance (Neon) into the site using Hindlll linkers. The kanomycin resistance gene allowed positive selection of recombinant clones. The Neo` fragment was removed by digestion with Hindlll 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 10 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 Lipof ectin* (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% non-essential amino acids and 2% penicillin/Streptomysin (MEM+). Controls consisted of MEM+ with no DNA, with several ug of HVT DNA, or with 4 out of 5 of the 20 subclones. Separately, 30 Al 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 C in Flo/M199 (1:1) media containing 1% non-essential amino acids, 2%
penicillin/streptomycin, and 5% fetal calf serum (CEF+). Cells were transfected at a confluence of 90 -30 95%. For transfection, wells were aspirated and rinsed 3 times with MEM+, and then incubated 4 hours at 39 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 35 were then examined daily for the appearance of plaques.
Lipofectin with control HVT DNA resulted in the * Trade-mark 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.
BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. When the foreign gene encoded the enzyme Q-galactosidase, the plaques that contained the gene were visualized more easily. The chemical Bluogal7" (Bethesda Research Labs) was incorporated at the level of 200-300 g/ml into the agarose overlay during the plaque assay, and the plaques that expressed active /3-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 they replaced the /3-galactosidase gene; in this instance non-blue plaques were picked for purification of the recombinant virus.
SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT BVT
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 C. Once plaques have developed, the agarose overlay is removed from the dish, the monolayer rinsed lx with PBS, fixed with 100% methanol for 10 minutes at room temperature and the cells air dried.
WO %/05291 PCTIUS95110245 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 MgC12), and then incubated 10 minutes to overnight at room temperature with freshly prepared substrate solution (0.3 mg/ml Nitro Blue tetrazolium +
0.15 mg/ml 5-Bromo-4-Chloro-3-Indolyi 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 HVT STOCKS. When no suitable immunological reagent exists to detect the presence of 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 -.50-100 plaques/10 cm.dish, overlaid with nutrient agarose media and incubated for 4-5 days at 39 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 NaCl and 0.5M
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 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 g/ml) for one hour at 65 C. Radio-labeled probe DNA (alpha 32P-dCTP) is then added and the membranes incubated at 65 C
overnight (-12 hours). After hybridization the NC
membranes are washed two times (30 minutes each) with 2X SSC at 65 C, followed by two additional washes at 65 C with 0.5X SSC. The NC membranes are then dried and exposed to X-ray film (Kodak X-OMAT;kAR) at -70 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 beta-galactosidase (IacZ) gene was inserted into the HVT
EcoRI # 7 fragment at the unique Stul site. The marker 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 SaII 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 PvuI1 restriction fragment of plasmid pJF751 (Ferrari et al., 1985).
Fragment 3 is an approximately 754 base pair Ndel to * Trade-mark Sall 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 5 g/ml Concanavalin A and incubated at 39"for 48 hours.
Total RNA was isolated from the cells using guanidine isothionate lysis reagents and protocols from the Promega RNA isolation kit (Promega Corporation, Madison WI). 4 g of total RNA was used in each 1st strand reaction containing the appropriate antisense primers and ANN 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 * Trade-mark 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 10 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
20 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 25 such that the UL43 ORF is in the opposite transcriptional orientation to the pSP64 0-lacatamase gene.
HOMOLOGY VECTOR 172-63.1. The plasmid 172-63.1 was 30 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
35 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 Hindlll 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 inserted as a Sall 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 Sall 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 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 AvaII 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 Sinai to BamHI restriction sub-fragment of the HSV-1 BamHI restriction fragment N. Fragment 6 is an approximately 1812 base pair BamHI to PstI 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 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 constructed for the purpose of generating recombinant HVT. It contains an approximately 38,850 base pair region of genomic HVT DNA (see Figure 8). 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 8). 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.
SUBGENOMIC 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 8). 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 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 Pi and P2 (described in Figure 8). A bacterial strain containing this cosmid has been deposited 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. 75430.
SUBGENOMIC 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 8). 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 accordingq 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 8). A bacterial strain containing this cosmid has been deposited on March 3, 1993 .10 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. 75427.
HOMOLOGY VECTOR 435-47.1. The plasmid 435-47.1 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 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 Hindlll linker (CAAGCTTG) was then inserted into thetiunique Stul site of the EcoRI #7 fragment.
SUBGENOMIC CLONE 437-26.24. Plasmid 437-26.24 was constructed for the purpose of generating recombinant 5 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 10 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 20 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.
25 SUBGENOMIC 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 30 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, 35 1989), by joining two' restriction fragments from the following sources. The first fragment is an approximately 2970 base pair HindIII to BamHI
restriction fragment of pSP64 (Promega). The second fragment is the approximately 15,300 base pair BamHI to Stul 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 Hindlll 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 Hindlll site.
The MDV genes were inserted at the blunt ended Hindlll site as a blunt ended PstI to EcoRI fragment (see Figures 10A and 10B). The Hindlll and EcoRI sites were blunted by the Klenow fill in reaction. The PstI site was blunted by the T4 DNA polymerase reaction. Note 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 Sall 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 Hindlil 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 Bc1I 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 Safi restriction sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi et al., 1984). Note that the fragments are oriented such that Bc1I and NdeI sites are contiguous.
HOMOLOGY VECTOR 528-11.43. The plasmid 528-11.43 was constructed for the purpose of inserting the infectious laryngotracheitis (ILT) virus gB gene (A.M. Grifin, 1991) into HVT. The gB gene was inserted as an EcoRI
fragment into the homology vector 435-47.1 at the unique Hindlll site. The gB gene was inserted at the blunt ended HindIII site as a blunt ended EcoRI
fragment. The Hindill 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 Hindlil restriction enzyme site into which foreign DNA may be inserted.
When a plasmid containing a foreign DNA insert at the = CA 02626498 2008-04-10 Hindlll 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 fragment of pSP64 (Promega). The second fragment is an approximately 1368 base pair PvuI to Sall restriction fragment of pSP65 (Promega). The third fragment is the approximately 3400 base pair XhoI to XhoI fragment of 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 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 (Bi strain) . The last fragment is an approximately 784 base pair SmaI to Smal 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 Hindlll 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 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 is an approximately 1811 base pair AvaII to NaeI restriction fragment of the full length NDV HN
cDNA clone (B1 strain). The third fragment is an approximately 754 base pair NdeI to Sall 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 (B1 strain). 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).
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 Hindill site located between the MDV gA
and gB genes (see Junction B, Figure 10A). The HN gene 5 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 Sall to BamHI restriction sub-fragment of the PRV BamHI
15 fragment #10 (Lomniczi, et al., 1984) The second fragment is an approximately 1811 base pair AvaII to NaeI restriction fragment of the full length NDV HN
cDNA clone (B1 strain). The last fragment is an approximately 754 base pair NdeI to Sall restriction 20 sub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984).
SUBGENOMIC CLONE 550-60.6. Plasmid 550-60.6 was constructed for the purpose of generating recombinant 25 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 30 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 35 approximately 4176 base pair EcoRV to BamHt 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 Hindlll and then resected with the Exolil 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 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, 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 Hindlll fragment into the homology vector 456-17.22 at the Hindlil 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 Hindlll 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 Hindlll 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 the synthetic DNA sequences indicated in Figures 11A
and 11B. Fragment 1 is an approximately 784 base pair SmaI to Sinai 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 Sall to NcoI sub-fragment of the MDV BglII 4.2 KB
genomic restriction fragment (Ross, et al., 1991).
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 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 Safi restriction sub-fragment of the PRV
BamHI restriction fragment #7 (Lomniczi, et al., 1984).
The fourth fragment is an approximately 1191 base pair Pstl to AvaII restriction sub-fragment of the HCMV
genomic XbaI E fragment (D.R. 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).
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 AvaII restriction sub-fragment of the HCMV genomic XbaI 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 AvaII and BclI sites are contiguous. The third fragment is an approximately 784 base pair Sinai 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 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.
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 1acZ 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 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 Bc1I restriction sub-fragment of the ILT KpnI genomic restriction fragment #8 (10.6 KB). 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). Note that the second and third fragments are oriented such that BclI
and NdeI 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
5 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 pWE15 (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 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 constructed for the purpose of generating recombinant HVT. It was isolated as an Ascl 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 .10 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 Xhol site (Nucleotide #1339-1344;=SEQ ID NO. 48) has been converted to a unique Pacl site using synthetic DNA
linkers. The Pacl site was used in insertion and expression of foreign genes in HVT. (See Figure 13A)..
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 686-63.A1. Plasmid 686-63.Al 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 14VT DNa. This region includes BamHI fragments 10 and 21, and approximately 1100 base pairs of fragment 6 and approxin~ately 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.
SUBGENOMIC 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 approximately 1100 base pair BamHI to AscI subfragment and an approximately 13,000 base pair AscI to NotI
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 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 #1. 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 Pacl site of plasmid 654-45.1. The IBDV VP2 gene uses the IBRV VP8 promoter and ILTV US3 polyadenylation signal. The cosmid was constructed utilizing standard recombinant DNA techniques (Sambrook, et al., 1989). The first fragment is a 208 base pair Hindlil 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 primer used for reverse transcription and PCR was 5'-CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3' (SEQ ID NO.
53). The sense primer used for PCR was 5'-CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3' (SEQ ID NO.
54). The DNA fragment generated by PCR was cloned into the PCR-DirectT" vector (Clontech Laboratories, Inc., Pali Alto, CA). The IBDV VP2 fragment was subcloned next tot he VP8 promoter using BclI sites generated by the PCR primers. The DNA sequence at this junction adds amino acids methionine, aspartate and glutamine 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 Pacl 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 Hindlll 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 Pacl 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 Sail 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 NaeI restriction fragment of the full length NDV HN
cDNA clone (B1 strain). The third fragment is an approximately 754 base pair NdeI to SalI restriction subfragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al., 1984). The fourth fragment is an approximately, 1191 base pair PstI to Avail restriction subfragment 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 (Bi strain; SEQ ID NO: 12). The sixth fragment is an approximately 784 base pair SmaI to Sinai 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 721-38.1) contains the MDV gA, gD and gB genes inserted into a StuI site in the HVT US2 gene converted to a WO 96/05291 PC.T/US95110245 unique Hindlll 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 5 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 10 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 Stul 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 10 and 11) . The fourth fragment is an approximately 14,500 base pair Hindill 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 25 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 Stul site in the HVT US2 gene converted to a unique Hindill site within the BamHI #1 fragment of the 30 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 35 restriction digest with BamHI of S-HVT-106 and isolation of an approximately 46,300 base pari 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 6100 base pair BamHI fragment from cosmid vector pSY1626 derived from pHC79 (Bethesda Research Labs, Inc.) and pWE15 (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 Stul 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 (Lomniczi et al., 1984), the HCMV immediate early promoter (D.R. 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 11B), the approximately 450 base pair ILTV US3 polyadenylation site, and the MDV gB gene (Figures 10A
and 10B). The fourth fragment is an approximately 14,500 base pair Stul 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 HVT. Plasmid 729-37.1 contains the ILTV gD and gB genes inserted into the NotI site of plasmid 686-63.Al. 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 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 .10 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 contians the entire Smal B fragment and two Smal 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 base 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 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 SmaI 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.A8 contains the chicken myelomonocytic growth factor (cGMF) gene inserted into the Pacl site of plasmid 654-45.1. The cMGF gene uses the HCN V 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 Pacl site of HVT subgenomic clone 654-45.1. The first fragment is an approximately 1191 base pair PstI to Avail restriction subfragment of the HCMV genomic XbaI E fragment (D.R. 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 5'-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 784 base pair Smal to Smal 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.Al contains the chicken interferon gene inserted into the Pacl 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 Pacl 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: 60).
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 162 amino acids of the mature protein encoding chicken interferon. The third fragment is an approximately 784 base pair Smal to Smal restriction subfragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).
5 S-HVT-001 is a herpesvirus of turkeys (HVT) that contains the E. coli /3-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 10 a starting point to engineer the insertion of foreign genes into HVT. The BamHI restriction map of HVT is shown in Figure 1A. From this data, several different regions of HVT DNA into which insertions of foreign genes could be made were targeted. The foreign gene 15 chosen for insertion was the E. coli (3-galactosidase (lacZ) gene , which was used in PRV. The promoter was the PRV gpX promoter. The ZacZ gene was inserted into the unique long region of HVT, specifically into the XhoI 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 BluogalTM
(Bethesda Research Labs). Similarly, the lacZ gene has been inserted into. the Sail 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).
S-HVT-003 is a herpesvirus of turkeys (HVT) that contains the E. coli (3-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 3') (SEQ ID
NO: 2), two of which are antigens to provide protection against IBDV infections of chickens. Expression of the genes for both 0-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 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
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 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 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 V153. 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, 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 #10 fragment as an approximately 800 base pair Sail 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 BalI fragment followed by an approximately 40 base pair Ava I to Sma I fragment. The third segment of DNA (Segment 3, .10 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 Sinai 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 BamHI, #10 fragment as an approximately 330 base pair NaeI to AluI fragment. Additionally, segment four contains approximately 36 base pairs of HSV TK 5'untranslated leader sequence as a PstI to Bg1II fragment in which the PstI site has been joined to the AluI 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 5, 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 Bg1II
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 3') is under the control of the gpX promoter (segment 4), 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 Smal fragment (obtained from Bernard Roizman, Univ. of Chicago). The HpaI site of segment five was fused to the Smal 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 (5' to 3') the PRV promoter, the gpX TATA box, the gpX cap site, the first seven amino acids of gpX, the E. coli Q-galactosidase (1acZ) 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, 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 was engineered such that it was flanked by two EcoRI
restriction endonuclease sites. As a result, an approximately 9100 base pair fragment containing both 1acZ 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/1acZ
cassette. The following procedures were used to construct S-HVT-003 by homologous recombination. The IBDV/1acZ 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 1acZ (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 7). 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 5 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 10 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 1acZ gene and. the IBDV gene incorporated into its genome.
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.
25 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-30 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 35 dominant region of the IBDV 40 kd (VP2) capsid protein.
The filters were washed and treated with [1211] 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 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 immunologically indistinguishable from the authentic viral VP2 protein. In addition, the VP2 protein produced from S-HVT-003 comigrates with the 40 kd species 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.
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 received 0.5 ml of an inactivated IBDV vaccine inoculated 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 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.
DAY
Vaccine Group Bird No. 28 31 35 38 42 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 IBDV' 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 IBDV 262 <2 <2 <2 <2 1:4 1:4 263 <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
chicks were vaccinated with S-HVT-003 (20 with 0.2m1 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 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/1acZ cassette into the XhoI site of BamHI #16 does not result in detectable attenuation of virus.
Subsequent experiments examining the recovered virus .10 by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS
procedure confirmed the in vivo stability of S-HVT-003, by demonstrating /3-galactosidase expression in 100% of the viruses.
Harvest Date Day 4 Day 7 Sample Pi P2 Pi P2 T 2 2+ 2+
T 3 2+ 2+
T 4 + 4+
T 5 3+ 3+
15 T 6 2+ contaminated T 7 + 5+
T 8 + 5+
T 8 + 5+
T 9 + 5+
20 T10 + 5+
N = control, T = vaccinated CPE ranged from negative (-) to 5+
25 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 30 five control and fourteen vaccinated chicks were challenged with virulent IBDV by bi-lateral eyedrop (103=8EID50) . All birds were sacrificed 6-days post challenge and bursa to body weight ratios were calculated. A summary of the results is shown in 35 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 40 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 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.
ELISA VN
Sample Grout) HVT IBDV IBDV
C-0 (n=3) 0 0 <100 C-4 (n=2) 0 0 nd T-4 (n=5) 0 0 nd C-7 (n=2) 0 0 <100 T-7 (n=5) 0 0 <100 C-14 (n=5) 0 0 nd T-14 (n=14) 0 0 <100 C-21 (n=5) 0 0 nd T-21 (n=14) 1 0 <100 C-27 (n=5) 0 0 nd CC-27 (n=5) 0 5 nd 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.
Sample Group Body wt. Bursa wt. BBR
Control (n=5) 258.8 1.5088 0.0058 Challenge 209 0.6502 0.0031 Control (n=5) Challenge 215.5 0.5944 0.0027 Treated (n=10) 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 0.2m1 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 and incubated. Tests were developed with Tris-malcate buffer (TMB) + H202 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.
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 (9-galactosidase and IBDV antigens) that are recognized in the host by an immune response directed to these proteins.
WO %/05291 PCT/US95/10245 TABLE 5ti Serology: Herpes/IBDV ELISA titer Bleed Date 5 Bird# 11/3 11/10 11/14 11/24 12/1 12/8 12/15 12/22 Vaccinated and Challenged Control Challenged only Maximum titer level is 9 Example 3 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 /3-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 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 MDV gA gene. S-HVT-004 is a recombinant virus that contains both the /3-galactosidase gene and the MDV gA
gene incorporated into the genome.
Figure 6C shows the structure of S-HVT-004.
Example 4 NEWCASTLE DISEASE VIRUS
Newcastle disease virus (NDV) is closely related to PI-3 in overall structure. Hemagglutinin (HN) and fusion (F) 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.
INFECTIOUS BRONCHITIS VIRUS
Infectious bronchitis virus (IBV) is a virus of chickens closely related in overall structure to TGE.
Major neutralizing antigen of TGE was engineered for expression in PRV. 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 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.
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, 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 EcoRI-SaII 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-SaII 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 Stul site within HVT US2, previously utilized for the construction of S-HVT-012. The site was initially altered by insertion of a unique Hindlll 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
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 Stul site in HVT
US2 gene.
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-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 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 clinical signs of Marek's disease, then necropsied and observed for Marek's lesions. This study demonstrated the ability of HVT-045 and HVT-047 to provide 100% protection against challenge (Table 1). The commercial vaccine gave 96% protection, and 79% of the non-vaccinated chicks developed Marek's disease.
PROTECT SUSCEPTIBLE CHICKS AGAINST VIRULENT MAREK'S
DISEASE VIRUS
Marek's Protection Vaccine Group MD-5 Challenge RB1B Challenge S-HVT-046 20/20 Not Tested S-HVT-047 Not Tested 24/24 HVTa Not Tested 24/25 Controls 2/20 5/24 a Commercial Example 6 S-HVT-012 is a recombinant herpesvirus of turkeys that contains the E. coli (3-galactosidase (1acZ) gene inserted into the short unique region. The 1acZ 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 Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2382.
For insertion into the genome of HVT, the f3-galactosidase gene was introduced into the unique Stul site of the. cloned EcoRI fragment #7 of HVT, i.e., the fragment containing the Stul 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 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 IacZ
gene. S-HVT-012 is a recombinant virus that contains the 1acZ gene incorporated into the genome at the Stul site within the US2 gene of HVT.
S-HVT-012 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.
Examine 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, #7, and #9 (see figure i)) 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 Sall in EcoRI fragment #6 and StuI in EcoRI fragment V. A
0-galactosidase (1acZ) 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 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 1acZ 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 1000, clearly defining an appropriate site for the insertion of foreign DNA. The three homology vectors used to define these sites are described below.
Example 7A
Homology 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 also determined (see SEQ ID NO: 3). 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 (gC). The XhoI site interrupts an ORF
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: 5). 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.
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 Hindlll restriction site into which foreign DNA may be cloned. The Hindlil restriction site in plasmid results from the insertion of a Hindlll linker into the naturally occurring Stul site of EcoRI fragment V.
Hindlll 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 Stul indicated that this fragment contains open reading frames coding for US10, US2, and US3. The Stul site interrupts US2 at approximately amino acid 124, suggesting that the US2 gene is non-essential for HVT replication.
Example 7C
Homolocry 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 XhoI
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).
Example 8 S-HVT-014 is a recombinant herpesvirus of turkeys that contains the E. coli 3-galactosidase (lacZ) gene inserted into the long unique region. The IacZ 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 /3-galactosidase gene was introduced into the unique XhoI
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 XhoI site within the BarHI #10 fragment used for construction recombinant herpesvirues of turkeys described in Examples 16 through 19. Flanking regions of EcoRI fragment #9 were used for" homologous recombination. HVT DNA and plasmid DNA 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. S-HVT-014 is a recombinant virus that contains the IacZ 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.
Example 9 S-HVT-005 is a recombinant herpesvirus of turkeys that contains the E. coli f3-galactosidase (lacZ) gene inserted into the long unique region. The 1acZ 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 a galactosidase gene was introduced into an approximately 1300 base pair deletion of the XhoI #9 fragment-of HVT.
The deletion which lies between the unique M1uI and EcoRV sites removes the complete coding region of the HVT gA gene (see SEQ ID NO: 3). Flanking regions of XhoI fragment #9 were used for homologous recombination. HVT DNA and plasmid DNA 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, the DNA was analyzed for the presence of the 1acZ
gene. S-HVT-005 is a recombinant virus that contains the 1acZ 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.
Example 10 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 5), S-HVT-046 (Example 10A), S-HVT-047 (Example 10B), S-HVT-062 (Example 10C).
Example 10A S-HVT-046 S-HVT-046 is a recombinant herpesvirus of turkeys that 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 MDV antigens are more likely to elicit the proper antigenic response than the HVT equivalent antigen.
S-HVT-046 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 HindIll, 437-26.26 with BamHI and Hindlll, and 456-17.22 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.
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 with NotI, 437-26.24 with BamHI and HindIll, 437-26.26 with BamHI and Hindlll, 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.(gD) 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
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 HindlIl, 556-60.6 with BamHI and Hindlll, 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 pathogen free (SPF) chicks were vaccinated with either S-HVT-045, S-HVT-046, or S-HVT-047. Five days post-vaccination, vaccinated chicks, and non-vaccinated, control chicks were challenged with MDV. Following a 6-week 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 non-vaccinated 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-i 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 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.
VIRULENT MAREK'S VIRUS CHALLENGE
Study Vaccine Group Dosea Protectionb 1 S-HVT-045 2.2 X 103 24/24 (1000) 1 S-HVT-046 2.2 X 103 20/20 (1000) 1 S-HVT-047 2.2 X 103 24/24 (1000) 1 Controls 7/44 (16%) 1 HVT/SB-1 24/25 (96%) 2 S-HVT-062 7.5 X 102 32/32 (100%) 2 S-HVT-062 1.5 X 10' 22/22 (100%) 2 Controls 0/20 (0%) 2 HVT` 7.5 X 102 17/21 (81%) 2 HVT/SB-1 7.5 X 102 21/22 (95%) a PFU/0.2 ml.
No. protected/Total; Challenge 5 days post-vaccination.
` Commercial vaccine.
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 u4 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 255-18.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 1001i of the plaques were blue.
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 with NotI, 437-26.24 with BamHI and HindIll, 437-26.26 with BamHI and Hindlll, 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 under the control of the PRV gX 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-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 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 Hindlil, and 549-62.10 uncut. Insertion of the appropriate DNA was confirmed by southern blot analysis.
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
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 Notl, 407-32.1C1 with NotI, 437-26.24 with BamHI and HindlIl, 437-26.26 with BamHI and Hindlll, 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.
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
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 HindIll, 437-26.26 with BamHI and Hindlll, 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 USDA-licensed, conventional vaccine comprised of NDV
131/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-050) gave complete protection against a challenge that caused Newcastle disease in 100% of non-vaccinated control chicks. Recombinant virus S-HVT-049 gave 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-i 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 the ability of S-HVT-050 to provide protection greater than the commercial Marek's disease vaccines.
AGAINST VIRULENT NEWCASTLE AND MAREK'S DISEASE VIRUS
CHALLENGE
Protection (%) Vaccine Study Group Dosea NDVb MDVc 1 S-HVT-048 4.0 X 104 19/19 (100) 1 S-HVT-049 3.0 X 104 4/20 (20) 1 S-HVT-050 1.5 X 104 20/20 (100) 1 Controls 0/20 (0) 1 NDV B1/Bld 18/18 (100) 2 S-HVT-050 7.5 X 102 13/14 (93) 2 S-HVT-050 1.5 X 103 16/17 (94) 2 Controls 5/23 (22) 2 HVTd 20/26 (77) 2 HVT/SB_ld 10/12 (83) a PFU/0.2 ml.
b No. protected/Total; Challenge 3 weeks post-vaccination.
c No. protected/Total; Challenge 5 days post-vaccination.
d Commercial vaccine.
Example 12 Bivalent Vaccines Against Infectious Laryncgotracheitis 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 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 with NotI, 437-26.24 with BamHI and HindIll, 437-26.26 with BamHI and Hindill, 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.
= CA 02626498 2008-04-10 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 HindIll, 437-26.26 with BamHI and Hindlil, 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
genes are inserted in the unique short region in the same transcriptional orientation as the US2 gene. An E. coli 1acZ 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 plasmid 634-29.16 were co-transfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS
procedure into primary chick embryo fibroblast (CEF) 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 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.
VIRULENT' INFECTIOUS LARYNGOTRACHEITIS VIRUS CHALLENGE
Vaccine Group Dosea Protectionb S-HVT-051 28/30 (93%) 2.1 X 103 S-HVT-052 1.7 X 103 29/29 (100%) S-HVT-051 + 2.1 X 103 24/24 (100%) S-HVT-052 1.7 X 103 Controls 2/30 (7%) ILT 29/30 (97%) a PFU/0.2 ml.
b No.protected/Total; Challenge 2-3 weeks post-vaccination.
Commercial vaccine.
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.
S-HVT-096 is a recombinant herpesvirus of turkeys that contains the IBDV VP2 gene, under the control of the 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 HindlIl, 556-60.6 with BamHI, and 602-57.F1 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.
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.
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 HindlIl, 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.
Example 15 Vaccines utilizing HVT to express antigens from various pathogens.
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, Pasteurella spp, Haemophilus spp, Chlamydia spp, Mycoplasma spp, Campylobacter spp, Bordetella spp, Poultry nematodes, cestodes, trematodes, Poultry 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 Stul site converted into a Hindlil site in the HVT US2 gene. The 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-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 Pacl 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.
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 Pacl site in the EcoRl #9 (BamHI #10) fragment of the HVT genome. 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 13A and 15; SEQ ID NO: 48, 50). S-HVT-139 further contains the MDV gA, gD, and gB genes are inserted into the unique Stul site converted into a HindIll 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.SG6 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 S-HVT-140 is a recombinant herpesvirus of turkeys that ti contains the ILT virus gD and gI genes inserted into a unique XhoI site converted to a Pacl 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 Stul site converted into a Hindlll site in the HVT US2 gene. The ILTV gD and gI genes are expressed as overlapping transcripts from their own respective endogenous ILTV
promoters, and the MDV genes are also expressed from 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 PRV gX promoter. S-HVT-140 is useful as a vaccine in poultry against Infectious Laryngotracheitis, Marek's 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.
Example 17A HVT-126 S-HVT-126 is a recombinant herpesvirus of turkeys that contains the IBDV VP2 gene inserted into an XhoI site converted to a Pacl site in the EcoRl #9 (BamHI #10) 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
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
site converted to a Pacl site in the EcoRl #9 (BamHI
#10) 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) 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.
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 Pacl site in the EcoRl #9 (BamHI
#10) fragment of the HVT genome (Figures 13 A 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). 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 Hindlll 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 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 NotI, 672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 722-60.E2 uncut.
= CA 02626498 2008-04-10 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 Pacl site in the EcoRl #9 (BamHI #10) fragment of the HVT genome (Figures 13A and 15). S-HVT-128 further contains the MDV gA, gD, and gB
genes inserted into a unique Stul site converted into a Hindlll 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 with NotI, 1.72-07.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 the unique short region (see HVT-062) and the PRV gX
promoter-lacZ gene inserted into an XhoI 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 Pacl site in the EcoRl #9 (BamHI
#10) fragment within the unique long region of HVT.
(Figure 14; SEQ ID NOs: 48 and 50) The NDV HN gene is 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 BamHT.
Example 19 S-HVT-145 HVT/MDV recombinant virus vaccine S-HVT-145 is a recombinant virus vaccine containing MDV
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
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 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 Pacl site or NotI site in the EcoRl #9 (BamHI #10) fragment within the uniuqe long region of HVT/MDV recombinant virus (Figures 13 and 15).
A cosmid was constructed containing the entir MDV
unique short region. MDV genomic DNa contains several Smal sites in the uniuqe long and internal and terminal repeats of the virus, but no Sinai 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 and cloned into a blunt ended site of the cosmid vector pWE15. 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
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 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.
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 unique long/internal repeat junction and the unique long/terminal repeat junction (55, 56, and 57).
SB-1 strain is an MDV serotype 1 with attenuated pathogenicity. Vaccination with a combination of HVT
and SB-i 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 non-virulent 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-i 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.
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-i genomic DNA. To determine the appropriate sub-infectious dose, transfection of the SB-i 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-i which.
recombine withthe HVT unique short subgenomic clones.
Therefore, a virus resulting from recombination between overlapping homologous regions of the SB-1 and HVT
subgenomic fragments is highly favored. Alternatively, SB-i 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 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, Newcastle's disease virus and infectious bursal disease virus.
Example 20 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 (58), and chicken interferon (cIFN) is homologous to mammalian type 1 interferon (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 15.
Example 20A S-HVT-144 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 Pacl site in the EcoRl #9 fragment within the unique long region of HVT. The cMGF gene is in the opposite 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 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 A recombinant herpesvirus of turkeys contains the chicken interferon (cIFN) gene inserted into an XhoI
site converted to a Pacl 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 with NotI, 761-07.A1 with Asc I, 415-09.BA1 with BamHI.
Recombinant HVT expressing avian cytokines is combined with HVT expressing genes for avian disease antigens to enhance immune response. Additional cytokines that are 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 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 il, 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 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 Pacl 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 Stul site converted into a Hindlll 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.
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 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 Pacl site in the EcoRl #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 Stul site converted into a Hindlll 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 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, 672-07.C40 with NotI, 672-01.A40 with NotI, 761-07.A1 with Asc I, 722-60.E2 uncut.
Example 20E Recombinant HVT expressing Marek's disease virus genes and chicken 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 Pacl 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 Stul site converted into a Hindlll 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 an enhanced immune response in poultry against Marek's N
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.
Example 20F Recombinant HVT expressing Marek's disease virus genes, Newcastle disease virus genes and chicken myelomonocytic growth factor gene.
A recombinant herpesvirus of turkeys contains the chicken myelomonocytic growth factor (cGMF) gene inserted into an XhoI site converted to a Pacl site in the EcoRl #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 Stul site converted into a Hindlll 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
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 limited to humans, equine, bovine, porcine, canine and feline.
Recombinant HVT is useful as a vaccine against equine diseases when foreign antigens from diseases or disease 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 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
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, 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 growth 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 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 i3-galactosidase. cIFN added to the cell culture media reduced both the expression of 9-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, 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 evasion mechanism involves poxvirus soluble receptors for interleukins, interferon, or tumor necrosis factors which inactive the cytokines and allow viral replication (60). 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 (61). in a further embodiment, an interferon-negative CEF cell line is constructed by 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 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.
References 1. Buckmaster et al., J. Gen. Virol. 69:2033, 1988.
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(1) GENERAL INFORMATION:
(i) APPLICANT: SYNTRO CORPORATION
(ii) TITLE OF INVENTION: Recombinant Herpesvirus of Turkeys And Uses Thereof (iii) NUMBER OF SEQUENCES: 60 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: John P. White (B) STREET: 1185 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: USA
(F) ZIP: 10036 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 09-AUG-1995 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: White, John P
(B) REGISTRATION NUMBER: 28,678 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212)278-0400 (B) TELEFAX: (212)391-0526 (C) TELEX: 422523 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3350 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 129..2522 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg Ser Leu Lei; Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Glu ACA CCC TGG AGA AGC ACA CTC TCA GGT CAG AGA CTG ACC TAC AAT .TTG 266 Thr Pro Trp Arg Ser Thr Leu Ser Gly Gln Arg Leu Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr Lys Phe Asp Arg Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Pro Gly Gly Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu Ser Val Gly Gly Glu Leu Val Phe Arg Thr Ser Val His Gly Leu Val Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Thr Val Ile Thr Arg Ala Val Ala Ala Asn Thr Gly Leu Thr Thr Gly Thr Asp Asn Leu Met Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gln Pro Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln Ala Gly Asp Gln Met Leu Trp Ser Ala Arg Gly Ser Leu Ala Val Thr Ile His Gly Gly Asn Tyr Pro Giy Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile Arg Ala Ile Arg Arg Ile Ala Val Pro Val Val Ser Thr Leu Phe Pro Pro Ala Ala Pro Leu Ala His Ala Ile Gly Glu Gly Val Asp Tyr Leu Leu Gly Asp Glu Ala Gln Ala Ala Ser Gly Thr Ala Arg Ala Ala Ser Gly Lys Ala Arg Ala Ala Ser Gly Arg Ile Arg Gln Leu Thr Leu Ala Ala Asp Lys Gly Tyr Glu Val Val Ala Asn Leu Phe Gln Val Pro Gln Asn Pro Val Val Asp Gly Ile Leu Ala Ser Pro Gly Val Leu Arg Gly Ala His Asn Leu Asp Cys Val Leu Arg Glu Gly Ala Thr Leu Phe Pro Val Val Ile Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu AAC AGC AAA ATG TTT GCT GTC ATT GAA GGC GTG"CGA GAA GAC CTC CAA 1898 Asn Ser Lys Met Phe Ala Val Ile Glu Gly Val Arg Glu Asp Leu Gln Pro Pro Ser Gln Arg Gly Ser Phe Ile Arg Thr Leu Ser Gly His Arg Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg Asp Tyr Thr Val Val Pro Ile Asp Asp Val Trp Asp Asp Ser Ile Met Leu Ser Lys Asp Pro Ile Pro Pro Ile Val Gly Asn Ser Gly Asn Leu Ala Ile Ala Tyr Met Asp Val Phe Arg Pro Lys Val Pro Ile His Val Ala Met Thr Gly Ala Leu Asn Ala Cys Gly Glu Ile Glu Lys Val Ser Phe Arg Ser Thr Lys Leu Ala Thr Ala His Arg Leu Gly Leu Lys Leu Ala Gly Pro Gly Ala Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe Ile Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Asn Ala Gly Arg Gln Tyr His Leu Ala Met Ala Ala Ser Glu Phe Lys Arg Pro Arg Thr Arg Glu Cys Arg Gln Ser Asn Gly Ser Ser Ser Gln Arg Gly Pro Thr Ile Pro Ile Cys Thr Gln Cys Val His Val Ala Gly Arg Glu Trp Asp Cys Asp (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 797 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg Ser=Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Glu Thr Pro Trp Arg Ser Thr Leu Ser Gly G1n-Arg Leu Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr Lys Phe Asp Arg Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Pro Gly Gly Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu Ser Val Gly Gly Glu Leu Val Phe Arg Thr Ser Val His Gly Leu Val Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Thr Val Ile Thr Arg Ala Val Ala Ala Asn Thr Gly Leu Thr Thr Gly Thr Asp Asn Leu MMe~ Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gln Pro Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln Ala Gly Asp Gln Met Leu Trp Ser Ala Arg Gly Ser Leu Ala Val Thr Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly'Ala Met Asn Tyr Thr Lys Leu Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile Arg Ala Ile Arg Arg Ile Ala Val Pro Val Val Ser Thr Leu Phe Pro Pro Ala Ala Pro Leu Ala His Ala Ile Gly Glu Gly Val Asp Tyr Leu Leu Giy Asp Glu Ala Gln Ala Ala Ser Gly Thr Ala Arg Ala Ala Ser Gly Lys Ala Arg Ala Ala Ser Gly Arg Ile Arg Gln Leu Thr Leu Ala Ala Asp Lys Gly Tyr Glu Val Val Ala Asn Leu Phe Gln Val Pro Gln Asn Pro Val Val Asp Gly Ile Leu Ala Ser Pro Gly Val Leu Arg Gly Ala His Asn Leu Asp Cys Val Leu Arg Glu Gly Ala Thr Leu Phe Pro Val Val Ile Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu Asn Ser Lys Met Phe Ala Val Ile Glu Gly Val Arg Glu Asp Leu Gln Pro Pro Ser Gln Arg Gly Ser Phe Ile Arg Thr Leu Ser Gly His Arg Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg Asp Tyr Thr Val Val Pro Ile Asp Asp Val Trp Asp Asp Ser Ile Met Leu Ser Lys Asp Pro Ile Pro Pro Ile Val Gly Asn Ser Gly Asn Leu Ala Ile Ala Tyr Met Asp Val Phe Arg Pro Lys Val Pro Ile His Val Ala Met Thr Gly Ala Leu Asn Ala Cys Gly Glu Ile Glu Lys Val Ser Phe Arg Ser Thr Lys Leu Ala Thr Ala His Arg Leu Gly Leu Lys Leu Ala Gly Pro Gly Ala Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe Ile Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Asn Ala Gly Arg Gln Tyr His Leu Ala Met Ala Ala Ser Glu Phe Lys Arg Pro Arg Thr Arg Glu Cys Arg Gln Ser Asn Gly Ser Ser Ser Gln Arg Gly Pro Thr Ile Pro Ile Cys Thr Gln Cys Val His Val Ala Gly Arg Glu Trp Asp Cys Asp (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5426 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 73..1182 (D) OTHE{. INFORMATION: /product= "HVT UL42"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1306..2574 (D) OTHER INFORMATION: /product= "HVT UL43"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2790..4259 (D) OTHER INFORMATION: /product= "HVT gA"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4701..5339 (D) OTHER INFORMATION: /product= "HVT UL45"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Met Ala Gly Ile Thr Val Ala Cys Asp His Thr Ala Gly Glu Ala His Thr Pro Glu Asp Met Gln Lys Lys Trp Arg Ile Ile Leu Ala Gly Glu Lys Phe Met Thr Ile Ser Ala Ser Leu Lys Ser Ile Val Ser Cys Val Lys Asn Pro Leu Leu Thr Phe Gly Ala Asp Gly Leu Ile Val Gln Gly Thr Val Cys Gly Gln Arg Ile Phe Val Pro Ile Asp Arg Asp Ser Phe Ser Glu Tyr Glu Trp His Gly Pro Thr Ala Met Phe Leu Ala Leu Thr Asp Ser Arg Arg Thr Leu Leu Asp Ala Phe Lys Cys Glu Lys Arg Arg Ala Ile Asp Val Ser Phe Thr Phe Ala Gly Glu Pro Pro Cys Arg His Leu Ile Gln Ala Val Thr Tyr Met Thr Asp Gly Gly Ser Val Ser Asn Thr Ile Ile Lys Tyr Glu Leu Trp Asn Ala Ser Thr Ile Phe Pro Gln Lys Thr Pro Asp Val Thr Phe Ser Leu Asn Lys Gin Gin Leu Asn Lys Ile Leu Ala Val Ala Ser Lys Leu Gln His Glu Glu Leu Val Phe Ser Leu Lys Pro Glu Gly Gly Phe Tyr Val Gly Thr Val Cys Thr Val Ile Ser Phe Glu Val Asp Gly Thr Ala Met Thr Gln Tyr Pro Tyr Asn Pro Pro Thr Ser Ala Thr Leu Ala Leu Val Val Ala Cys Arg Lys Lys Lys Ala Asn Lys Asn Thr Ile Leu Thr Ala Tyr Gly Ser Gly Lys Pro Phe Cys Val Ala Leu Glu Asp Thr Ser Ala Phe Arg Asn Ile Val Asn Lys Ile Lys Ala Gly Thr Ser Gly Val Asp Leu Gly Phe Tyr Thr Thr Cys Asp Pro Pro Met Leu Cys Ile Arg Pro His Ala Phe Gly Ser Pro Thr Ala Phe Leu Phe Cys Asn Thr Asp Cys Met Thr Ile Tyr Glu Leu Glu Glu Val Ser Ala Val Asp Gly Ala Ile Arg Ala Lys Arg Ile Asn Glu Tyr Phe Pro Thr Val Ser Gln Ala Thr Ser Lys Lys Arg Lys Gln Ser Pro Pro Pro Ile Glu Arg Glu Arg Lys Thr Thr Arg Ala Asp Thr Gln Met Pro Ser Gly Ala Ser Ser Ser Pro Pro Pro Ala Tyr Thr Ser Ala Ala Pro Leu Glu Thr Tyr Asn Ser Trp Leu Ser Ala Phe Ser Cys Ala Tyr Pro Gln Cys Thr Ala Gly Arg Gly His Arg Gln Asn Gly Lys Lys Cys Ile Arg Cys Ile Val Ile Ser Val Cys Ser Leu Val Cys Ile Ala Ala His Leu Ala Val Thr Val Ser Gly Val Ala Leu Ile Pro Leu Ile Asp Gln Asn Arg Ala Tyr Gly Asn Cys Thr Val Cys Val Ile Ala Gly Phe Ile Ala Thr Phe Ala Ala Arg Leu Thr Ile Arg Leu Ser Glu Thr Leu Met Leu Val Gly Lys Pro Ala Gln Phe Ile Phe Ala Ile Ile Ala Ser Val Ala Glu Thr Leu Ile Asn Asn Glu Ala Leu Ala Ile Ser Asn Thr Thr Tyr Lys Thr Ala Leu Arg Ile Ile Glu Val Thr Ser Leu Ala Cys Phe Val Met Leu Gly Ala Ile Ile Thr Ser His Asn Tyr Val Cys Ile Ser Thr Ala Gly Asp Leu Thr Trp Lys Gly Gly Ile Phe His Ala Tyr His Gly Thr Leu Leu Gly Ile Thr ATA CCA AAC ATA CAC CCA ATC CCT.CTC GCG GGG TTT CTT GCA GTC TAT 1956 Ile Pro Asn Ile His Pro Ile Pro Leu Ala Gly Phe Leu Ala Val Tyr Thr Ile Leu Ala Ile Asn Ile Ala Arg Asp Ala Ser Ala Thr Leu Leu Ser Thr Cys Tyr Tyr Arg Asn Cys Arg Glu Arg Thr Ile Leu Arg Pro Ser Arg Leu Gly His Gly Tyr Thr Ile Pro Ser Pro Gly Ala Asp Met Leu Tyr Glu Glu Asp Val Tyr Ser Phe Asp Ala Ala Lys Gly His Tyr Ser Ser Ile Phe Leu Cys Tyr Ala Met Gly Leu Thr Thr Pro Leu Ile Ile Ala Leu His Lys Tyr Met Ala Gly Ile Lys Asn Ser Ser Asp Trp Thr Ala Thr Leu Gln Gly Met Tyr Gly Leu Val Leu Gly Ser Leu Ser Ser Leu Cys Ile Pro Ser Ser Asn Asn Asp Ala Leu Ile Arg Pro Ile Gln Ile Leu Ile Leu Ile Ile Gly Ala Leu Ala Ile Ala Leu Ala Gly Cys Gly Gln Ile Ile Gly Pro Thr Leu Phe Ala Ala Ser Ser Ala Ala Met Ser Cys Phe Thr Cys Ile Asn Ile Arg Ala Thr Asn Lys Gly Val Asn Lys Leu Ala Ala Ala Ser Val Val Lys Ser Val Leu Gly Phe Ile Ile Ser Gly Met Leu Thr Cys Val Leu Leu Pro Leu Ser Met Val Ser Asn Met Arg Val Leu Arg Val Leu Arg Leu Thr Gly Trp Val Gly Ile Phe Leu Val Leu Ser Leu Gln Gln Thr Ser Cys Ala Gly Leu Pro His Asn Val Asp Thr His His Ile Leu Thr Phe Asn Pro Ser Pro Ile Ser Ala Asp Gly Val Pro Leu Ser Glu Val Pro Asn Ser Pro Thr Thr Glu Leu Ser Thr Thr Val Ala Thr Lvs Thr Ala Val Pro Thr Thr Glu Ser Thr Ser Ser Ser Glu Ala His Arg Asn Ser Ser His Lys Ile Pro Asp Ile Ile Cys Asp Arg Glu Glu Val Phe Val Phe Leu Asn Asn Thr Gly Arg Ile Leu Cys Asp Leu Ile Val Asp Pro Pro Ser Asp Asp Glu Trp Ser Asn Phe Ala Leu Asp Val Thr Phe Asn Pro Ile Glu Tyr His Ala Asn Glu Lys Asn Val Glu Val Ala Arg Val Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Lys Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Ser Phe Trp Thr Ser Pro Thr Pro Arg Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Met His Thr Met Gly Val Glu Val Arg Asn Val Asp Tyr Lys Asp Asn Gly Tyr Phe Gln Val Ile Leu Arg Asp Arg Phe Asn Arg Pro Leu Val Glu Lys His Ile Tyr Met Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Arg Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Giy Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Leu Glu Ser Pro Pro Lys Val Ser Cys Leu Val Ala Trp Arg Gln Gly Asp Met Ile Ser Thr Ser Asn Ala Thr Ala Val Pro Thr Val Tyr Tyr His Pro Arg Ile Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Gly Ile Thr Val Arg Trp Leu Val His Asp Glu Pro Gln Pro Asn Thr Thr Tyr Asp Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg Tyr Arg Asn Leu Ala Ser Arg Ile Pro Val Gln Asp Asn Trp Ala Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Val Asp Arg Phe Gln Asn Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Met .Pro Met Ile Val Thr Ile Thr Ala Val Leu Gly Leu Ala Leu Phe Leu Gly Ile Gly Ile Ile Ile Thr Ala Leu Cys Phe Tyr Leu Pro Gly Arg Asn Met Met Ser Pro Thr Pro Glu Asp Asp Arg Asp Leu Val Val Val Arg Gly Arg Leu Arg Met Met Asp Ser Gly Thr Glu Thr Asp Arg Glu Gln Arg His Pro Arg Thr Thr Trp Arg Ser Ile Cys Cys Gly Cys Thr Ile Gly Met Val Phe Thr Ile Phe Val Leu Val Ala Ala Val Leu Leu Gly Ser Leu Phe Thr Val Ser Tyr Met Ala Met Glu Ser Giy Thr Cys Pro Asp Glu Trp Ile Gly Leu Gly Tyr Ser Cys Met Arg Val Ala Gly Lys Asn Ala Thr Asp Leu Glu Ala Leu Asp Thr'gys Ala Arg His Asn Ser Lys Leu Ile Asp Phe Ala Asn Ala Lys Val Leu Val Glu Ala Ile Ala Pro Phe Gly Val Pro Asn Ala Ala Tyr Gly Glu Val Phe Arg Leu Arg Asp Ser Lys Thr Thr Cys Ile Arg Pro Thr Met Gly Gly Pro Val Ser Ala Asp Cys Pro Val Thr Cys Thr Val Ile Cys Gln Arg Pro Arg Pro Leu Ser Thr Met Ser Ser Ile Ile Arg Asp Ala Arg Val Tyr Leu His Leu Glu Arg Arg Asp Tyr Tyr Glu Val Tyr Ala Ser Val Leu Ser Asn Ala Met Ser Lys (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 369 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Met Ala Gly Ile Thr Val Ala Cys Asp His Thr Ala Gly Glu Ala His Thr Pro Glu Asp Met Gln Lys Lys Trp Arg Ile Ile Leu Ala Gly Glu Lys Phe Met Thr Ile Ser Ala Ser Leu Lys Ser Ile Val Ser Cys Val Lys Asn Pro Leu Leu Thr Phe Gly Ala Asp Gly Leu Ile Val Gln Gly Thr Val Cys Gly Gln Arg Ile Phe Val Pro Ile Asp Arg Asp Ser Phe Ser Glu Tyr Glu Trp His Gly Pro Thr Ala Met Phe Leu Ala Leu Thr Asp Ser Arg Arg Thr Leu Leu Asp Ala Phe Lys Cys Glu Lys Arg Arg Ala Ile Asp Val Ser Phe Thr Phe Ala Gly Glu Pro Pro Cys Arg His Leu Ile Gln Ala Val Thr Tyr Met Thr Asp Gly Gly Ser Val Ser Asn Thr Ile Ile Lys Tyr Glu Leu Trp Asn Ala Ser Thr Ile Phe Pro Gin Lys Thr Pro Asp Val Thr Phe Ser Leu Asn Lys Gln Gln Leu Asn Lys Ile Leu Ala Val Ala Ser Lys Leu Gln His Glu Glu Leu Val Phe Ser Leu Lys Pro Glu Gly Gly Phe Tyr Val Gly Thr Val Cys Thr Val Ile Ser Phe Glu Val Asp Gly Thr Ala Met Thr Gln Tyr Pro Tyr Asn Pro Pro lhr Ser Ala Thr Leu Ala Leu Val Val Ala Cys Arg Lys Lys Lys Ala Asn Lys Asn Thr Ile Leu Thr Ala Tyr Gly Ser Gly Lys Pro Phe Cys Val Ala Leu Glu Asp Thr Ser Ala Phe Arg Asn Ile Val Asn Lys Ile Lys Ala Gly Thr Ser Gly Val Asp Leu Gly Phe Tyr Thr Thr Cys Asp Pro Pro Met Leu Cys Ile Arg Pro His Ala Phe Gly Ser Pro Thr Ala Phe Leu Phe Cys Asn Thr Asp Cys Met Thr Ile Tyr Glu Leu Glu Glu Val Ser Ala Val Asp Gly Ala Ile Arg Ala Lys Arg Ile Asn Glu Tyr Phe Pro Thr Val Ser Gln Ala Thr Ser Lys Lys Arg Lys Gln Ser Pro Pro Pro Ile Glu Arg Glu Arg Lys Thr Thr Arg Ala Asp Thr Gln (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 422 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S:
Met Pro Ser Gly Ala Ser Ser Ser Pro Pro Pro Ala Tyr Thr Ser Ala Ala Pro Leu Glu Thr Tyr Asn Ser Trp Leu Ser Ala Phe Ser Cys Ala Tyr Pro Gln Cys Thr Ala Gly Arg Gly His Arg Gln Asn Gly Lys Lys 35 ti 40 45 Cys Ile Arg Cys Ile Val Ile Ser Val Cys Ser Leu Val Cys Ile Ala Ala His Leu Ala Val Thr Val Ser Gly Val Ala Leu Ile Pro Leu Ile Asp Gln Asn Arg Ala Tyr Gly Asn Cys Thr Val Cys Val Ile Ala Gly Phe Ile Ala Thr Phe Ala Ala Arg Leu Thr Ile Arg Leu Ser Glu Thr Leu Met Leu Val Gly Lys Pro Ala Gln Phe Ile Phe Ala Ile Ile Ala Ser Val Ala Glu Thr Leu Ile Asn Asn Glu Ala Leu Ala Ile Ser Asn Thr Thr Tyr Lys Thr Ala Leu Arg Ile Ile Glu Val Thr Ser Leu Ala Cys Phe Val Met Leu Gly Ala Ile Ile Thr Ser His Asn Tyr Val Cys 11e Ser Thr Ala Gly Asp Leu Thr Trp Lys Gly Gly Ile Phe His Ala Tyr His Gly Thr Leu Leu Gly Ile Thr Ile Pro Asn Ile His Pro Ile Pro Leu Ala Gly Phe Leu Ala Val Tyr Thr Ile Leu Ala Ile Asn Ile Ala Arg Asp Ala Ser Ala Thr Leu Leu Ser Thr Cys Tyr Tyr Arg Asn Cys Arg Glu Arg Thr Ile Leu Arg Pro Ser Arg Leu Gly His Gly Tyr Thr Ile Pro Ser Pro Gly Ala Asp Met Leu Tyr Glu Glu Asp Val Tyr Ser Phe Asp Ala Ala Lys Gly His Tyr Ser Ser Ile Phe Leu Cys Tyr Ala Met Gly Leu Thr Thr Pro Leu Ile Ile Ala Leu His Lys Tyr Met Ala Gly Ile Lys Asn Ser Ser Asp Trp Thr Ala Thr Leu Gln Gly Met Tyr Gly Leu Val Leu Gly Ser Leu Ser Ser Leu Cys Ile Pro Ser Ser Asn Asn Asp Ala Leu Ile Arg Pro Ile Gln Ile Leu Ile Leu Ile Ile Gly Ala Leu Ala Ile Ala Leu Ala Gly Cys Gly Gin Ile Ile Gly Pro Thr Leu Phe Ala Ala Ser Ser Ala Ala Met Ser Cys Phe Thr Cys Ile Asn Ile Arg Ala Thr Asn Lys Gly Val Asn Lys Leu Ala Ala Ala Ser Val Val Lys Ser Val Leu Gly Phe Ile Ile Ser Gly Met Leu Thr Cys Val Leu Leu Pro Leu Ser (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 489 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Val Sar Asn Met Arg Val Leu Arg Val Leu Arg Leu Thr Gly Trp Val Gly Ile Phe Leu Val Leu Ser Leu Gln Gln Thr Ser Cys Ala Gly Leu Pro His Asn Val Asp Thr His His Ile Leu Thr Phe Asn Pro Ser Pro Ile Ser Ala Asp Gly Val Pro Leu Ser Glu Val Pro Asn Ser Pro Thr Thr Glu Leu Ser Thr Thr Val Ala Thr Lys Thr Ala Val Pro Thr Thr Glu Ser Thr Ser Ser Ser Glu Ala His Arg Asn Ser Ser His Lys Ile Pro Asp Ile Ile Cys Asp Arg Glu Glu Val Phe Val Phe Leu Asn Asn Thr Gly Arg Ile Leu Cys Asp Leu Ile Val Asp Pro Pro Ser Asp Asp Glu Trp Ser Asn Phe Ala Leu Asp Val Thr Phe Asn Pro Ile Glu Tyr His Ala Asn Glu Lys Asn Val Glu Val Ala Arg Val Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Lys Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Ser Phe Trp Thr Ser Pro Thr Pro Arg Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Met His Thr Met Gly Val Glu Val Arg Asn Val Asp Tyr Lys Asp Asn Gly Tyr Phe Gln Val Ile Leu Arg Asp Arg Phe Asn Arg Pro Leu Val Glu Lys His Ile Tyr Met Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Arg Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Gly Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Leu Glu Ser Pro Pro Lys Val Ser Cys Leu Val Ala Trp Arg Gln Gly Asp Met Ile Ser Thr Ser Asn Ala Thr Ala Val Pro Thr Val Tyr Tyr His Pro Arg Ile Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Gly Ile Thr Val Arg Trp Leu Val His Asp Glu Pro Gln Pro Asn Thr Thr Tyr Asp Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg Tyr Arg Asn Leu Ala Ser Arg Ile Pro Val Gln Asp Asn Trp Ala Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Val Asp Arg Phe Gln Asn Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Met Pro Met Ile Val Thr Ile Thr Ala Val Leu Gly Leu Ala Leu Phe Leu Gly Ile Gly Ile Ile Ile Thr Ala Leu Cys Phe Tyr Leu Pro Gly Arg Asn (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 amino acids (B) TYPE: amino acid (D) 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 Gly Arg Leu Arg Met Met Asp Ser Gly Thr Glu Thr Asp Arg Glu Gln Arg His Pro Arg Thr Thr Trp Arg Ser Ile Cys Cys Gly Cys Thr Ile Gly Met Val Phe Thr Ile Phe Val Leu Val Ala Ala Val Leu Leu Gly Ser Leu Phe Thr Val Ser Tyr Met Ala Met Glu Ser Gly Thr Cys Pro Asp Glu Trp Ile Gly Leu Gly Tyr Ser Cys Met Arg Val Ala Gly Lys Asn Ala Thr Asp Leu Glu Ala Leu Asp Thr Cys Ala Arg His Asn Ser Lys Leu Ile Asp Phe Ala Asn Ala Lys Val Leu Val Glu Ala Ile Ala Pro Phe Gly Val Pro Asn Ala Ala Tyr Gly Glu Val Phe Arg Leu Arg Asp Ser Lys Thr Thr Cys Ile Arg Pro Thr Met Gly Gly Pro Val Ser Ala Asp Cys Pro Val Thr Cys Thr Val Ile Cys Gln Arg Pro Arg Pro Leu Ser Thr Met Ser Ser Ile Ile Arg Asp Ala Arg Val Tyr Leu His Leu Glu Arg Arg Asp Tyr Tyr Glu Val Tyr Ala Ser Val Leu Ser Asn Ala Met Ser Lys (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1506 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1506 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Leu Thr Pro Arg Val Leu Arg Ala Leu Gly Trp Thr Gly Leu Phe Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Gly Ala Ser Leu Ser Arg Asp Leu Glu Thr Pro Pro Phe Leu Ser Phe Asp Pro Ser Asn Ile Ser Ile Asn Gly Ala Pro Leu Thr Glu Val Pro His Ala Pro Ser Thr Glu Ser Val Ser Thr Asn Ser Glu Ser Thr Asn Glu His Thr Ile Thr Glu Thr Thr Gly Lys Asn Ala Tyr Ile His Asn Asn Ala Ser Thr Asp Lys Gln Asn Ala Asn Asp Thr His Lys Thr Pro Asn Ile Leu Cys Asp Thr G1'i Glu Val Phe Val Phe Leu Asn Glu Thr Gly Arg Phe Val Cys Thr Leu Lys Val Asp Pro Pro Ser Asp Ser Glu Trp Ser Asn Phe Val Leu Asp Leu Ile Phe Asn Pro Ile Glu Tyr His Ala Asn Glu Lys Asn Val Glu Ala Ala Arg Ile Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Gln Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Thr Phe Trp Thr Ser Pro Ser Pro His Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Thr Asn Thr Met Gly Val Glu Ile Arg Asn Val Asp Tyr Ala Asp Asn Gly Tyr Met Gln Val Ile Met Arg Asp His Phe Asn Arg Pro Leu Ile Asp Lys His Ile Tyr Ile Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Gln Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Gly Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Ile Asp Phe Pro Pro Lys Ile Ser Cys Leu Val Ala Trp Lys Gln Gly Asp Met Ile Ser Thr Thr Asn Ala Thr Ala Ile Pro Thr Val Tyr His His Pro Arg Leu Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Glu Ile Thr Val Arg Trp Leu Val His Asp Glu Ala Gln Pro Asn Thr Thr Tyr Asn Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg His Arg Asn Leu Leu Ser Arg Ile Pro Val Trp Asp Asn Trp Thr Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Glu Asp Lys Phe Gln Asp Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Thr Pro Met Val Ile Thr Val Thr Ala Val Leu Gly Leu Ala Val Ile Leu Gly Met Gly Ile Ile Met Thr Ala Leu Cys Leu Tyr Asn Ser Thr Arg Lys Asn Ile Arg Leu (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 amino acids (B) TYPE: amino acid (D) 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 Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Gly Ala Ser Leu Ser Arg Asp Leu Glu Thr Pro Pro Phe Leu Ser Phe Asp Pro Ser-Asn Ile Ser Ile Asn Gly Ala Pro Leu Thr Glu Val Pro His Ala Pro Ser Thr Glu Ser Val Ser Thr Asn Ser Glu Ser Thr Asn Glu His Thr Ile Thr Glu Thr Thr Gly Lys Asn Ala Tyr Ile His Asn Asn Ala Ser Thr Asp Lys Gln Asn Ala Asn Asp Thr His Lys Thr Pro Asn Ile Leu Cys Asp Thr Glu Glu Val Phe Val Phe Leu Asn Glu Thr Gly Arg Phe Val Cys Thr .115 120 125 Leu Lys Val Asp Pro Pro Ser Asp Ser Glu Trp Ser Asn Phe Val Leu Asp Leu Ile Phe Asn Pro Ile Glu Tyr His Ala Asn Glu_Lys Asn Val Glu Ala Ala Arg Ile Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Gln Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Thr Phe Trp Thr Ser Pro Ser Pro His Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Thr Asn Thr Met Gly Val Glu Ile Arg Asn Val Asp Tyr Ala Asp Asn Gly Tyr Met Gln Val Ile Met Arg Asp His Phe Asn Arg Pro Leu Ile Asp Lys His Ile Tyr Ile Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Gln Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Gly Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Ile Asp Phe Pro Pro Lys Ile Ser Cys Leu Val Ala Trp Lys Gln Gly Asp Met Ile Ser Thr Thr Asn Ala Thr Ala Ile Pro Thr Val Tyr His His Pro Arg Leu Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Glu Ile Thr Val Arg Trp Leu Val His Asp Glu Ala Gln Pro Asn Thr Thr Tyr Asn Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg His Arg Asn Leu Leu Ser Arg Ile Pro Val Trp Asp Asn Trp Thr Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Glu Asp Lys Phe Gln Asp Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Thr Pro Met Val Ile Thr Val Thr Ala Val Leu Gly Leu Ala Val Ile Leu Gly Met Gly Ile Ile Met Thr Ala Leu Cys Leu Tyr Asn Ser Thr Arg Lys Asn Ile Arg Leu (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1734 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1734 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn Asp Glu Arg Glu Ala Lys. Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu Thr Val.Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn Gln Asp Val Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile.Thr Ser Leu Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gln Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro TCA TTT GAC ATG AGT GCT ACC CAT TAC TGC.TAC ACC CAT AAT GTA ATA 576 Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Ala Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr Arg Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr Gly Gly Leu Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Glu Gly Lys Tyr Val Ile Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe Tyr Arg Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Gly Glu Gln Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg Glu Ala Arg Ser Gly (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 577 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn Asp Glu Arg Glu Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn Gln Asp Val Ial Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser Leu Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gin Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Ala Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser.Ala Val Pro Thr Arg Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr Gly Gly Leu Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Glu Gly Lys Tyr Val Ile Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe Tyr Arg Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Gly Glu Gln .'1_a Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg Glu Ala Arg Ser Gly (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1662 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1662 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Gly Ser Arg Pr6.,Ser Thr Lys Asn Pro Ala Pro Met Met Leu Thr Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly AGA CAG GGG CGC CTT ATA GGC GCC ATT ATT GGC GGT GTG GCT CTT GGG' 384 Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala ACC AAT GAG GCT GTG CAT GAG GTC. ACT GAC GGA TTA TCG CAA CTA GCA 528 Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala GTG GCA GTT GGG AAG ATG CAG CAG TTC GTT AAT GAC CAA TTT AAT,AAA 576 Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala Gln Gln Val Gly Val Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr Val Phe Gly Pro Gln Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro Lys Val Val Thr Arg Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val 405 410 415.
Ser Leu Ile Asp Lys Gln Ser Cys Asn Val Leu Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gly Asn Leu Asp Ile Ser Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asn Lys Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Val Asn Val Lys Leu Thr Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Ile Ile Ser Leu Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys Tyr Leu Met Tyr Lys Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu Asp Gln Met Arg Ala Thr Thr Lys Met (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 553 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Ala Pro Met Met Leu Thr Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala Gln Gln Val Gly Val Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr Val Phe Gly Pro Gln Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly=
Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro Lys Val Val Thr Arg Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met ?70 375 380 Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val Lau Ile Asp Lys Gln Ser Cys Asn Val Leu Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gly Asn Leu Asp Ile Ser Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asn Lys Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Val Asn Val Lys Leu Thr Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Ile Ile Ser Leu Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys Tyr Leu Met Tyr Lys Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu Asp Gln Met Arg Ala Thr Thr Lys Met (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3489 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..3489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gin Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly Cys Ile Val Gly Thr Ile His Gly Gly Arg Val Val Asn Ala Ser Ser Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln' Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Thr Gly Met Leu Gin Lys Asn Phe Leu Arg Val Ser Ala Met. Lys Asn Gly Gln Leu Phe Tyr Asn Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Lys Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tvr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gln Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln Asn Ile Leu Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gin Ser Val Phe Ser Gly Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile 515 520 525.
Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Asn Val Ala Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gin Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn Ser Ile Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr GGT GAG TTT AAT ATT TCT CTT CTG.TTA ACA ACT CCT AGT AGT CCT AGA 2064 Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu GGT TTT. CTT AAG GAC CTT GCG TGT GCT CGT GAA TAT AAT GGT TTG CTT 2208 Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gin Gin Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala 850. 855 860 Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu His Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val ACT GAA CAA AAC=AGA CCT AAA AAG TCT GTT TAA 3489 Thr Glu Gln Asn Arg Pro Lys Lys Ser Val (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1162 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly Cys Ile Val Gly Thr Ile His Gly Gly Arg Val Val Asn Ala Ser Ser Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Thr Gly Met Leu Gln Lys Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Lys Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala Gin Asp Val Ile Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gln Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gin Asn Ile Leu Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Giu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Giy Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Asn Val Ala Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Asn Cys Lau Gln Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn Cer Iie Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu His Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn x:45 950 955 960 Ala Ser Gin Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arq Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala hsn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr Glu Gln Asn Arg Pro Lys Lys Ser Val (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1846 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear N
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1846 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Phe Ala Leu Cys Sir Ala Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser Ala Phe Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val. A.sn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser Val Ala Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser TTT TGT ACA GCT CAC TGT AAT TTT.ACT TCT TAT ATA GTG TTT GTT ACA 336 Phe Cys Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr Pro Gly His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Lys Phe Arg Ser Leu Gln Cys Val Asn Asn His Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr Ser Asn Tyr Thr Glu Asp Val Val Ala Ala Gly Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro .275 280 285 Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Ala Cys Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Trp Ser Asn Ser Leu Ser Val Ser Leu Ile Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala Thr Gln Pro Pro Val Leu Thr Gin Asn Phe Tyr Asn Asn Ile Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu Ala Glu Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gin Phe Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser GTT AAT GAA AAT.GTT ACG AAT TGC CCT TAT GTT AGT TAT GGC AAG TTT 1680 Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Arg (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 615 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Phe Ala Leu Cys Ser Ala Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser Ala Phe Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val Asn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser Val Ala Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser Phe Cys Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr Pro Gly His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Lys Phe Arg Ser Leu Gin Cys Val Asn Asn His Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr Ser Asn Tyr Thr Glu Asp Val Val Ala Ala Gly Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr Fsr Thr fly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Ala Cys Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Trp Ser Asn Ser Leu Ser Val Ser Leu Ile Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala Thr Gln Pro Pro Val Leu Thr Gln Asn Phe Tyr Asn Asn Ile Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu 450 ~.* 455 460 Ala Glu Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu Cys Glu Asp Val Asn Gln Gin Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Arg (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2116 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear.
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNN[NNNNN 420 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 705 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Tyr Asn Tyr Leu Ala Asp Ala Gly Met Ala Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Ala Gln Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Ala Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr Arg Arg Ser Arg Arg Ser Ile Thr Ala Asn Val Thr Asn Xaa Pro Tyr Val Ser Tyr Gly Lys Phe Cys Leu Lys Pro Asp Gly Ser Xaa Ser Xaa Ile Ala Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Cys Gly Asn Ser Leu Asp Cys Arg Lys Leu Xaa Gln Gln Tyr Gly Pro Val Xaa Asp Asn Ile Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Leu Tyr Ser Ser Thr Lys Pro Ser Gly Phe Asn Thr Pro Val Phe Ser Asn Leu Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Val Asp Thr Ser Ser Ser Thr Thr Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Xaa Xaa Xaa Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Gln Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gln Gln Val Xaa Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Xaa Gly Ala Ile Ser Ser Val Ile Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala Gin Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn_Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu Ser Phe Xaa Asn Val Thr Ala Ile Val Gly Phe Cys Lys Ala Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Ser Ile Gin Val Asn Gly Ser His Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Xaa Lys Thr Val Ile Thr Thr Xaa Val Asp Asn Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Gly Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Xaa Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Leu Asp Asn Asp Val Val Thr Glu Gln Xaa Arg Pro Lys (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gln Arg Arg (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) 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 Gln Arg Arg (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
(2) INF071MATION FOR SEQ ID NO : 24 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
tiv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 16..66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gln Arg Arg Asp Trp (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gln Arg Arg Asp Trp (2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys Asp Leu Glu (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) 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 Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys Asp Leu Glu (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE'DESCRIPTION: SEQ ID NO:33:
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65.base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAA NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:.
(A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
96/05291 PCT/US95/10245 ,,:., (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE. NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE : DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
(2) !NFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2681 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 146..481 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: complement (602..1402) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1599..2135 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: complement (2308..2634) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
CTGCATTTTC TGCTGCCGAA CAAACTTCAT CGCTATGCAA AGAGATGCGT GTGTACACGC '2100 (2) INFORMATION FOR SEQ ID NO:49:
.(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 111 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) 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 Glu Asp Met Leu Arg Arg Ser Ser Pro Glu Asp Ile Thr Asp Ser Leu Thr Met Cys Leu Ile Met Leu Ser Arg Ile Arg Arg Thr Met Arg Thr Ala Gly Asn Lys Tyr Ser Tyr Met Ile Asp Pro Met Asn Arg Met Ser Asn Tyr Thr Pro Gly Glu Cys Met Thr Gly Ile Leu Arg Tyr Ile Asp Glu His Ala Arg Arg Cys Pro Asp His Ile Cys Asn Leu Tyr Ile Thr Cys Thr Leu Met Pro Met Tyr Val His Gly Arg Tyr Phe Tyr Cys Asn Ser Phe Phe Xaa (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Mat His Phe Pro Phe Asn Lys Lys Cys Arg Glu Asn Thr Gln Pro Leu Trp Met Tyr Pro Arg Glu Leu Pro Arg Arg Gly Ala Tyr Gly Val Ala Ala Arg Pro Ile Leu Ile Thr Asp Met Gly Pro Thr Ile Asn Ile Leu Leu Val Gly Gly Trp Lys Gly Arg Leu Val Thr Ala Lys Ser Ser Val Gln His Arg Ala Val Leu Trp Ile Ser Gly Gly Gln Arg Glu Asn Ala Ser Gln Tyr Ser Phe Asn Arg Glu Asp Ala Asn Thr His Gly Trp Phe Leu Thr Leu Gly Ala Pro Trp Ile Val Gly Arg Asn Ile Leu Pro Glu Asp Arg Leu Leu Phe Ile Thr Asn His Cys Ser Gly Glu Leu Gln Ser Val Phe Ala Gly Met Pro Val Ala Leu Lys Thr Val Leu Glu His Leu His Leu Lys Arg Glu Ser Gly Leu Gln Pro Ala Leu Leu Met Asp Pro Arg Arg Phe Leu Glu Met Arg Asp Pro Arg Lys Ile Ile Cys Met Cys Glu Leu Asp Ile Arg Asp Ser Pro Gly Ala His Gly Ile Leu Val Asn Cys Cys Cys Gly Arg Pro Gly Gly Leu Gin Cys Leu Thr Phe Ile Arg lie Ile Glu Thr Leu Val Asn His Phe Leu Ser Ser Thr Asp Leu Thr Cys Leu Asn Leu Thr Cys Arg His Ser Asp Val Pro Thr Thr Val Ala Ser Thr Leu Ser Glu Asp Gly Phe Glu His Asp Xaa Glu Tyr Ser Ile Ser Thr Ile Ser Glu Glu Ala Glu Val Tyr (2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) 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 Arg Val Gln Asn Val Trp His Val Val Asp Pro Leu Pro Ile Val Ala Asn Tyr Lys Ser Asp Arg His Asp Arg Asp Lys Pro Val Pro Cys Gly Ile Arg Val Thr Ser Arg Ser Tyr Ile Ile Arg Pro Thr Cys Glu Ser Ser Lys Asp Leu His Ala Phe Phe Phe Gly Leu Ser Thr Gly Ser Ser Glu Ser Met Pro Ile Ser Gly Val Glu Thr Ile Val Arg Leu Met Asn Asn Lys Ala Ala Cys His Gly Lys Glu Gly Cys Arg Ser Pro Phe Ser His Ala Thr Ile Leu Asp Ala Val Asp Asp Asn Tyr Thr Met Asn Ile Glu Gly Val Cys Phe His Cys His Cys Asp Asp Lys Phe Ser Pro Asp Cys Trp Ile Ser Ala Phe Ser Ala Ala Glu Gln Thr Ser Ser Leu Cys Lys Glu Met Arg Val Tyr Thr Arg Arg Trp Ser Ile Arg Glu Thr Lys Cys Ser (2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Met Gly Leu Tyr Met Glu Pro Xaa Asn Xaa Val Ser Leu Gln Phe Leu Arg Gly Gly Ile Tyr Met Ile Ser Xaa Pro Lys Arg Gly Met Xaa Gln Arg Asp Val Met Val Ile Xaa Gly Lys Phe Phe Arg Ser Glu Ile Thr Gln Leu Pro Lys His Arg Ser Arg Leu Lys Glu Lys Ser Asp Gly Ser Ile Arg Thr Cys Met Asp Ser Val Arg Ile Asn His Asn Arg Ser Thr Vai Gly His Phe Gly Asn Ser Asn Ala Lys Arg Cys Thr Ser Ala Ile Thr Thr Pro Thr Met His Ile Val Thr Pro Ala Ser (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Oligonucleotide Primer (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Oligonucleotide Primer (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE;. nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
ix/; ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
40. L.E. Post and B. Roizman, Cell 25, 227-232, 1981.
41. K.L. Poffenberger et al., Proc. Natl. Acad. Sci.
USA 80, 2690-2694, 1981.
42. M.G. Gibson and P.G. Spear, Journal of Virology 48, 396-404, 1983.
43. G.T.-Y. Lee et al., Proc. Natl. Acad. Sci. USA
79, 6612-6616, 1982.
44. M.-F. Shih et al., Proc. Natl. 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 Technology Route to Virus Vaccines", American Society for Microbiology, Washington D.C., 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.
48. S.B. Mohanty and S.K. Dutta, Veterinary Virology, Lea and Febiger, pubs., Philadelphia, 1981.
49. A.M. Griffin, Journal of General Virology 72, 393-398, 1991.
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).
52. Kibenge, F.S., Jackwood, D.J., Mercado, C.C., Journal of General Virology 71 569-577 (1990).
53. Fukuchi et al., J. Virologu 51 102-109, 1984.
54. Fukuchi et al., J. Virologu 53 994-997, 1985.
55. Ross, N., et al., Virus Genes 7 33-51, 1993.
56. Maotani, K.A., et al., J. Virology 58: 657-659, 1986.
57. Ross, L.J.N., et al., J. General Virology 64:2785-2790, 1983.
58. A. Leutz, et al., EMBO Journal 8: 175-182 (1989).
59. M.J. Sekellick, et al., Journal of Interferon Research 14: 71-79 (1994).
60. G.L. Smith, Journal of General Virology 74, 1725-1740 (1993).
61. B. Scgumacher, et al., Virology 203, 144-148 (1994) SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: SYNTRO CORPORATION
(ii) TITLE OF INVENTION: Recombinant Herpesvirus of Turkeys And Uses Thereof (iii) NUMBER OF SEQUENCES: 60 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: John P. White (B) STREET: 1185 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: USA
(F) ZIP: 10036 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 09-AUG-1995 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: White, John P
(B) REGISTRATION NUMBER: 28,678 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212)278-0400 (B) TELEFAX: (212)391-0526 (C) TELEX: 422523 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3350 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 129..2522 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg Ser Leu Lei; Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Glu ACA CCC TGG AGA AGC ACA CTC TCA GGT CAG AGA CTG ACC TAC AAT .TTG 266 Thr Pro Trp Arg Ser Thr Leu Ser Gly Gln Arg Leu Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr Lys Phe Asp Arg Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Pro Gly Gly Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu Ser Val Gly Gly Glu Leu Val Phe Arg Thr Ser Val His Gly Leu Val Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Thr Val Ile Thr Arg Ala Val Ala Ala Asn Thr Gly Leu Thr Thr Gly Thr Asp Asn Leu Met Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gln Pro Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln Ala Gly Asp Gln Met Leu Trp Ser Ala Arg Gly Ser Leu Ala Val Thr Ile His Gly Gly Asn Tyr Pro Giy Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile Arg Ala Ile Arg Arg Ile Ala Val Pro Val Val Ser Thr Leu Phe Pro Pro Ala Ala Pro Leu Ala His Ala Ile Gly Glu Gly Val Asp Tyr Leu Leu Gly Asp Glu Ala Gln Ala Ala Ser Gly Thr Ala Arg Ala Ala Ser Gly Lys Ala Arg Ala Ala Ser Gly Arg Ile Arg Gln Leu Thr Leu Ala Ala Asp Lys Gly Tyr Glu Val Val Ala Asn Leu Phe Gln Val Pro Gln Asn Pro Val Val Asp Gly Ile Leu Ala Ser Pro Gly Val Leu Arg Gly Ala His Asn Leu Asp Cys Val Leu Arg Glu Gly Ala Thr Leu Phe Pro Val Val Ile Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu AAC AGC AAA ATG TTT GCT GTC ATT GAA GGC GTG"CGA GAA GAC CTC CAA 1898 Asn Ser Lys Met Phe Ala Val Ile Glu Gly Val Arg Glu Asp Leu Gln Pro Pro Ser Gln Arg Gly Ser Phe Ile Arg Thr Leu Ser Gly His Arg Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg Asp Tyr Thr Val Val Pro Ile Asp Asp Val Trp Asp Asp Ser Ile Met Leu Ser Lys Asp Pro Ile Pro Pro Ile Val Gly Asn Ser Gly Asn Leu Ala Ile Ala Tyr Met Asp Val Phe Arg Pro Lys Val Pro Ile His Val Ala Met Thr Gly Ala Leu Asn Ala Cys Gly Glu Ile Glu Lys Val Ser Phe Arg Ser Thr Lys Leu Ala Thr Ala His Arg Leu Gly Leu Lys Leu Ala Gly Pro Gly Ala Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe Ile Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Asn Ala Gly Arg Gln Tyr His Leu Ala Met Ala Ala Ser Glu Phe Lys Arg Pro Arg Thr Arg Glu Cys Arg Gln Ser Asn Gly Ser Ser Ser Gln Arg Gly Pro Thr Ile Pro Ile Cys Thr Gln Cys Val His Val Ala Gly Arg Glu Trp Asp Cys Asp (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 797 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg Ser=Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Glu Thr Pro Trp Arg Ser Thr Leu Ser Gly G1n-Arg Leu Thr Tyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr Lys Phe Asp Arg Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Pro Gly Gly Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu Ser Val Gly Gly Glu Leu Val Phe Arg Thr Ser Val His Gly Leu Val Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Thr Val Ile Thr Arg Ala Val Ala Ala Asn Thr Gly Leu Thr Thr Gly Thr Asp Asn Leu MMe~ Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gln Pro Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln Ala Gly Asp Gln Met Leu Trp Ser Ala Arg Gly Ser Leu Ala Val Thr Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly'Ala Met Asn Tyr Thr Lys Leu Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile Arg Ala Ile Arg Arg Ile Ala Val Pro Val Val Ser Thr Leu Phe Pro Pro Ala Ala Pro Leu Ala His Ala Ile Gly Glu Gly Val Asp Tyr Leu Leu Giy Asp Glu Ala Gln Ala Ala Ser Gly Thr Ala Arg Ala Ala Ser Gly Lys Ala Arg Ala Ala Ser Gly Arg Ile Arg Gln Leu Thr Leu Ala Ala Asp Lys Gly Tyr Glu Val Val Ala Asn Leu Phe Gln Val Pro Gln Asn Pro Val Val Asp Gly Ile Leu Ala Ser Pro Gly Val Leu Arg Gly Ala His Asn Leu Asp Cys Val Leu Arg Glu Gly Ala Thr Leu Phe Pro Val Val Ile Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu Asn Ser Lys Met Phe Ala Val Ile Glu Gly Val Arg Glu Asp Leu Gln Pro Pro Ser Gln Arg Gly Ser Phe Ile Arg Thr Leu Ser Gly His Arg Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Gly Arg Asp Tyr Thr Val Val Pro Ile Asp Asp Val Trp Asp Asp Ser Ile Met Leu Ser Lys Asp Pro Ile Pro Pro Ile Val Gly Asn Ser Gly Asn Leu Ala Ile Ala Tyr Met Asp Val Phe Arg Pro Lys Val Pro Ile His Val Ala Met Thr Gly Ala Leu Asn Ala Cys Gly Glu Ile Glu Lys Val Ser Phe Arg Ser Thr Lys Leu Ala Thr Ala His Arg Leu Gly Leu Lys Leu Ala Gly Pro Gly Ala Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe Ile Lys Arg Phe Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Asn Ala Gly Arg Gln Tyr His Leu Ala Met Ala Ala Ser Glu Phe Lys Arg Pro Arg Thr Arg Glu Cys Arg Gln Ser Asn Gly Ser Ser Ser Gln Arg Gly Pro Thr Ile Pro Ile Cys Thr Gln Cys Val His Val Ala Gly Arg Glu Trp Asp Cys Asp (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5426 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 73..1182 (D) OTHE{. INFORMATION: /product= "HVT UL42"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1306..2574 (D) OTHER INFORMATION: /product= "HVT UL43"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2790..4259 (D) OTHER INFORMATION: /product= "HVT gA"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4701..5339 (D) OTHER INFORMATION: /product= "HVT UL45"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Met Ala Gly Ile Thr Val Ala Cys Asp His Thr Ala Gly Glu Ala His Thr Pro Glu Asp Met Gln Lys Lys Trp Arg Ile Ile Leu Ala Gly Glu Lys Phe Met Thr Ile Ser Ala Ser Leu Lys Ser Ile Val Ser Cys Val Lys Asn Pro Leu Leu Thr Phe Gly Ala Asp Gly Leu Ile Val Gln Gly Thr Val Cys Gly Gln Arg Ile Phe Val Pro Ile Asp Arg Asp Ser Phe Ser Glu Tyr Glu Trp His Gly Pro Thr Ala Met Phe Leu Ala Leu Thr Asp Ser Arg Arg Thr Leu Leu Asp Ala Phe Lys Cys Glu Lys Arg Arg Ala Ile Asp Val Ser Phe Thr Phe Ala Gly Glu Pro Pro Cys Arg His Leu Ile Gln Ala Val Thr Tyr Met Thr Asp Gly Gly Ser Val Ser Asn Thr Ile Ile Lys Tyr Glu Leu Trp Asn Ala Ser Thr Ile Phe Pro Gln Lys Thr Pro Asp Val Thr Phe Ser Leu Asn Lys Gin Gin Leu Asn Lys Ile Leu Ala Val Ala Ser Lys Leu Gln His Glu Glu Leu Val Phe Ser Leu Lys Pro Glu Gly Gly Phe Tyr Val Gly Thr Val Cys Thr Val Ile Ser Phe Glu Val Asp Gly Thr Ala Met Thr Gln Tyr Pro Tyr Asn Pro Pro Thr Ser Ala Thr Leu Ala Leu Val Val Ala Cys Arg Lys Lys Lys Ala Asn Lys Asn Thr Ile Leu Thr Ala Tyr Gly Ser Gly Lys Pro Phe Cys Val Ala Leu Glu Asp Thr Ser Ala Phe Arg Asn Ile Val Asn Lys Ile Lys Ala Gly Thr Ser Gly Val Asp Leu Gly Phe Tyr Thr Thr Cys Asp Pro Pro Met Leu Cys Ile Arg Pro His Ala Phe Gly Ser Pro Thr Ala Phe Leu Phe Cys Asn Thr Asp Cys Met Thr Ile Tyr Glu Leu Glu Glu Val Ser Ala Val Asp Gly Ala Ile Arg Ala Lys Arg Ile Asn Glu Tyr Phe Pro Thr Val Ser Gln Ala Thr Ser Lys Lys Arg Lys Gln Ser Pro Pro Pro Ile Glu Arg Glu Arg Lys Thr Thr Arg Ala Asp Thr Gln Met Pro Ser Gly Ala Ser Ser Ser Pro Pro Pro Ala Tyr Thr Ser Ala Ala Pro Leu Glu Thr Tyr Asn Ser Trp Leu Ser Ala Phe Ser Cys Ala Tyr Pro Gln Cys Thr Ala Gly Arg Gly His Arg Gln Asn Gly Lys Lys Cys Ile Arg Cys Ile Val Ile Ser Val Cys Ser Leu Val Cys Ile Ala Ala His Leu Ala Val Thr Val Ser Gly Val Ala Leu Ile Pro Leu Ile Asp Gln Asn Arg Ala Tyr Gly Asn Cys Thr Val Cys Val Ile Ala Gly Phe Ile Ala Thr Phe Ala Ala Arg Leu Thr Ile Arg Leu Ser Glu Thr Leu Met Leu Val Gly Lys Pro Ala Gln Phe Ile Phe Ala Ile Ile Ala Ser Val Ala Glu Thr Leu Ile Asn Asn Glu Ala Leu Ala Ile Ser Asn Thr Thr Tyr Lys Thr Ala Leu Arg Ile Ile Glu Val Thr Ser Leu Ala Cys Phe Val Met Leu Gly Ala Ile Ile Thr Ser His Asn Tyr Val Cys Ile Ser Thr Ala Gly Asp Leu Thr Trp Lys Gly Gly Ile Phe His Ala Tyr His Gly Thr Leu Leu Gly Ile Thr ATA CCA AAC ATA CAC CCA ATC CCT.CTC GCG GGG TTT CTT GCA GTC TAT 1956 Ile Pro Asn Ile His Pro Ile Pro Leu Ala Gly Phe Leu Ala Val Tyr Thr Ile Leu Ala Ile Asn Ile Ala Arg Asp Ala Ser Ala Thr Leu Leu Ser Thr Cys Tyr Tyr Arg Asn Cys Arg Glu Arg Thr Ile Leu Arg Pro Ser Arg Leu Gly His Gly Tyr Thr Ile Pro Ser Pro Gly Ala Asp Met Leu Tyr Glu Glu Asp Val Tyr Ser Phe Asp Ala Ala Lys Gly His Tyr Ser Ser Ile Phe Leu Cys Tyr Ala Met Gly Leu Thr Thr Pro Leu Ile Ile Ala Leu His Lys Tyr Met Ala Gly Ile Lys Asn Ser Ser Asp Trp Thr Ala Thr Leu Gln Gly Met Tyr Gly Leu Val Leu Gly Ser Leu Ser Ser Leu Cys Ile Pro Ser Ser Asn Asn Asp Ala Leu Ile Arg Pro Ile Gln Ile Leu Ile Leu Ile Ile Gly Ala Leu Ala Ile Ala Leu Ala Gly Cys Gly Gln Ile Ile Gly Pro Thr Leu Phe Ala Ala Ser Ser Ala Ala Met Ser Cys Phe Thr Cys Ile Asn Ile Arg Ala Thr Asn Lys Gly Val Asn Lys Leu Ala Ala Ala Ser Val Val Lys Ser Val Leu Gly Phe Ile Ile Ser Gly Met Leu Thr Cys Val Leu Leu Pro Leu Ser Met Val Ser Asn Met Arg Val Leu Arg Val Leu Arg Leu Thr Gly Trp Val Gly Ile Phe Leu Val Leu Ser Leu Gln Gln Thr Ser Cys Ala Gly Leu Pro His Asn Val Asp Thr His His Ile Leu Thr Phe Asn Pro Ser Pro Ile Ser Ala Asp Gly Val Pro Leu Ser Glu Val Pro Asn Ser Pro Thr Thr Glu Leu Ser Thr Thr Val Ala Thr Lvs Thr Ala Val Pro Thr Thr Glu Ser Thr Ser Ser Ser Glu Ala His Arg Asn Ser Ser His Lys Ile Pro Asp Ile Ile Cys Asp Arg Glu Glu Val Phe Val Phe Leu Asn Asn Thr Gly Arg Ile Leu Cys Asp Leu Ile Val Asp Pro Pro Ser Asp Asp Glu Trp Ser Asn Phe Ala Leu Asp Val Thr Phe Asn Pro Ile Glu Tyr His Ala Asn Glu Lys Asn Val Glu Val Ala Arg Val Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Lys Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Ser Phe Trp Thr Ser Pro Thr Pro Arg Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Met His Thr Met Gly Val Glu Val Arg Asn Val Asp Tyr Lys Asp Asn Gly Tyr Phe Gln Val Ile Leu Arg Asp Arg Phe Asn Arg Pro Leu Val Glu Lys His Ile Tyr Met Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Arg Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Giy Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Leu Glu Ser Pro Pro Lys Val Ser Cys Leu Val Ala Trp Arg Gln Gly Asp Met Ile Ser Thr Ser Asn Ala Thr Ala Val Pro Thr Val Tyr Tyr His Pro Arg Ile Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Gly Ile Thr Val Arg Trp Leu Val His Asp Glu Pro Gln Pro Asn Thr Thr Tyr Asp Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg Tyr Arg Asn Leu Ala Ser Arg Ile Pro Val Gln Asp Asn Trp Ala Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Val Asp Arg Phe Gln Asn Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Met .Pro Met Ile Val Thr Ile Thr Ala Val Leu Gly Leu Ala Leu Phe Leu Gly Ile Gly Ile Ile Ile Thr Ala Leu Cys Phe Tyr Leu Pro Gly Arg Asn Met Met Ser Pro Thr Pro Glu Asp Asp Arg Asp Leu Val Val Val Arg Gly Arg Leu Arg Met Met Asp Ser Gly Thr Glu Thr Asp Arg Glu Gln Arg His Pro Arg Thr Thr Trp Arg Ser Ile Cys Cys Gly Cys Thr Ile Gly Met Val Phe Thr Ile Phe Val Leu Val Ala Ala Val Leu Leu Gly Ser Leu Phe Thr Val Ser Tyr Met Ala Met Glu Ser Giy Thr Cys Pro Asp Glu Trp Ile Gly Leu Gly Tyr Ser Cys Met Arg Val Ala Gly Lys Asn Ala Thr Asp Leu Glu Ala Leu Asp Thr'gys Ala Arg His Asn Ser Lys Leu Ile Asp Phe Ala Asn Ala Lys Val Leu Val Glu Ala Ile Ala Pro Phe Gly Val Pro Asn Ala Ala Tyr Gly Glu Val Phe Arg Leu Arg Asp Ser Lys Thr Thr Cys Ile Arg Pro Thr Met Gly Gly Pro Val Ser Ala Asp Cys Pro Val Thr Cys Thr Val Ile Cys Gln Arg Pro Arg Pro Leu Ser Thr Met Ser Ser Ile Ile Arg Asp Ala Arg Val Tyr Leu His Leu Glu Arg Arg Asp Tyr Tyr Glu Val Tyr Ala Ser Val Leu Ser Asn Ala Met Ser Lys (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 369 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Met Ala Gly Ile Thr Val Ala Cys Asp His Thr Ala Gly Glu Ala His Thr Pro Glu Asp Met Gln Lys Lys Trp Arg Ile Ile Leu Ala Gly Glu Lys Phe Met Thr Ile Ser Ala Ser Leu Lys Ser Ile Val Ser Cys Val Lys Asn Pro Leu Leu Thr Phe Gly Ala Asp Gly Leu Ile Val Gln Gly Thr Val Cys Gly Gln Arg Ile Phe Val Pro Ile Asp Arg Asp Ser Phe Ser Glu Tyr Glu Trp His Gly Pro Thr Ala Met Phe Leu Ala Leu Thr Asp Ser Arg Arg Thr Leu Leu Asp Ala Phe Lys Cys Glu Lys Arg Arg Ala Ile Asp Val Ser Phe Thr Phe Ala Gly Glu Pro Pro Cys Arg His Leu Ile Gln Ala Val Thr Tyr Met Thr Asp Gly Gly Ser Val Ser Asn Thr Ile Ile Lys Tyr Glu Leu Trp Asn Ala Ser Thr Ile Phe Pro Gin Lys Thr Pro Asp Val Thr Phe Ser Leu Asn Lys Gln Gln Leu Asn Lys Ile Leu Ala Val Ala Ser Lys Leu Gln His Glu Glu Leu Val Phe Ser Leu Lys Pro Glu Gly Gly Phe Tyr Val Gly Thr Val Cys Thr Val Ile Ser Phe Glu Val Asp Gly Thr Ala Met Thr Gln Tyr Pro Tyr Asn Pro Pro lhr Ser Ala Thr Leu Ala Leu Val Val Ala Cys Arg Lys Lys Lys Ala Asn Lys Asn Thr Ile Leu Thr Ala Tyr Gly Ser Gly Lys Pro Phe Cys Val Ala Leu Glu Asp Thr Ser Ala Phe Arg Asn Ile Val Asn Lys Ile Lys Ala Gly Thr Ser Gly Val Asp Leu Gly Phe Tyr Thr Thr Cys Asp Pro Pro Met Leu Cys Ile Arg Pro His Ala Phe Gly Ser Pro Thr Ala Phe Leu Phe Cys Asn Thr Asp Cys Met Thr Ile Tyr Glu Leu Glu Glu Val Ser Ala Val Asp Gly Ala Ile Arg Ala Lys Arg Ile Asn Glu Tyr Phe Pro Thr Val Ser Gln Ala Thr Ser Lys Lys Arg Lys Gln Ser Pro Pro Pro Ile Glu Arg Glu Arg Lys Thr Thr Arg Ala Asp Thr Gln (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 422 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S:
Met Pro Ser Gly Ala Ser Ser Ser Pro Pro Pro Ala Tyr Thr Ser Ala Ala Pro Leu Glu Thr Tyr Asn Ser Trp Leu Ser Ala Phe Ser Cys Ala Tyr Pro Gln Cys Thr Ala Gly Arg Gly His Arg Gln Asn Gly Lys Lys 35 ti 40 45 Cys Ile Arg Cys Ile Val Ile Ser Val Cys Ser Leu Val Cys Ile Ala Ala His Leu Ala Val Thr Val Ser Gly Val Ala Leu Ile Pro Leu Ile Asp Gln Asn Arg Ala Tyr Gly Asn Cys Thr Val Cys Val Ile Ala Gly Phe Ile Ala Thr Phe Ala Ala Arg Leu Thr Ile Arg Leu Ser Glu Thr Leu Met Leu Val Gly Lys Pro Ala Gln Phe Ile Phe Ala Ile Ile Ala Ser Val Ala Glu Thr Leu Ile Asn Asn Glu Ala Leu Ala Ile Ser Asn Thr Thr Tyr Lys Thr Ala Leu Arg Ile Ile Glu Val Thr Ser Leu Ala Cys Phe Val Met Leu Gly Ala Ile Ile Thr Ser His Asn Tyr Val Cys 11e Ser Thr Ala Gly Asp Leu Thr Trp Lys Gly Gly Ile Phe His Ala Tyr His Gly Thr Leu Leu Gly Ile Thr Ile Pro Asn Ile His Pro Ile Pro Leu Ala Gly Phe Leu Ala Val Tyr Thr Ile Leu Ala Ile Asn Ile Ala Arg Asp Ala Ser Ala Thr Leu Leu Ser Thr Cys Tyr Tyr Arg Asn Cys Arg Glu Arg Thr Ile Leu Arg Pro Ser Arg Leu Gly His Gly Tyr Thr Ile Pro Ser Pro Gly Ala Asp Met Leu Tyr Glu Glu Asp Val Tyr Ser Phe Asp Ala Ala Lys Gly His Tyr Ser Ser Ile Phe Leu Cys Tyr Ala Met Gly Leu Thr Thr Pro Leu Ile Ile Ala Leu His Lys Tyr Met Ala Gly Ile Lys Asn Ser Ser Asp Trp Thr Ala Thr Leu Gln Gly Met Tyr Gly Leu Val Leu Gly Ser Leu Ser Ser Leu Cys Ile Pro Ser Ser Asn Asn Asp Ala Leu Ile Arg Pro Ile Gln Ile Leu Ile Leu Ile Ile Gly Ala Leu Ala Ile Ala Leu Ala Gly Cys Gly Gin Ile Ile Gly Pro Thr Leu Phe Ala Ala Ser Ser Ala Ala Met Ser Cys Phe Thr Cys Ile Asn Ile Arg Ala Thr Asn Lys Gly Val Asn Lys Leu Ala Ala Ala Ser Val Val Lys Ser Val Leu Gly Phe Ile Ile Ser Gly Met Leu Thr Cys Val Leu Leu Pro Leu Ser (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 489 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Val Sar Asn Met Arg Val Leu Arg Val Leu Arg Leu Thr Gly Trp Val Gly Ile Phe Leu Val Leu Ser Leu Gln Gln Thr Ser Cys Ala Gly Leu Pro His Asn Val Asp Thr His His Ile Leu Thr Phe Asn Pro Ser Pro Ile Ser Ala Asp Gly Val Pro Leu Ser Glu Val Pro Asn Ser Pro Thr Thr Glu Leu Ser Thr Thr Val Ala Thr Lys Thr Ala Val Pro Thr Thr Glu Ser Thr Ser Ser Ser Glu Ala His Arg Asn Ser Ser His Lys Ile Pro Asp Ile Ile Cys Asp Arg Glu Glu Val Phe Val Phe Leu Asn Asn Thr Gly Arg Ile Leu Cys Asp Leu Ile Val Asp Pro Pro Ser Asp Asp Glu Trp Ser Asn Phe Ala Leu Asp Val Thr Phe Asn Pro Ile Glu Tyr His Ala Asn Glu Lys Asn Val Glu Val Ala Arg Val Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Lys Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Ser Phe Trp Thr Ser Pro Thr Pro Arg Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Met His Thr Met Gly Val Glu Val Arg Asn Val Asp Tyr Lys Asp Asn Gly Tyr Phe Gln Val Ile Leu Arg Asp Arg Phe Asn Arg Pro Leu Val Glu Lys His Ile Tyr Met Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Arg Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Gly Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Leu Glu Ser Pro Pro Lys Val Ser Cys Leu Val Ala Trp Arg Gln Gly Asp Met Ile Ser Thr Ser Asn Ala Thr Ala Val Pro Thr Val Tyr Tyr His Pro Arg Ile Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Gly Ile Thr Val Arg Trp Leu Val His Asp Glu Pro Gln Pro Asn Thr Thr Tyr Asp Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg Tyr Arg Asn Leu Ala Ser Arg Ile Pro Val Gln Asp Asn Trp Ala Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Val Asp Arg Phe Gln Asn Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Met Pro Met Ile Val Thr Ile Thr Ala Val Leu Gly Leu Ala Leu Phe Leu Gly Ile Gly Ile Ile Ile Thr Ala Leu Cys Phe Tyr Leu Pro Gly Arg Asn (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 amino acids (B) TYPE: amino acid (D) 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 Gly Arg Leu Arg Met Met Asp Ser Gly Thr Glu Thr Asp Arg Glu Gln Arg His Pro Arg Thr Thr Trp Arg Ser Ile Cys Cys Gly Cys Thr Ile Gly Met Val Phe Thr Ile Phe Val Leu Val Ala Ala Val Leu Leu Gly Ser Leu Phe Thr Val Ser Tyr Met Ala Met Glu Ser Gly Thr Cys Pro Asp Glu Trp Ile Gly Leu Gly Tyr Ser Cys Met Arg Val Ala Gly Lys Asn Ala Thr Asp Leu Glu Ala Leu Asp Thr Cys Ala Arg His Asn Ser Lys Leu Ile Asp Phe Ala Asn Ala Lys Val Leu Val Glu Ala Ile Ala Pro Phe Gly Val Pro Asn Ala Ala Tyr Gly Glu Val Phe Arg Leu Arg Asp Ser Lys Thr Thr Cys Ile Arg Pro Thr Met Gly Gly Pro Val Ser Ala Asp Cys Pro Val Thr Cys Thr Val Ile Cys Gln Arg Pro Arg Pro Leu Ser Thr Met Ser Ser Ile Ile Arg Asp Ala Arg Val Tyr Leu His Leu Glu Arg Arg Asp Tyr Tyr Glu Val Tyr Ala Ser Val Leu Ser Asn Ala Met Ser Lys (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1506 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1506 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Leu Thr Pro Arg Val Leu Arg Ala Leu Gly Trp Thr Gly Leu Phe Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Gly Ala Ser Leu Ser Arg Asp Leu Glu Thr Pro Pro Phe Leu Ser Phe Asp Pro Ser Asn Ile Ser Ile Asn Gly Ala Pro Leu Thr Glu Val Pro His Ala Pro Ser Thr Glu Ser Val Ser Thr Asn Ser Glu Ser Thr Asn Glu His Thr Ile Thr Glu Thr Thr Gly Lys Asn Ala Tyr Ile His Asn Asn Ala Ser Thr Asp Lys Gln Asn Ala Asn Asp Thr His Lys Thr Pro Asn Ile Leu Cys Asp Thr G1'i Glu Val Phe Val Phe Leu Asn Glu Thr Gly Arg Phe Val Cys Thr Leu Lys Val Asp Pro Pro Ser Asp Ser Glu Trp Ser Asn Phe Val Leu Asp Leu Ile Phe Asn Pro Ile Glu Tyr His Ala Asn Glu Lys Asn Val Glu Ala Ala Arg Ile Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Gln Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Thr Phe Trp Thr Ser Pro Ser Pro His Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Thr Asn Thr Met Gly Val Glu Ile Arg Asn Val Asp Tyr Ala Asp Asn Gly Tyr Met Gln Val Ile Met Arg Asp His Phe Asn Arg Pro Leu Ile Asp Lys His Ile Tyr Ile Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Gln Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Gly Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Ile Asp Phe Pro Pro Lys Ile Ser Cys Leu Val Ala Trp Lys Gln Gly Asp Met Ile Ser Thr Thr Asn Ala Thr Ala Ile Pro Thr Val Tyr His His Pro Arg Leu Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Glu Ile Thr Val Arg Trp Leu Val His Asp Glu Ala Gln Pro Asn Thr Thr Tyr Asn Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg His Arg Asn Leu Leu Ser Arg Ile Pro Val Trp Asp Asn Trp Thr Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Glu Asp Lys Phe Gln Asp Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Thr Pro Met Val Ile Thr Val Thr Ala Val Leu Gly Leu Ala Val Ile Leu Gly Met Gly Ile Ile Met Thr Ala Leu Cys Leu Tyr Asn Ser Thr Arg Lys Asn Ile Arg Leu (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 amino acids (B) TYPE: amino acid (D) 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 Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Gly Ala Ser Leu Ser Arg Asp Leu Glu Thr Pro Pro Phe Leu Ser Phe Asp Pro Ser-Asn Ile Ser Ile Asn Gly Ala Pro Leu Thr Glu Val Pro His Ala Pro Ser Thr Glu Ser Val Ser Thr Asn Ser Glu Ser Thr Asn Glu His Thr Ile Thr Glu Thr Thr Gly Lys Asn Ala Tyr Ile His Asn Asn Ala Ser Thr Asp Lys Gln Asn Ala Asn Asp Thr His Lys Thr Pro Asn Ile Leu Cys Asp Thr Glu Glu Val Phe Val Phe Leu Asn Glu Thr Gly Arg Phe Val Cys Thr .115 120 125 Leu Lys Val Asp Pro Pro Ser Asp Ser Glu Trp Ser Asn Phe Val Leu Asp Leu Ile Phe Asn Pro Ile Glu Tyr His Ala Asn Glu_Lys Asn Val Glu Ala Ala Arg Ile Ala Gly Leu Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pro Arg Gln Ser Glu Leu Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Thr Phe Trp Thr Ser Pro Ser Pro His Gly Asn Lys Tyr Phe Ile Trp Ile Asn Lys Thr Thr Asn Thr Met Gly Val Glu Ile Arg Asn Val Asp Tyr Ala Asp Asn Gly Tyr Met Gln Val Ile Met Arg Asp His Phe Asn Arg Pro Leu Ile Asp Lys His Ile Tyr Ile Arg Val Cys Gln Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Lys Ala Ser Cys Ile Val Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Val Ser Trp Arg Gln Asn Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Ser Gly Arg Gly Ala Thr Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gly Ile Asp Phe Pro Pro Lys Ile Ser Cys Leu Val Ala Trp Lys Gln Gly Asp Met Ile Ser Thr Thr Asn Ala Thr Ala Ile Pro Thr Val Tyr His His Pro Arg Leu Ser Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Thr Ile Glu Cys Val Pro Ser Glu Ile Thr Val Arg Trp Leu Val His Asp Glu Ala Gln Pro Asn Thr Thr Tyr Asn Thr Val Val Thr Gly Leu Cys Arg Thr Ile Asp Arg His Arg Asn Leu Leu Ser Arg Ile Pro Val Trp Asp Asn Trp Thr Lys Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pro Phe Asp Glu Asp Lys Phe Gln Asp Ser Glu Tyr Tyr Asp Ala Thr Pro Ser Ala Arg Gly Thr Pro Met Val Ile Thr Val Thr Ala Val Leu Gly Leu Ala Val Ile Leu Gly Met Gly Ile Ile Met Thr Ala Leu Cys Leu Tyr Asn Ser Thr Arg Lys Asn Ile Arg Leu (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1734 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1734 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn Asp Glu Arg Glu Ala Lys. Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu Thr Val.Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn Gln Asp Val Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile.Thr Ser Leu Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gln Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro TCA TTT GAC ATG AGT GCT ACC CAT TAC TGC.TAC ACC CAT AAT GTA ATA 576 Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Ala Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr Arg Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr Gly Gly Leu Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Glu Gly Lys Tyr Val Ile Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe Tyr Arg Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Gly Glu Gln Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg Glu Ala Arg Ser Gly (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 577 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn Asp Glu Arg Glu Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn Gln Asp Val Ial Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser Leu Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gin Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Ala Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser.Ala Val Pro Thr Arg Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr Gly Gly Leu Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Glu Gly Lys Tyr Val Ile Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe Tyr Arg Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Gly Glu Gln .'1_a Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg Glu Ala Arg Ser Gly (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1662 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1662 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Gly Ser Arg Pr6.,Ser Thr Lys Asn Pro Ala Pro Met Met Leu Thr Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly AGA CAG GGG CGC CTT ATA GGC GCC ATT ATT GGC GGT GTG GCT CTT GGG' 384 Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala ACC AAT GAG GCT GTG CAT GAG GTC. ACT GAC GGA TTA TCG CAA CTA GCA 528 Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala GTG GCA GTT GGG AAG ATG CAG CAG TTC GTT AAT GAC CAA TTT AAT,AAA 576 Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala Gln Gln Val Gly Val Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr Val Phe Gly Pro Gln Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro Lys Val Val Thr Arg Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val 405 410 415.
Ser Leu Ile Asp Lys Gln Ser Cys Asn Val Leu Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gly Asn Leu Asp Ile Ser Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asn Lys Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Val Asn Val Lys Leu Thr Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Ile Ile Ser Leu Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys Tyr Leu Met Tyr Lys Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu Asp Gln Met Arg Ala Thr Thr Lys Met (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 553 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Ala Pro Met Met Leu Thr Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala Gln Gln Val Gly Val Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr Val Phe Gly Pro Gln Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly=
Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro Lys Val Val Thr Arg Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met ?70 375 380 Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val Lau Ile Asp Lys Gln Ser Cys Asn Val Leu Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gly Asn Leu Asp Ile Ser Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asn Lys Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Val Asn Val Lys Leu Thr Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Ile Ile Ser Leu Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys Tyr Leu Met Tyr Lys Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu Asp Gln Met Arg Ala Thr Thr Lys Met (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3489 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..3489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gin Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly Cys Ile Val Gly Thr Ile His Gly Gly Arg Val Val Asn Ala Ser Ser Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln' Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Thr Gly Met Leu Gin Lys Asn Phe Leu Arg Val Ser Ala Met. Lys Asn Gly Gln Leu Phe Tyr Asn Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Lys Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tvr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gln Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln Asn Ile Leu Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gin Ser Val Phe Ser Gly Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile 515 520 525.
Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Asn Val Ala Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gin Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn Ser Ile Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr GGT GAG TTT AAT ATT TCT CTT CTG.TTA ACA ACT CCT AGT AGT CCT AGA 2064 Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu GGT TTT. CTT AAG GAC CTT GCG TGT GCT CGT GAA TAT AAT GGT TTG CTT 2208 Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gin Gin Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala 850. 855 860 Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu His Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val ACT GAA CAA AAC=AGA CCT AAA AAG TCT GTT TAA 3489 Thr Glu Gln Asn Arg Pro Lys Lys Ser Val (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1162 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly Cys Ile Val Gly Thr Ile His Gly Gly Arg Val Val Asn Ala Ser Ser Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Thr Gly Met Leu Gln Lys Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Lys Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala Gin Asp Val Ile Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gln Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gin Asn Ile Leu Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Giu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Giy Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Asn Val Ala Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Asn Cys Lau Gln Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn Cer Iie Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu His Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn x:45 950 955 960 Ala Ser Gin Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arq Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala hsn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr Glu Gln Asn Arg Pro Lys Lys Ser Val (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1846 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear N
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1846 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Phe Ala Leu Cys Sir Ala Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser Ala Phe Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val. A.sn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser Val Ala Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser TTT TGT ACA GCT CAC TGT AAT TTT.ACT TCT TAT ATA GTG TTT GTT ACA 336 Phe Cys Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr Pro Gly His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Lys Phe Arg Ser Leu Gln Cys Val Asn Asn His Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr Ser Asn Tyr Thr Glu Asp Val Val Ala Ala Gly Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro .275 280 285 Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Ala Cys Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Trp Ser Asn Ser Leu Ser Val Ser Leu Ile Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala Thr Gln Pro Pro Val Leu Thr Gin Asn Phe Tyr Asn Asn Ile Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu Ala Glu Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gin Phe Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser GTT AAT GAA AAT.GTT ACG AAT TGC CCT TAT GTT AGT TAT GGC AAG TTT 1680 Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Arg (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 615 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Phe Ala Leu Cys Ser Ala Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser Ala Phe Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val Asn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser Val Ala Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser Phe Cys Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr Pro Gly His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Lys Phe Arg Ser Leu Gin Cys Val Asn Asn His Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr Ser Asn Tyr Thr Glu Asp Val Val Ala Ala Gly Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr Fsr Thr fly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Ala Cys Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Trp Ser Asn Ser Leu Ser Val Ser Leu Ile Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala Thr Gln Pro Pro Val Leu Thr Gln Asn Phe Tyr Asn Asn Ile Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu 450 ~.* 455 460 Ala Glu Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu Cys Glu Asp Val Asn Gln Gin Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Arg (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2116 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear.
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNN[NNNNN 420 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 705 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Tyr Asn Tyr Leu Ala Asp Ala Gly Met Ala Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Ala Gln Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Ala Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr Arg Arg Ser Arg Arg Ser Ile Thr Ala Asn Val Thr Asn Xaa Pro Tyr Val Ser Tyr Gly Lys Phe Cys Leu Lys Pro Asp Gly Ser Xaa Ser Xaa Ile Ala Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Cys Gly Asn Ser Leu Asp Cys Arg Lys Leu Xaa Gln Gln Tyr Gly Pro Val Xaa Asp Asn Ile Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Leu Tyr Ser Ser Thr Lys Pro Ser Gly Phe Asn Thr Pro Val Phe Ser Asn Leu Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Val Asp Thr Ser Ser Ser Thr Thr Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Xaa Xaa Xaa Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Gln Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gln Gln Val Xaa Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Xaa Gly Ala Ile Ser Ser Val Ile Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala Gin Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn_Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu Ser Phe Xaa Asn Val Thr Ala Ile Val Gly Phe Cys Lys Ala Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Ser Ile Gin Val Asn Gly Ser His Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Xaa Lys Thr Val Ile Thr Thr Xaa Val Asp Asn Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Gly Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Xaa Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Leu Asp Asn Asp Val Val Thr Glu Gln Xaa Arg Pro Lys (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gln Arg Arg (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) 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 Gln Arg Arg (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
(2) INF071MATION FOR SEQ ID NO : 24 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
tiv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:28:
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(2) INFORMATION FOR SEQ ID NO:29:
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(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 16..66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gln Arg Arg Asp Trp (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Met Lys Trp Ala Thr Trp Ile Asp Pro Val Val Leu Gln Arg Arg Asp Trp (2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys Asp Leu Glu (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
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Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys Asp Leu Glu (2) INFORMATION FOR SEQ ID NO:33:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:.
(A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
(2) INFORMATION FOR SEQ ID NO:37:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
(2) INFORMATION FOR SEQ ID NO:38:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
96/05291 PCT/US95/10245 ,,:., (2) INFORMATION FOR SEQ ID NO:39:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
(2) INFORMATION FOR SEQ ID NO:40:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
(2) INFORMATION FOR SEQ ID NO:43:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE. NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
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(2) !NFORMATION FOR SEQ ID NO:47:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2681 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 146..481 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: complement (602..1402) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1599..2135 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: complement (2308..2634) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
CTGCATTTTC TGCTGCCGAA CAAACTTCAT CGCTATGCAA AGAGATGCGT GTGTACACGC '2100 (2) INFORMATION FOR SEQ ID NO:49:
.(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Met Cys Arg Tyr Asn Thr Xaa Xaa Arg Ser Met Glu Asp Met Leu Arg Arg Ser Ser Pro Glu Asp Ile Thr Asp Ser Leu Thr Met Cys Leu Ile Met Leu Ser Arg Ile Arg Arg Thr Met Arg Thr Ala Gly Asn Lys Tyr Ser Tyr Met Ile Asp Pro Met Asn Arg Met Ser Asn Tyr Thr Pro Gly Glu Cys Met Thr Gly Ile Leu Arg Tyr Ile Asp Glu His Ala Arg Arg Cys Pro Asp His Ile Cys Asn Leu Tyr Ile Thr Cys Thr Leu Met Pro Met Tyr Val His Gly Arg Tyr Phe Tyr Cys Asn Ser Phe Phe Xaa (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Mat His Phe Pro Phe Asn Lys Lys Cys Arg Glu Asn Thr Gln Pro Leu Trp Met Tyr Pro Arg Glu Leu Pro Arg Arg Gly Ala Tyr Gly Val Ala Ala Arg Pro Ile Leu Ile Thr Asp Met Gly Pro Thr Ile Asn Ile Leu Leu Val Gly Gly Trp Lys Gly Arg Leu Val Thr Ala Lys Ser Ser Val Gln His Arg Ala Val Leu Trp Ile Ser Gly Gly Gln Arg Glu Asn Ala Ser Gln Tyr Ser Phe Asn Arg Glu Asp Ala Asn Thr His Gly Trp Phe Leu Thr Leu Gly Ala Pro Trp Ile Val Gly Arg Asn Ile Leu Pro Glu Asp Arg Leu Leu Phe Ile Thr Asn His Cys Ser Gly Glu Leu Gln Ser Val Phe Ala Gly Met Pro Val Ala Leu Lys Thr Val Leu Glu His Leu His Leu Lys Arg Glu Ser Gly Leu Gln Pro Ala Leu Leu Met Asp Pro Arg Arg Phe Leu Glu Met Arg Asp Pro Arg Lys Ile Ile Cys Met Cys Glu Leu Asp Ile Arg Asp Ser Pro Gly Ala His Gly Ile Leu Val Asn Cys Cys Cys Gly Arg Pro Gly Gly Leu Gin Cys Leu Thr Phe Ile Arg lie Ile Glu Thr Leu Val Asn His Phe Leu Ser Ser Thr Asp Leu Thr Cys Leu Asn Leu Thr Cys Arg His Ser Asp Val Pro Thr Thr Val Ala Ser Thr Leu Ser Glu Asp Gly Phe Glu His Asp Xaa Glu Tyr Ser Ile Ser Thr Ile Ser Glu Glu Ala Glu Val Tyr (2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
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(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 Arg Val Gln Asn Val Trp His Val Val Asp Pro Leu Pro Ile Val Ala Asn Tyr Lys Ser Asp Arg His Asp Arg Asp Lys Pro Val Pro Cys Gly Ile Arg Val Thr Ser Arg Ser Tyr Ile Ile Arg Pro Thr Cys Glu Ser Ser Lys Asp Leu His Ala Phe Phe Phe Gly Leu Ser Thr Gly Ser Ser Glu Ser Met Pro Ile Ser Gly Val Glu Thr Ile Val Arg Leu Met Asn Asn Lys Ala Ala Cys His Gly Lys Glu Gly Cys Arg Ser Pro Phe Ser His Ala Thr Ile Leu Asp Ala Val Asp Asp Asn Tyr Thr Met Asn Ile Glu Gly Val Cys Phe His Cys His Cys Asp Asp Lys Phe Ser Pro Asp Cys Trp Ile Ser Ala Phe Ser Ala Ala Glu Gln Thr Ser Ser Leu Cys Lys Glu Met Arg Val Tyr Thr Arg Arg Trp Ser Ile Arg Glu Thr Lys Cys Ser (2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Met Gly Leu Tyr Met Glu Pro Xaa Asn Xaa Val Ser Leu Gln Phe Leu Arg Gly Gly Ile Tyr Met Ile Ser Xaa Pro Lys Arg Gly Met Xaa Gln Arg Asp Val Met Val Ile Xaa Gly Lys Phe Phe Arg Ser Glu Ile Thr Gln Leu Pro Lys His Arg Ser Arg Leu Lys Glu Lys Ser Asp Gly Ser Ile Arg Thr Cys Met Asp Ser Val Arg Ile Asn His Asn Arg Ser Thr Vai Gly His Phe Gly Asn Ser Asn Ala Lys Arg Cys Thr Ser Ala Ile Thr Thr Pro Thr Met His Ile Val Thr Pro Ala Ser (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Oligonucleotide Primer (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Oligonucleotide Primer (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE;. nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
ix/; ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Claims (29)
1. A recombinant chimeric virus comprising a unique long viral genomic region which naturally occurs in a herpesvirus of turkeys and a unique short viral genomic region which naturally occurs in a Marek's disease virus.
2. The recombinant chimeric virus of claim 1, wherein a foreign DNA is inserted within a non essential region of the chimeric virus such that the foreign DNA expressed in a host cell.
3. The recombinant chimeric virus of claim 2, wherein the non-essential region is an EcoR1 #9 fragment of the unique long viral genomic region which naturally occurs in a herpesvirus of turkeys.
4. The recombinant chimeric virus of claim 2, wherein the foreign DNA
encodes a polypeptide.
encodes a polypeptide.
5. The recombinant chimeric virus of claim 2, wherein the foreign DNA
encodes a cytokine.
encodes a cytokine.
6. The recombinant chimeric virus of claim 5, wherein the cytokine is a chicken mylemonocytic growth factor (cMGF) or a chicken interferon (cIFN).
7. The recombinant chimeric virus of claim 2, wherein the foreign DNA
encodes E. coli beta-galactosidase.
encodes E. coli beta-galactosidase.
8. The recombinant chimeric virus of claim 4, wherein the polypeptide is obtained from Marek's Disease Virus, Newcastle Disease Virus, Infectious Laryngotracheitis Virus, Infectious Bronchitis Virus or Infectious Bursal Disease Virus.
9. The recombinant chimeric virus of claim 8, wherein the polypeptide is Marek's Disease Virus glycoprotein A, Marek's Disease Virus glycoprotein B, or Marek's Disease Virus glycoprotein D.
10. The recombinant chimeric virus of claim 8, wherein the polypeptide is a fusion protein of Newcastle disease virus or hemagglutinin-neuraminidase of Newcastle disease virus.
11. The recombinant chimeric virus of claim 10, wherein the polypeptide is the fusion protein of Newcastle disease virus.
12. The recombinant chimeric virus of claim 10, wherein the polypeptide is the hemagglutinin-neuraminidase of Newcastle disease virus.
13. The recombinant chimeric virus of claim 8, wherein the polypeptide is Infectious Laryngotracheitis Virus glycoprotein B, Infectious Laryngotracheitis Virus glycoprotein I, or Infectious Laryngotracheitis Virus glycoprotein D.
14. The recombinant chimeric virus of claim 8, wherein the polypeptide is the infectious laryngotracheitis virus glycoprotein D (gD).
15. The recombinant chimeric virus of claim 8, wherein the polypeptide is the infectious laryngotracheitis glycoprotein I (gI).
16. The recombinant chimeric virus of claim 8, wherein the polypeptide is the infectious laryngotracheitis glycoprotein D (gD) and glycoprotein I (gI).
17. The recombinant chimeric virus of claim 8, wherein the polypeptide is the infectious laryngotracheitis glycoprotein B (gB).
18. The recombinant chimeric virus of claim 8, wherein the polypeptide is Infectious Bronchitis Virus spike protein or Infectious Bronchitis Virus matrix protein.
19. The recombinant chimeric virus of claim 8, wherein the polypeptide is Infectious Bursal Disease Virus VP2, Infectious Bursal Disease Virus VP3 or Infectious Bursal Disease Virus VP4.
20. The recombinant chimeric virus of claim 4, wherein the polypeptide is from a pathogen selected from the group consisting of 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., Bardetella spp., Eimeria spp., Histomonas spp., Trichomonas spp., poultry nematodes, poultry cestodes, poultry trematodes, poultry mites/lice and poultry protozoa.
21. The recombinant chimeric virus of claim 2, wherein the foreign DNA is under the control of a herpesvirus promoter.
22. The recombinant chimeric virus of claim 2, wherein the foreign DNA is under the control of a heterologous promoter.
23. The recombinant chimeric virus of claim 21, wherein the promoter is obtained from an endogenous upstream sequence of a gene that encodes a protein selected from the group consisting of Pseudorabies Virus glycoprotein X, Herpesvirus Simplex Virus type -- 1 alpha 4, Marek's Disease Virus glycoprotein A, Marek's Disease Virus glycoprotein B, Marek's Disease Virus glycoprotein D, Infectious Laryngotracheitis Virus glycoprotein B, Bovine Herpesvirus 1.1 VP8, and Infectious Laryngotracheitis Virus glycoprotein D.
24. The recombinant chimeric virus of claim 22 wherein the heterologous promoter is Human Cytomegalovirus Immediate Early or chicken anemia virus promoter.
25. The recombinant chimeric virus of claim 22, wherein the promoter is obtained from the human cytomegalovirus immediate early promoter.
26. The recombinant chimeric virus of claim 22, wherein the promoter is obtained from the infectious laryngotracheitis virus glycoprotein D.
27. The recombinant chimeric virus of claim 22, wherein the promoter is obtained from the infectious laryngotracheitis virus glycoprotein I.
28. A vaccine which comprises an effective immunizing amount of the recombinant chimeric virus of any one of claims 2 to 27 and a suitable carrier.
29. Use of the vaccine of claim 28 for immunizing a bird against an avian pathogen.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/288,065 US5961982A (en) | 1985-09-06 | 1994-08-09 | Recombinant herpesvirus of turkeys and uses thereof |
| US08/288,065 | 1994-08-09 | ||
| US08/362,240 US5965138A (en) | 1985-09-06 | 1994-12-22 | Recombinant chimeric virus and uses thereof |
| US08/362,240 | 1994-12-22 | ||
| CA2196570A CA2196570C (en) | 1994-08-09 | 1995-08-09 | Recombinant herpesvirus of turkeys and uses thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2196570A Division CA2196570C (en) | 1994-08-09 | 1995-08-09 | Recombinant herpesvirus of turkeys and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2626498A1 CA2626498A1 (en) | 1996-02-22 |
| CA2626498C true CA2626498C (en) | 2011-04-12 |
Family
ID=39688993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2626498A Expired - Fee Related CA2626498C (en) | 1994-08-09 | 1995-08-09 | Recombinant chimeric virus |
Country Status (3)
| Country | Link |
|---|---|
| CA (1) | CA2626498C (en) |
| ES (1) | ES2360298T3 (en) |
| PT (1) | PT776361E (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| UA118651C2 (en) * | 2011-11-30 | 2019-02-25 | Меріал Інк | Recombinant hvt vectors expressing antigens of avian pathogens and uses thereof |
-
1995
- 1995-08-09 ES ES06026820T patent/ES2360298T3/en not_active Expired - Lifetime
- 1995-08-09 CA CA2626498A patent/CA2626498C/en not_active Expired - Fee Related
- 1995-08-09 PT PT95930814T patent/PT776361E/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CA2626498A1 (en) | 1996-02-22 |
| ES2360298T3 (en) | 2011-06-02 |
| PT776361E (en) | 2007-04-30 |
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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20140811 |