Recombinant swinepox virus

BACKGROUND OF THE INVENTION
Swinepox virus (SPV) belongs to the family poxviridae. Viruses belonging to this group are large, double-stranded DNA viruses that characteristically develop in the cytoplasm of the host cell. SPV is the only member of the genus Suipoxvirus. Several features distinguish SPV from other poxviruses. SPV exhibits species specificity (18) compared to other poxviruses such as vaccinia which exhibit a broad host range. SPV infection of tissue culture cell lines also differs dramatically from other poxviruses (24). It has also been demonstrated that SPV does not exhibit antigenic cross-reactivity with vaccinia virus and shows no gross detectable homology at the DNA level with the ortho, lepori, avi or entomopox virus groups (24). Accordingly, what is known and described in the prior art regarding other poxviruses does not pertain a priori to swinepox virus. SPV is only mildly pathogenic, being characterized by a self-limiting infection with lesions detected only in the skin and regional lymph nodes. Although the SPV infection is quite limited, pigs which have recovered from SPV are refractory to challenge with SPV, indicating development of active immunity (18).
The present invention concerns the use of SPV as a vector for the delivery of vaccine antigens and therapeutic agents to swine. The following properties of SPV support this rationale: SPV is only mildly pathogenic in swine, SPV is species specific, and SPV elicits a protective immune response. Accordingly, SPV is an excellent candidate for a viral vector delivery system, having little intrinsic risk which must be balanced against the benefit contributed by the vector's vaccine and therapeutic properties.
The prior art for this invention stems first from the ability to clone and analyze DNA while in bacterial plasmids. The techniques that are available are detailed for the most part in Maniatis et al., 1983 and Sambrook et al., 1989. These publications teach state of the art general recombinant DNA techniques.
Among the poxviruses, five (vaccinia, fowlpox, canarypox, pigeon, and raccoon pox) have been engineered, previous to this disclosure, to contain foreign DNA sequences. Vaccinia virus has been used extensively to vector foreign genes (25) and is the subject of U.S. Pat. Nos. 4,603,112 and 4,722,848. Similarly, fowlpox has been used to vector foreign genes and is the subject of several patent applications EPA 0 284 416, PCT WO 89/03429, and PCT WO 89/12684. Raccoon pox (10) and Canarypox (31) have been utilized to express antigens from the rabies virus. These examples of insertions of foreign genes into poxviruses do not include an example from the genus Suipoxvirus. Thus, they do not teach methods to genetically engineer swinepox viruses, that is, where to make insertions and how to get expression in swinepox virus.
The idea of using live viruses as delivery systems for antigens has a very long history going back to the first live virus vaccines. The antigens delivered were not foreign but were naturally expressed by the live virus in the vaccines. The use of viruses to deliver foreign antigens in the modern sense became obvious with the recombinant vaccinia virus studies. The vaccinia virus was the vector and various antigens from other disease causing viruses were the foreign antigens, and the vaccine was created by genetic engineering. While the concept became obvious with these disclosures, what was not obvious was the answer to a more practical question of what makes the best candidate virus vector. In answering this question, details of the pathogenicity of the virus, its site of replication, the kind of immune response it elicits, the potential it has to express foreign antigens, its suitability for genetic engineering, its probability of being licensed by regulatory agencies, etc, are all factors in the selection. The prior art does not teach these questions of utility.
The prior art relating to the use of poxviruses to deliver therapeutic agents relates to the use of a vaccinia virus to deliver interleukin-2 (12). In this case, although the interleukin-2 had an attenuating effect on the vaccinia vector, the host did not demonstrate any therapeutic benefit.
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 (16), ribozymes (34), suppressor tRNAs (2), interferon-inducing double stranded RNA and numerous examples of protein therapeutics, from hormones, e.g., insulin, to lymphokines, e.g., interferons and interleukins, to natural 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
The invention provides a recombinant swinepox virus capable of replication which comprises swinepox viral DNA and foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant swinepox virus is introduced. The foreign DNA is inserted into the swinepox viral DNA at a site which is not essential for replication of the swinepox virus and is under the control of a promoter.
This invention provides a homology vector for producing a recombinant swinepox virus by inserting foreign DNA into the genomic DNA of a swinepox virus which comprises a double-stranded DNA molecule. This molecule consists essentially of double-stranded foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant swinepox virus is introduced. At one end of this foreign DNA is double-stranded swinepox viral DNA homologous to genomic DNA located at one side of a site on the genomic DNA which is not essential for replication of the swinepox virus. At the other end of the foreign DNA is double-stranded swinepox viral DNA homologous to genomic DNA located at the other side of the same site on the genomic DNA.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Details of the SPV Kasza Strain. Diagram of SPV genomic DNA showing the unique long and Terminal repeat (TR) regions. A restriction map for the enzyme HindIII is indicated (23). Fragments are lettered in order of decreasing size. Note that the terminal repeats are greater than 2.1 kb but less than 9.7 kb in size.
FIGS. 2A-2B DNA sequence from homology vector 515-85.1. The sequence of two regions of the homology vector 515-85.1 are shown. The first region (FIG. 2A) (SEQ ID NO:1) covers a 599 base pair sequence which flanks the unique AccI site as indicated in FIGS. 3A-3B. The beginning (Met) and end (Val) of a 115 amino acid ORF is indicated by the translation of amino acids below the DNA sequence. The second region (FIG. 2B) (SEQ ID NO:3) covers the 899 base pairs upstream of the unique HindIII site as indicated in FIGS. 3A-3B. The beginning (Asp) and end (Ile) of a 220 amino acid ORF is indicated by the translation of amino acids below the DNA sequence.
FIGS. 3A-3B Homology between the 515.85.1 ORF and the Vaccinia virus 01L ORF. The first line shows a restriction map of the SPV HindIII M fragment. The second map shows a restriction map of the DNA insertion in plasmid 515-85.1. The location of the 515-85.1 �VV 01L-like! ORF is indicated on the map. The locations of the DNA sequences shown in FIGS. 2A-2B are indicated below the map by heavy bars. The third line shows the homology between the VV 01L ORF (SEQ ID NO:5) and the 515-85.1 ORF (SEQ ID NO:6) at their respective N-termini. The fourth line shows the homology between the VV 01L ORF (SEQ ID NO:7) and the 515-85.1 ORF (SEQ ID NO:8) at their respective C-termini.
FIGS. 4A-4C Detailed description of the DNA insertion in Homology Vector 520-17.5. Diagram showing the orientation of DNA fragments assembled in plasmid 520-17.5. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID NO's: 9, 10, 13, and 16). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements are also given. The following two conventions are used: 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, swinepox virus (SPV), early promoter 1 (EP1), late promoter 2 (LP2), lactose operon Z gene (lacZ), and Escherichia coli (E. coli).
FIGS. 5A-5D Detailed description of the DNA insertion in Homology Vector 538-46.16. Diagram showing the orientation of DNA fragments assembled in plasmid 538-46.16. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 17, 18, 21, 26, and 28). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: 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, swinepox virus (SPV), pseudorabies virus (PRV), g50 (gpD), glycoprotein 63 (gp63), early promoter 1 (EP1), late promoter 1 (LP1) (SEQ ID NO: 46), late promoter 2 (LP2), lactose operon Z gene (lacZ), and Escherichia coli (E. coli).
FIG. 6 Western blot of lysates from recombinant SPV infected cells with anti-serum to PRV. Lanes (A) uninfected Vero cell lysate, (B) S-PRV-000 (pseudorabies virus S62/26) infected cell lysate, (C) pre-stained molecular weight markers, (D) uninfected EMSK cell lysate, (E) S-SPV-000 infected cell lysate, (F) S-SPV-003 infected cell lysate, (G) S-SPV-008 infected cell lysate. Cell lysates were prepared as described in the PREPARATION OF INFECTED CELL LYSATES. Approximately 1/5 of the total lysate sample was loaded in each lane.
FIG. 7 DNA sequence of NDV Hemagglutinin-Neuraminidase gene (HN) (SEQ ID NO: 29). The sequence of 1907 base pairs of the NDV HN cDNA clone are shown. The translational start and stop of the HN gene is indicated by the amino acid translation below the DNA sequence.
FIGS. 8A-8C Detailed description of the DNA insertion in Homology Vector 538-46.26. Diagram showing the orientation of DNA fragments assembled in plasmid 538-46.26. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID NO's: 31, 32, 34, 37, and 40). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: 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, swinepox virus (SPV), Newcastle Disease virus (NDV), hemagglutinin-neuraminidase (HN), early promoter 1 (EP1), late promoter 1 (LP1), late promoter 2 (LP2), lactose operon Z gene (lacZ), and Escherichia coli (E. coli).
FIGS. 9A-9C Detailed description of Swinepox Virus S-SPV-010 and the DNA insertion in Homology Vector 561-36.26. Diagram showing the orientation of DNA fragments assembled in plasmid 561-36.26. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID. NO: 47, 48, 49, 50, 51, 52). The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, � !, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), Escherichia coli (E. coli), thymidine kinase (TK), pox synthetic late promoter 1 (LP1), base pairs (BP).
FIGS. 10A-10D Detailed description of Swinepox Virus S-SPV-011 and the DNA insertion in Homology Vector 570-91.21. Diagram showing the orientation of DNA fragments assembled in plasmid 570-91.21. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID NO: 53, 54, 55, 56, 57, 58, 59, 60). The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, � !, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), pseudorabies virus (PRV), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), pox synthetic early promoter 2 (EP2) (SEQ ID NO: 45), gIII (gpC), base pairs (BP).
FIGS. 11A-11D Detailed description of Swinepox Virus S-SPV-012 and the DNA insertion in Homology Vector 570-91.41. Diagram showing the orientation of DNA fragments assembled in plasmid 570-91.41. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68). The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites brackets, � !, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), pseudorabies virus (PRV), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), pox synthetic early promoter 1 late promoter 2 (EP1LP2) (SEQ ID NO: 43), gIII (gpC), base pairs (BP).
FIGS. 12A-12D Detailed description of Swinepox Virus S-PRV-013 and the DNA insertion in Homology Vector 570-91.64. Diagram showing the orientation of DNA fragments assembled in plasmid 570-91.64. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID NO: 69, 70, 71, 72, 73, 74, 75, 76). The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, � !, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), pseudorabies virus (PRV), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2EP2) (SEQ ID NO: 44), gIII (gpC) base pairs (BP).
FIGS. 13A-13D Detailed description of Swinepox Virus S-PRV-014 and the DNA insertion in Homology Vector 599-65.25. Diagram showing the orientation of DNA fragments assembled in plasmid 599-65.25. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID NO: 77, 78, 79, 80, 81, 82, 83, 84). The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, � !, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), infectious laryngotracheitis virus (ILT), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), pox synthetic early promoter 1 late promoter 2 (EP1LP2), glycoprotein G (gpG), polymerase chain reaction (PCR), base pairs (BP).
FIGS. 14A-14D Detailed description of Swinepox Virus S-SPV-016 and the DNA insertion in Homology Vector 624-20.1C. Diagram showing the orientation of DNA fragments assembled in plasmid 624-20.1C. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown (SEQ ID NO: 85, 86, 87, 88, 89, 90, 91, 92, 93). The restriction sites are used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, � !, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), infectious laryngotracheitis virus (ILT), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2EP2), glycoprotein I (gpI), polymerase chain reaction (PCR), base pairs (BP).
FIGS. 15A-15D Detailed description of Swinepox Virus S-SPV-017 and the DNA insertion in Homology Vector 614-83.18. Diagram showing the orientation of DNA fragments assembled in plasmid 614-83.18. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown, (SEQ ID NO: 94, 95, 96, 97, 98, 99, 100, 101, 102). The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, � !, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), infectious bovine rhinotracheitis virus (IBR), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2 EP2), glycoprotein G (gpG), polymerase chain reaction (PCR), base pairs (BP).
FIG. 16 Western blot of lysates from recombinant SPV infected cells with polyclonal goat anti-PRV gIII (gpc). Lanes (A) S-PRV-002 (U.S. Pat. No. 4,877,737, issued Oct. 31, 1989) infected cell lysate, (B) molecular weight markers, (C) mock-infected EMSK cell lysate, (D) S-SPV-003 infected cell lysate, (E) S-SPV-008 infected cell lysate, (F) S-SPV-011 infected cell lysate, (G) S-SPV-012 infected cell lysate, (H) S-SPV-013 infected cell lysate. Cell lysates are prepared as described in the PREPARATION OF INFECTED CELL LYSATES. Approximately 1/5 of the total lysates sample is loaded in each lane.

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a recombinant swinepox virus (SPV) capable of replication in an animal into which the recombinant swinepox virus is introduced which comprises swinepox viral DNA and foreign DNA encoding RNA which does not naturally occur in the animal into which the recombinant swinepox virus is introduced, the foreign DNA being inserted into the swinepox viral DNA at an insertion site which is not essential for replication of the swinepox virus and being under the control of a promoter.
For purposes of this invention, "a recombinant swinepox virus capable of replication" is a live swinepox virus which has been generated by the recombinant methods well known to those of skill in the art, e.g., the methods set forth in HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV in Materials and Methods and has not had genetic material essential for the replication of the recombinant swinepox virus deleted.
For purposes of this invention, "an insertion site which is not essential for replication of the swinepox virus" is a location in the swinepox viral genome where a sequence of DNA is not necessary for viral replication, for example, complex protein binding sequences, sequences which code for reverse transcriptase or an essential glycoprotein, DNA sequences necessary for packaging, etc. For purposes of this invention, a "promoter" is a specific DNA sequence on the DNA molecule to which the foreign RNA polymerase attaches and at which transcription of the foreign RNA is initiated.
The invention further provides foreign RNA which encodes a polypeptide. Preferably, the polypeptide is antigenic in the animal. Preferably, this antigenic polypeptide is a linear polymer of more than 10 amino acids linked by peptide bonds which stimulates the animal to produce antibodies.
The invention further provides an insertion site present within the larger HindIII to BglII subfragment of the HindIII M fragment of swinepox viral DNA. Preferably, the insertion site is within an open reading frame contained in the HindIII to BglII subfragment. Preferably, the insertion site is the AccI restriction endonuclease site located in the HindIII to BglII subfragment.
The invention further provides an insertion site within an open reading frame encoding swinepox thymidine kinase. Preferably, the insertion site is the NdeI restriction endonuclease site located within the swinepox virus thymidine kinase gene.
For purposes of this invention, 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 a recombinant swinepox virus capable of replication which contains a foreign DNA encoding a polypeptide which is a detectable marker. Preferably the detectable marker is the polypeptide E. coli .beta.-galactosidase. Preferably, the insertion site for the foreign DNA encoding E. coli .beta.-galactosidase is the AccI restriction endonuclease site located within the HindIII M fragment of the swinepox viral DNA. Preferably, this recombinant swinepox virus is designated S-SPV-003 (ATCC Accession No. VR 2335). The S-SPV-003 swinepox virus 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, Md. 20852 U.S.A. under ATCC Accession No. VR 2335. For purposes of this invention, a "polypeptide which is a detectable marker" includes the bimer, trimer and tetramer form of the polypeptide. E. coli .beta.-galactosidase is a tetramer composed of four polypeptides or monomer sub-units.
The invention further provides a recombinant swinepox virus capable of replication which contains foreign DNA encoding an antigenic polypeptide which is or is from pseudorabies virus (PRV) g50 (gpD), pseudorabies virus (PRV) II (gpB), Pseudorabies virus (PRV) gIII (gpC), Pseudorabies virus (PRV) glycoprotein H, Transmissible gastroenteritis (TGE) glycoprotein 195, Transmissible gastroenteritis (TGE) matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, Bovine Viral Diarrhea (BVD) glycoprotein 55, Newcastle Disease Virus (NDV) hemagglutinin-neuraminidase, swine flu hemagglutinin or swine flu neuraminidase. Preferably, the antigenic polypeptide is Pseudorabies Virus (PRV) g50 (gpD). Preferably, the antigenic protein is Newcastle Disease Virus (NDV) hemagglutinin-neuraminidase.
The invention further provides a recombinant swinepox virus capable of replication which contains foreign DNA encoding an antigenic polypeptide which is or is from Serpulina hyodysenteriae, Foot and Mouth Disease Virus, Hog Cholera Virus, Swine Influenza Virus, African Swine Fever Virus or Mycoplasma hyopneumoniae.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) g50 (gpD). This recombinant swinepox virus can be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli B-galactosidase. A preferred site within the swinepox viral genome for insertion of the foreign DNA encoding PRV g50 (gpD) and E. coli B-galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA. Preferably, this recombinant swinepox virus is designated S-SPV-008 (ATCC Accession No. VR 2339). The S-SPV-008 swinepox virus 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, Md. 20852 U.S.A. under ATCC Accession No. VR 2339.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) gIII (gpC). This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli B-galactosidase. A preferred site within the swinepox viral DNA for insertion of the foreign DNA encoding PRV C gene and E. coli B-galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA. Preferably, this recombinant swinepox virus is designated S-SPV-011, S-SPV-012, or S-SPV-013. The swinepox virus designated S-SPV-013 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, Md. 20852 U.S.A. under ATCC Accession No. VR 2418.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) gII (gpB). This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli B-galactosidase. A preferred site within the swinepox viral DNA for insertion of the foreign DNA encoding PRV gII (gpB) and E. coli B-galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA. Preferably, this recombinant swinepox virus is designated S-SPV-015.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) g50 (gpD) and foreign DNA encoding pseudorabies virus (PRV) gIII (gpC). This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli B-galactosidase. A preferred site within the swinepox viral DNA for insertion of the foreign DNA encoding PRV g50 (gpD), PRV gIII (gpC) and E. coli B-galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) g50 (gpD) and foreign DNA encoding pseudorabies virus (PRV) gII (gpB). This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli B-galactosidase. A preferred site within the swinepox viral genome for insertion of foreign DNA encoding PRV g50 (gpD), PRV gII (gpB) and E. coli B-galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) gIII (gpC) and foreign DNA encoding pseudorabies virus (PRV) gII (gpB). This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli B-galactosidase. A preferred site within the swinepox viral genome for insertion of foreign DNA encoding PRV gIII (gpC), PRV gII (gpB) and E. coli B-galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) g50 (gpD), foreign DNA encoding pseudorabies virus (PRV) gIII (gpC), and foreign DNA encoding pseudorabies virus (PRV) gII (gpB). This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli B-galactosidase.
A preferred site within the swinepox viral genome for insertion of foreign DNA encoding PRV g50 (gpD), PRV gIII (gpC), PRV gII (gpB) and E. coli B-galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA.
The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding RNA encoding the antigenic polypeptide Newcastle Disease Virus (NDV) hemagglutinin-neuraminidase further comprising foreign DNA encoding a polypeptide which is a detectable marker. Preferably, this recombinant swinepox virus is designated S-SPV-009 (ATCC Accession No. VR 2344). The S-SPV-009 swinepox virus 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, Md. 20852 U.S.A. under ATCC Accession No. VR 2344.
The invention further provides that the inserted foreign DNA is under the control of a promoter. Preferably, the promoter is a swinepox viral promoter. Preferably, the promoter is a synthetic pox viral promoter. For purposes of this invention, the promoters were generated by methods well known to those of skill in the art, for example, as set forth in the STRATEGY FOR THE CONSTRUCTION OF SYNTHETIC POX VIRAL PROMOTERS in Materials and Methods. For purposes of this invention, a synthetic pox promoter includes a synthetic late pox promoter, a synthetic early pox promoter or a synthetic early/late pox promoter.
The invention provides for a homology vector for producing a recombinant swinepox virus by inserting foreign DNA into the genomic DNA of a swinepox virus. The homology vector comprises a double-stranded DNA molecule consisting essentially of a double-stranded foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant swinepox virus is introduced, with at one end of the foreign DNA, double-stranded swinepox viral DNA homologous to genomic DNA located at one side of a site on the genomic DNA which is not essential for replication of the swinepox virus, and at the other end of the foreign DNA, double-stranded swinepox viral DNA homologous to genomic DNA located at the other side of the same site on the genomic DNA. Preferably, the RNA encodes a polypeptide.
In one embodiment, the polypeptide is a detectable marker. Preferably, the polypeptide which is a detectable marker is E. coli .beta.-galactosidase.
In one embodiment, the polypeptide is antigenic in the animal. Preferably, the antigenic polypeptide is or is from pseudorabies virus (PRV) g50 (gpD), pseudorabies virus (PRV) gII (gpB), Pseudorabies virus (PRV) gIII (gpC), Pseudorabies virus (PRV) glycoprotein H, Transmissible gastroenteritis (TGE) glycoprotein 195, Transmissible gastroenteritis (TGE) matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, Bovine Viral Diarrhea (BVD) glycoprotein 55, Newcastle Disease Virus (NDV) hemagglutinin-neuraminidase, swine flu hemagglutinin or swine flu neuraminidase. Preferably, the antigenic polypeptide is or is from Serpulina hyodysenteriae, Foot and Mouth Disease Virus, Hog Cholera Virus, Swine Influenza Virus, African Swine Fever Virus or Mycoplasma hyopneumoniae.
In one embodiment of the invention, the double-stranded swinepox viral DNA is homologous to genomic DNA present within the larger HindIII to BglII subfragment of the HindIII M fragment of swinepox virus. Preferably, the double-stranded swinepox viral DNA is homologous to genomic DNA present within the open reading frame contained in this HindIII to BglII subfragment. Preferably, the double-stranded swinepox viral DNA is homologous to genomic DNA present within the AccI restriction endonuclease site located in this HindIII to BglII subfragment.
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 swinepox virus.
In one embodiment of the invention, the double-stranded swinepox viral DNA in the homology vector is homologous to genomic DNA present within the open reading frame encoding swinepox thymidine kinase. Preferably, the double-stranded swinepox viral DNA is homologous to genomic DNA present within the NdeI restriction endonuclease site located in the open reading frame encoding swinepox thymidine kinase.
The invention further provides a homology vector where foreign DNA further comprises a synthetic pox viral promoter.
The invention further provides a vaccine which comprises an effective immunizing amount of a recombinant swinepox virus of the present invention and a suitable carrier.
Suitable carriers for the pseudorabies virus are well known in the art and include proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as stabilized, hydrolyzed proteins, lactose, etc.
For purposes of this invention, an "effective immunizing amount" of the recombinant swinepox virus of the present invention is within the range of 10.sup.3 to 10.sup.9 PFU/dose.
The present invention also provides a method of immunizing an animal, wherein the animal is a swine, bovine, equine, caprine or ovine. For purposes of this invention, this includes immunizing the animal against the virus or viruses which cause the disease or diseases pseudorabies, transmissible gastroenteritis, swine rotavirus, swine parvovirus, Serpulina hyodysenteriae, bovine viral diarrhea, Newcastle disease, swine flu, foot and mouth disease, hog cholera, African swine fever or Mycoplasma hyopneumoniae. 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.
The present invention also provides a method for testing a swine to determine whether the swine has been vaccinated with the vaccine of the present invention, particularly the embodiment which contains the recombinant swinepox virus S-SPV-008 (ATCC Accession No. VR 2339), or is infected with a naturally-occurring, wild-type pseudorabies virus. This method comprises obtaining from the swine to be tested a sample of a suitable body fluid, detecting in the sample the presence of antibodies to pseudorabies virus, the absence of such antibodies indicating that the swine has been neither vaccinated nor infected, and for the swine in which antibodies to pseudorabies virus are present, detecting in the sample the absence of antibodies to pseudorabies virus antigens which are normally present in the body fluid of a swine infected by the naturally-occurring pseudorabies virus but which are not present in a vaccinated swine indicating that the swine was vaccinated and is not infected.
The present invention also provides a host cell infected with a recombinant swinepox virus capable of replication. In one embodiment, the host cell is a mammalian cell.
Preferably, the mammalian cell is a Vero cell. Preferably, the mammalian cell is an ESK-4 cell, PK-15 cell or EMSK cell.
For purposes of this invention a "host cell" is a cell used to propagate a vector and its insert. Infecting the cells was accomplished by methods well known to those of skill in the art, for example, as set forth in INFECTION--TRANSFECTION PROCEDURE in Material and Methods.
Methods for constructing, selecting and purifying recombinant swinepox virus, including S-SPV-003, S-SPV-008, S-SPV-011, S-SPV-012, S-SPV-013 and S-SPV-015 are detailed below in Materials and Methods.
MATERIALS AND METHODS
PREPARATION OF SWINEPOX VIRUS STOCK SAMPLES. Swinepox virus (SPV) samples were prepared by infecting embryonic swine kidney (EMSK) cells, ESK-4 cells, PK-15 cells or Vero cells at a multiplicity of infection of 0.01 PFU/cell in a 1:1 mixture of Iscove's Modified Dulbecco's Medium (IMDM) and RPMI 1640 medium containing 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components were obtained from Sigma or equivalent supplier, and hereafter are referred to as EMSK negative medium). Prior to infection, the cell monolayers were washed once with EMSK negative medium to remove traces of fetal bovine serum. The SPV contained in the initial inoculum (0.5 ml for 10 cm plate; 10 ml for T175 cm flask) was then allowed to absorb onto the cell monolayer for two hours, being redistributed every half hour. After this period, the original inoculum was brought up to the recommended volume with the addition of complete EMSK medium (EMSK negative medium plus 5% fetal bovine serum). The plates were incubated at 37.degree. C. in 5% CO.sub.2 until cytopathic effect was complete. The medium and cells were harvested and frozen in a 50 ml conical screw cap tube at -70.degree. C. Upon thawing at 37.degree. C., the virus stock was aliquoted into 1.0 ml vials and refrozen at -70.degree. C. The titers were usually about 10.sup.6 PFU/ml.
PREPARATION OF SPV DNA. For swinepox virus DNA isolation, a confluent monolayer of EMSK cells in a T175 cm.sup.2 flask was infected at a multiplicity of 0.1 and incubated 4-6 days until the cells were showing 100% cytopathic effect. The infected cells were then harvested by scraping the cells into the medium and centrifuging at 3000 rpm for 5 minutes in a clinical centrifuge. The medium was decanted, and the cell pellet was gently resuspended in 1.0 ml Phosphate Buffer Saline (PBS: 1.5 g Na.sub.2 HPO.sub.4, 0.2 g KH.sub.2 PO.sub.4, 0.8 g NaCL and 0.2 g KCl per liter H.sub.2 O) (per T175) and subjected to two successive freeze-thaws (-70.degree. C. to 37.degree. C.). Upon the last thaw, the cells (on ice) were sonicated two times for 30 seconds each with 45 seconds cooling time in between. Cellular debris was then removed by centrifuging (Sorvall RC-5B superspeed centrifuge) at 3000 rpm for 5 minutes in a HB4 rotor at 4.degree. C. SPV virions, present in the supernatant, were then pelleted by centrifugation at 15,000 rpm for 20 minutes at 4.degree. C. in a SS34 rotor (Sorvall) and resuspended in 10 mM Tris (pH 7.5). This fraction was then layered onto a 36% sucrose gradient (w/v in 10 mM tris pH 7.5) and centrifuged (Beckman L8-70M Ultracentrifuge) at 18,000 rpm for 60 minutes in a SW41 rotor (Beckman) at 4.degree. C. The virion pellet was resuspended in 1.0 ml of 10 mM tris pH 7.5 and sonicated on ice for 30 seconds. This fraction was layered onto a 20% to 50% continuous sucrose gradient and centrifuged 16,000 rpm for 60 minutes in a SW41 rotor at 4.degree. C. The SPV virion band located about three quarters down the gradient was harvested, diluted with 20% sucrose and pelleted by centrifugation at 18,000 rpm for 60 minutes in a SW41 rotor at 4.degree. C. The resultant pellet was then washed once with 10 mM Tris pH 7.5 to remove traces of sucrose and finally resuspended in 10 mM Tris pH 7.5. SPV DNA was then extracted from the purified virions by lysis (4 hours at 60.degree. C.) induced by the addition of EDTA, SDS, and proteinase K to final concentrations of 20 mM, 0.5% and 0.5 mg/ml, respectively. After digestion, three phenol:chloroform (1:1) extractions were conducted and the sample precipitated by the addition of two volumes of absolute ethanol and incubation at -20.degree. C. for 30 minutes. The sample was then centrifuged in an Eppendorf minifuge for 5 minutes at full speed. The supernatant was decanted, and the pellet air dried and rehydrated in 0.01M Tris pH 7.5, 1 mM EDTA at 4.degree. C.
PREPARATION OF INFECTED CELL LYSATES. For cell lysate preparation, serum free medium was used. A confluent monolayer of cells (EMSK, ESK-4, PK-15 or Vero for SPV or VERO for PRV) in a 25 cm.sup.2 flask or a 60 mm petri dish was infected with 100 .mu.l of virus sample. 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. The cell pellet was resuspended in 250 .mu.l of disruption buffer (2% sodium dodecyl sulfate, 2% .beta.-mercapto-ethanol). The samples were sonicated for 30 seconds on ice and stored at -20.degree. C.
WESTERN BLOTTING PROCEDURE. Samples of lysates and protein standards were run on a polyacrylamide gel according to the procedure of Laemnli (1970). After gel electrophoresis the proteins were transferred and processed according to Sambrook et al. (1982). The primary antibody was a swine anti-PRV serum (Shope strain; lot370, PDV8201, NVSL, Ames, IA) diluted 1:100 with 5% non-fat dry milk in Tris-sodium chloride, and sodium Azide (TSA: 6.61 g Tris-HCl, 0.97 g Tris-base, 9.0 g NaCl and 2.0 g Sodium Azide per liter H.sub.2 O). The secondary antibody was a goat anti-swine alkaline phosphatase conjugate diluted 1:1000 with TSA.
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). Except as noted, these were used with minor variation.
DNA SEQUENCING. Sequencing was performed using the USB Sequenase Kit and .sup.35 S-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.TM. and Supersee.TM. programs from Coral Software.
CLONING WITH THE POLYMERASE CHAIN REACTION. 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. The primers used in each case are detailed in the descriptions of the construction of homology vectors below.
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. This method relies upon the homologous recombination between the swinepox virus DNA and the plasmid homology vector DNA which occurs in the tissue culture cells containing both swinepox virus DNA and transfected plasmid homology vector. For homologous recombination to occur, the monolayers of EMSK cells are infected with S-SPV-001 (Kasza SPV strain, 17) at a multiplicity of infection of 0.01 PFU/cell to introduce replicating SPV (i.e. DNA synthesis) into the cells. The plasmid homology vector DNA is then transfected into these cells according to the INFECTION--TRANSFECTION PROCEDURE. The construction of homology vectors used in this procedure is described below
INFECTION--TRANSFECTION PROCEDURE. 6 cm plates of EMSK cells (about 80% confluent) were infected with S-SPV-001 at a multiplicity of infection of 0.01 PFU/cell in EMSK negative medium and incubated at 37.degree. C. in a humidified 5% CO.sub.2 environment for 5 hours. The transfection procedure used is essentially that recommended for Lipofectin.TM. Reagent (BRL). Briefly, for each 6 cm plate, 15 .mu.g of plasmid DNA was diluted up to 100 .mu.l with H.sub.2 O. Separately, 50 micrograms of Lipofectin Reagent was diluted to 100 .mu.l with H.sub.2 O. The 100 .mu.l of diluted Lipofectin Reagent was then added dropwise to the diluted plasmid DNA contained in a polystyrene 5 ml snap cap tube and mixed gently. The mixture was then incubated for 15-20 minutes at room temperature. During this time, the virus inoculum was removed from the 6 cm plates and the cell monolayers washed once with EMSK negative medium. Three ml of EMSK negative medium was then added to the plasmid DNA/lipofectin mixture and the contents pipetted onto the cell monolayer. The cells were incubated overnight (about 16 hours) at 37.degree. C. in a humidified 5% CO.sub.2 environment. The next day the 3 ml of EMSK negative medium was removed and replaced with 5 ml EMSK complete medium. The cells were incubated at 37.degree. C. in 5% CO.sub.2 for 3-7 days until cytopathic effect from the virus was 80-100%. Virus was harvested as described above for the preparation of virus stocks. This stock was referred to as a transfection stock and was subsequently screened for recombinant virus by the BLUOGAL SCREEN FOR RECOMBINANT SWINEPOX VIRUS OR CPRG SCREEN FOR RECOMBINANT SWINEPOX VIRUS.
SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). When the E. coli .beta.-galactosidase (lacZ) marker gene was incorporated into a recombinant virus the plaques containing the recombinants were visualized by one of two simple methods. In the first method, the chemical Bluogal.TM. (Bethesda Research Labs) was incorporated (200 .mu.g/ml) into the agarose overlay during the plaque assay, and plaques expressing active .beta.-galactosidase turned blue. The blue plaques were then picked onto fresh cells (EMSK) and purified by further blue plaque isolation. In the second method, CPRG (Boehringer Mannheim) was incorporated (400 .mu.g/ml) into the agarose overlay during the plaque assay, and plaques expressing active .beta.-galactosidase turned red. The red plaques were then picked onto fresh cells (EMSK) and purified by further red plaque isolation. In both cases viruses were typically purified with three rounds of plaque purification.
SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV USING BLACK PLAQUE ASSAYS. To analyze expression of foreign antigens expressed by recombinant swinepox viruses, monolayers of EMSK cells were infected with recombinant SPV, overlayed with nutrient agarose media and incubated for 6-7 days at 37.degree. C. for plaque development to occur. The agarose overlay was then removed from the dish, the cells fixed with 100% methanol for 10 minutes at room temperature and the cells air dried. Fixation of the cells results in cytoplasmic antigen as well as surface antigen detection whereas specific surface antigen expression can be detected using non-fixed cells. The primary antibody was then diluted to the appropriate dilution with PBS and incubated on the cell monolayer for 2 hours at room temperature. To detect PRV g50 (gpD) expression from S-SPV-008, swine anti-PRV serum (Shope strain; lot370, PDV8201, NVSL, Ames, IA) was used (diluted 1:100). To detect NDV HN expression from S-SPV-009, a rabbit antiserum specific for the HN protein (rabbit anti-NDV#2) was used (diluted 1:1000). Unbound antibody was then removed by washing the cells three times with PBS at room temperature. The secondary antibody, either a goat anti-swine (PRV g50 (gpD); S-SPV-008) or goat anti-rabbit (NDV HN; S-SPV-009), horseradish peroxidase conjugate was diluted 1:250 with PBS and incubated with the cells for 2 hours at room temperature. Unbound secondary antibody was then removed by washing the cells three times with PBS at room temperature. The cells were then incubated 15-30 minutes at room temperature with freshly prepared substrate solution (100 .mu.g/ml 4-chloro-1-naphthol, 0.003% H.sub.2 O.sub.2 in PBS). Plaques expressing the correct antigen stain black.
PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS. Viral glycoproteins are purified using antibody affinity columns. To produce monoclonal antibodies, 8 to 10 week old BALB/c female mice are vaccinated intraperitoneally seven times at two to four week intervals with 10.sup.7 PFU of S-SPV-009, -014, -016, -017, -018, or -019. Three weeks after the last vaccination, mice are injected intraperitoneally with 40 mg of the corresponding viral glycoprotein. Spleens are removed from the mice three days after the last antigen dose.
Splenocytes are fused with mouse NS1/Ag4 plasmacytoma cells by the procedure modified from Oi and Herzenberg, (41). Splenocytes and plasmacytoma cells are pelleted together by centrifugation at 300.times.g for 10 minutes. One ml of a 50% solution of polyethylene glycol (m.w. 1300-1600) is added to the cell pellet with stirring over one minute. Dulbecco's modified Eagles's medium (5 ml) is added to the cells over three minutes. Cells are pelleted by centrifugation at 300.times.g for 10 minutes and resuspended in medium with 10% fetal bovine serum and containing 100 mM hypoxanthine, 0.4 mM aminopterin and 16 mM thymidine (HAT). Cells (100 ml) are added to the wells of eight to ten 96-well tissue culture plates containing 100 ml of normal spleen feeder layer cells and incubated at 37.degree. C. Cells are fed with fresh HAT medium every three to four days.
Hybridoma culture supernatants are tested by the ELISA ASSAY in 96-well microtiter plates coated with 100 ng of viral glycoprotein. Supernatants from reactive hybridomas are further analyzed by black-plaque assay and by Western Blot. Selected hybridomas are cloned twice by limiting dilution. Ascetic fluid is produced by intraperitoneal injection of 5.times.10.sup.6 hybridoma cells into pristane-treated BALB/c mice.
Cell lysates from S-SPV-009, -014, -016, -017, -018, or -019 are obtained as described in PREPARATION OF INFECTED CELL LYSATES. The glycoprotein-containing cell lysates (100 mls) are passed through a 2-ml agarose affinity resin to which 20 mg of glycoprotein monoclonal antibody has been immobilized according to manufacturer's instructions (AFC Medium, New Brunswick Scientific, Edison, N.J.). The column is washed with 100 ml of 0.1% Nonidet P-40 in phosphate-buffered saline (PBS) to remove nonspecifically bound material. Bound glycoprotein is eluted with 100 mM carbonate buffer, pH 10.6 (40). Pre- and posteluted fractions are monitored for purity by reactivity to the SPV monoclonal antibodies in an ELISA system.
ELISA ASSAY. A standard enzyme-linked immunosorbent assay (ELISA) protocol is used to determine the immune status of cattle following vaccination and challenge.
A glycoprotein antigen solution (100 ml at ng/ml in PBS) is allowed to absorb to the wells of microtiter dishes for 18 hours at 4.degree. C. The coated wells are rinsed one time with PBS. Wells are blocked by adding 250 ml of PBS containing 1% BSA (Sigma) and incubating 1 hour at 37.degree. C. The blocked wells are rinsed one time with PBS containing 0.02% Tween 20. 50 ml of test serum (previously diluted 1:2 in PBS containing 1% BSA) are added to the wells and incubated 1 hour at 37.degree. C. The antiserum is removed and the wells are washed 3 times with PBS containing 0.02% Tween 20. 50 ml of a solution containing anti-bovine IgG coupled to horseradish peroxidase (diluted 1:500 in PBS containing 1% BSA, Kirkegaard and Perry Laboratories, Inc.) is added to visualize the wells containing antibody against the specific antigen. The solution is incubated 1 hour at 37.degree. C., then removed and the wells are washed 3 times with PBS containing 0.02% Tween 20. 100 ml of substrate solution (ATBS, Kirkegaard and Perry Laboratories, Inc.) are added to each well and color is allowed to develop for 15 minutes. The reaction is terminated by addition of 0.1M oxalic acid. The color is read at absorbance 410 nm on an automatic plate reader.
STRATEGY FOR THE CONSTRUCTION OF SYNTHETIC POX VIRAL PROMOTERS. For recombinant swinepox vectors synthetic pox promoters offer several advantages including the ability to control the strength and timing of foreign gene expression. We chose to design three promoter cassettes LP1, EP1 and LP2 based on promoters that have been defined in the vaccinia virus (1, 7 and 8). Each cassette was designed to contain the DNA sequences defined in vaccinia flanked by restriction sites which could be used to combine the cassettes in any order or combination. Initiator methionines were also designed into each cassette such that inframe fusions could be made at either EcoRI or BamHI sites. A set of translational stop codons in all three reading frames and an early transcriptional termination signal (9) were also engineered downstream of the inframe fusion site. DNA encoding each cassette was synthesized according to standard techniques and cloned into the appropriate homology vectors (see FIGS. 4A-4C, 5A-5D, and 8A-8C).
VACCINATION STUDIES IN SWINE USING RECOMBINANT SWINEPOX VIRUS CONTAINING PSEUDORABIES VIRUS GLYCOPROTEIN GENES: Young weaned pigs from pseudorabies-free herd are used to test the efficacy of the recombinant swinepox virus containing one or more of the pseudorabies virus glycoprotein genes (SPV/PRV). The piglets are inoculated intradermally or orally about 10.sup.3 to 10.sup.7 plaque forming units (PFU) of the recombinant SPV/PRV viruses.
Immunity is determined by measuring PRV serum antibody levels and by challenging the vaccinated pigs with virulent strain of pseudorabies virus. Three to four weeks post-vaccination, both vaccinated and non-vaccinated groups of pigs are challenged with virulent strain of pseudorabies virus (VDL4892). Post challenge, the pigs are observed daily for 14 days for clinical signs of pseudorabies.
Serum samples are obtained at the time of vaccination, challenge, and at weekly intervals for two to three weeks post-vaccination and assayed for serum neutralizing antibody.
HOMOLOGY VECTOR 515-85.1. The plasmid 515-85.1 was constructed for the purpose of inserting foreign DNA into SPV. It contains a unique AccI restriction enzyme site into which foreign DNA may be inserted. When a plasmid, containing a foreign DNA insert at the AccI site, is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing the foreign DNA will result. A restriction map of the DNA insert in homology vector 515-85.1 is given in FIGS. 4A-4C. It may be constructed utilizing standard recombinant DNA techniques (22 and 29), by joining two restriction fragments from the following sources. The first fragment is an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). The second fragment is an approximately 3302 base pair HindIII to BglII restriction sub-fragment of the SPV HindIII restriction fragment M (23).
HOMOLOGY VECTOR 520-17.5. The plasmid 520-17.5 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli .beta.-galactosidase (lacZ) marker gene flanked by SPV DNA. Upstream of the marker gene is an approximately 2156 base pair fragment of SPV DNA. Downstream of the marker gene is an approximately 1146 base pair fragment of SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the marker gene will result. Note that the .beta.-galactosidase (lacZ) marker gene is under the control of a synthetic early/late pox promoter. A detailed description of the plasmid is given in FIGS. 4A-4C. It may be constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 4. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 2156 base pair HindIII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 3006 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 3 is an approximately 1146 base pair AccI to BglII restriction sub-fragment of the SPV HindIII fragment M (23).
HOMOLOGY VECTOR 538-46.16. The plasmid 538-46.16 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli .beta.-galactosidase (lacZ) marker gene and the PRV g50 (gpD) gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1146 base pair fragment of SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes will result. Note that the .beta.-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the g50 (gpD) gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in FIGS. 5A-5D. It may be constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGS. 5A-5D. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 2156 base pair HindIII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 3006 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 3 is an approximately 1571 base pair EcoRI to StuI restriction sub-fragment of the PRV BamHI fragment 7 (21). Note that the EcoRI site was introduced in to this fragment by PCR cloning. In this procedure the primers described below were used along with a template consisting of a PRV BamHI #7 fragment subcloned into pSP64. The first primer 87.03 (5'-CGCGAATTCGCTCG CAGCGCTATTGGC-3') (SEQ ID NO:41) sits down on the PRV g50 (gpD) sequence (26) at approximately amino acid 3 priming toward the 3' end of the gene. The second primer 87.06 (5'-GTAGGAGTGGCTGCTGAAG-3') (SEQ ID NO:42) sits down on the opposite strand at approximately amino acid 174 priming toward the 5' end of the gene. The PCR product may be digested with EcoRI and SalII to produce an approximately 509 base pair fragment. The approximately 1049 base pair SalI to StuI sub-fragment of PRV BamHI #7 may then be ligated to the approximately 509 base pair EcoRI to SalI fragment to generate the approximately 1558 base pair EcoRI to StuI fragment 3. Fragment 4 is an approximately 1146 base pair AccI to BglII restriction sub-fragment of the SPV HindIII fragment M (23).
HOMOLOGY VECTOR 570-91.21. The plasmid 570-91.21 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the PRV gIII (gpC) gene flanked by SPV DNA. Upstream of the foreign DNA genes is an approximately 1146 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the .beta.-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the gIII (gpC) gene is under the control of a synthetic early pox promoter (EP2). A detailed description of the plasmid is given in FIGS. 10A-10D. It may be constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGS. 10A-10D. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1146 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 3 is an approximately 2378 base pair NcoI to NcoI fragment of plasmid 251-41.A, a subfragment of PRV BamHI #2 and #9. EcoRI linkers have replaced the NcoI and NcoI sites at the ends of this fragment. Fragment 4 is an approximately 2156 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII fragment M (23). The AccI sites in fragments 1 and 4 have been converted to PstI sites using synthetic DNA linkers.
HOMOLOGY VECTOR 570-91.41. The plasmid 570-91.41 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the PRV gIII (gpC) gene flanked by SPV DNA. Upstream of the foreign DNA genes is an approximately 2156 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1146 base pair fragment of SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the .beta.-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the gIII (gpC) gene is under the control of a synthetic early late pox promoter (EP1LP2). A detailed description of the plasmid is given in FIGS. 11A-11D. It may be constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGS. 11A-11D. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1146 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 3 is an approximately 2378 base pair NcoI to NcoI fragment of plasmid 251-41.A, a subfragment of PRV BamHI #2 and #9. EcoRI linkers have replaced the NcoI and NcoI sites at the ends of this fragment. Fragment 4 is an approximately 2156 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII fragment M (23). The AccI sites in fragments 1 and 4 have been converted to PstI sites using synthetic DNA linkers.
HOMOLOGY VECTOR 570-91.64. The plasmid 570-91.64 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the PRV gIII (gpC) gene flanked by SPV DNA. Upstream of the foreign DNA genes is an approximately 1146 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the .beta.-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the gIII (gpC) gene is under the control of a synthetic late early pox promoter (LP2EP2). A detailed description of the plasmid is given in FIGS. 12A-12D. It may be constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGS. 12A-12D. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1146 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 3 is an approximately 2378 base pair NcoI to NcoI fragment of plasmid 25-41.A, a subfragment of PRV BamHI #2 and #9. EcoRI linkers have replaced the NcoI and NcoI sites at the ends of this fragment. Fragment 4 is an approximately 2156 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII fragment M (23). The AccI sites in fragments 1 and 4 have been converted to PstI sites using synthetic DNA linkers.
HOMOLOGY VECTOR 538-46.26. The plasmid 538-46.26 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E.coli .beta.-galactosidase (lacZ) marker gene and the Newcastle Disease Virus (NDV) hemagglutinin-Neuraminidase (HN) gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1146 base pair fragment of SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes will result. Note that the .beta.-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the HN gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in FIGS. 8A-8C. It may be constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGS. 8A-8C. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 2156 base pair HindIII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 1810 base pair AvaII to NaeI restriction fragment of a NDV HN cDNA clone. The sequence of the HN cDNA clone is given in FIG. 7. The cDNA clone was generated from the B1 strain of NDV using standard cDNA cloning techniques (14). Fragment 3 is an approximately 3006 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 4 is an approximately 1146 base pair AccI to BglII restriction sub-fragment of the SPV HindIII fragment M (23).
HOMOLOGY VECTOR 599-65.25. The plasmid 599-65.25 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the ILT gpG gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1146 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. When the plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the B-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the ILT gpG gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in FIG. 13. It may be constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 13. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1146 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 1073 base pair EcoRI to MboI fragment. Note that the EcoRI site was introduced by PCR cloning. In this procedure, the primers described below were used with a template consisting of a 2.6 kb Sst I to Asp718I subfragment of a 5.1 kbAsp718I fragment of ILT virus genome. The first primer 91.13 (5'-CCGAATTCCGGCTTCAGTAACATAGGATCG -3') (SEQ ID NO: 103) sits down on the ILT gpG sequence at amino acid 2. It adds an additional asparagine residue between amino acids 1 and 2 and also introduces an EcoRI restriction site. The second primer 91.14 (5'-GTACCCATACTGGTCGTGGC-3') (SEQ ID NO: 104) sits down on the opposite strand at approximately amino acid 196 priming toward the 5' end of the gene. The PCR product is digested with EcoRI and BamHI to produce an approximately 454 base pair fragment. The approximately 485 base pair MboI sub-fragment of ILT Asp718I (5.1 kb) fragment is ligated to the approximately 454 base pair EcoRI to BamHI fragment to generate fragment 2 from EcoRI to MboI which is approximately 939 base pairs (293 amino acids) in length. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 4 is an approximately 2156 base pair AccI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites of fragments 1 and 4 have been converted to PstI sites using synthetic DNA linkers.
HOMOLOGY VECTOR 624-20.1C. The plasmid 624-20.1C was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the ILT gpI gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1146 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. When the plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the B-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the ILT gpI gene is under the control of a synthetic late/early pox promoter (LP2EP2). A detailed description of the plasmid is given in FIGS. 14A-14D. It may be constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGS. 14A-14D. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1146 base pair Bgl II to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 1090 base pair fragment with EcoRI and BamHI restriction sites at the ends synthesized by PCR cloning and containing the entire amino acid coding sequence of the ILT gpI gene. The ILT gpI gene was synthesized in two separate PCR reactions. In this procedure, the primers described below were used with a template consisting the 8.0 kb ILT Asp 718I fragment. The first primer 103.6 (5'-CCGGAATTCGCTACTT GGAACTCTGG-3') (SEQ ID NO 105) sits down on the ILT gpI sequence at amino acid number 2 and introduces an EcoRI site at the 5' end of the ILT gpI gene. The second primer 103.3 (5'-CATTGTCCCGAGACGGACAG-3') (SEQ ID NO. 106) sits down on the ILT gpI sequence at approximately amino acid 269 on the opposite strand to primer 103.6 and primes toward the 5' end of the gene. The PCR product was digested with EcoRI and BglI (BglI is located approximately at amino acid 209 which is 179 base pairs 5' to primer 2) to yield a fragment 625 base pairs in length corresponding to the 5' end of the ILT gpI gene. The third primer 103.4 (5'-CGCGATCCAACTATCGGTG-3') (SEQ ID NO. 107) sits down on the ILT gpI gene at approximately amino acid 153 priming toward the 3' end of the gene. The fourth primer 103.5 (5' GCGGATCCACATTCAG ACTTAATCAC-3') (SEQ ID NO. 108) sits down at the 3' end of the ILT gpI gene 14 base pairs beyond the UGA stop codon, introducing a BamHI restriction site and priming toward the 5' end of the gene. The PCR product is digested with Bgl I (at amino acid 209) and BamHI to yield a fragment 476 base pairs in length corresponding to the 3' end of the ILT gpI gene. Fragment 2 consists of the products of the two PCR reactions ligated together to yield an ILT gpI gene which is a EcoRI to BamHI fragment approximately 1101 base pairs (361 amino acids) in length. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 4 is an approximately 2156 base pair AccI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 4 were converted to unique NotI sites using NotI linkers.
HOMOLOGY VECTOR 614-83.18. The plasmid 614-83.18 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the IBR gpG gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1146 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. When the plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the B-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the IBR gG gene is under the control of a synthetic late/early pox promoter (LP2EP2). A detailed description of the plasmid is given in FIGS. 15A-15D. It may be constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGS. 15A-15D. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1146 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is an approximately 1085 base pair fragment synthesized by PCR cloning with EcoRI and BamHI restriction sites at the ends and containing the amino acid coding sequence from amino acids 2 to 362 of the IBR gpG gene. In the PCR cloning procedure, the primers described below were used with a template consisting of the IBR-000 virus (Cooper strain). The first primer 106.9 (5'-ATGAATTCCCCTGCCGCCCGGACCGGCACC-3') (SEQ ID NO. 109) sits down on the IBR gpG sequence at amino acid number 1 and introduces an EcoRI site at the 5' end of the IBR gpG gene and two additional amino acids between amino acids 1 and 2. The second primer 106.8 (5'-CATGGATCCCGCTCGAGGCGAGCGGGCTCC-3') (SEQ ID NO. 110) sits down on the IBR gpG sequence at approximately amino acid 362 on the opposite strand to primer 1 and primes synthesis toward the 5' end of the IBR gpG gene. Fragment 2 was generated by digesting the PCR product with EcoRI and BamHI to yield a fragment 1085 base pairs in length corresponding to the amino terminal 362 amino acids (approximately 80%) of the IBR gpG gene. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 4 is an approximately 2156 base pair AccI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 4 were converted to unique NotI sites using NotI linkers.
HOMOLOGY VECTOR FOR CONSTRUCTING S-SPV-019 (LacZ/IBR gpE HOMOLOGY VECTOR):
This lacZ/IBR gpE homology vector is used to insert foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the IBR gpE gene flanked by SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes will result. Note that the B-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter and the gpE gene is under the control of a synthetic late/early pox promoter. The homology vector may be constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the appropriate synthetic DNA sequences. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). The upstream SPV homology is an approximately 1146 base pair BglIII to AccI restriction sub-fragment of the SPV HindIII fragment M (23). The IBR gE gene is an approximately 1888 base pair fragment synthesized by PCR cloning with EcoRI and BamHI ends. In the PCR cloning procedure, the primers described below were used with a template consisting of the IBR-000 VIRUS (Cooper strain). The first primer 4/93.17DR (5'-CTGGTTCGGCCCAGAATTCTATGGGTCTCGCGCGGCTCGTGG-3' (SEQ ID NO. 111) sits down on the IBR gpE gene at amino acid number 1 and introduces an EcoRI site at the 5' end of the IBR gpE gene and adds two additional amino acids at the amino terminus of the protein. The second primer 4/93.18DR (5'-CTCGCTCGCCCAGGATCCCTAGCGGAGGATGGACTTGAGTCG-3') (SEQ ID NO. 112) sits down on the IBR gpE sequence at approximately amino acid 648 on the opposite strand to primer 1 and primes synthesis toward the 5' end of the IBR gpE gene. The lacZ promoter and marker gene is identical to the one used in plasmid 520-17.5. The downstream SPV homology is an approximately 2156 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII restriction fragment M (23). The AccI site in the SPV homology vector is converted to a unique XbaI site.
HOMOLOGY VECTOR FOR CONSTRUCTING S-SPV-018 (LacZ/PRV gpE HOMOLOGY VECTOR):
This homology vector is constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli B-galactosidase (lacZ) marker gene and the PRV gpE gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1146 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2156 base pair fragment of SPV DNA. When the plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing the DNA coding for the foreign genes results. Note that the B-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the PRV gpE gene is under the control of a synthetic early/late pox promoter (EP1LP2). The homology vector is constructed utilizing standard recombinant DNA techniques (22,30), by joining restriction fragments from the following sources with synthetic DNA sequences. The plasmid vector is derived from an approximately 2972 base pair HindIII to BamHI restriction fragment pSP64 (Promega). Fragment 1 is an approximately 1146 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2 is the lacZ promoter and marker gene which is identical to the one used in plasmid 520-17.5. Fragment 3 is an approximately 2484 base pair DraI to MluI sub-fragment of PRV derived from the PRV BamHI #7 DNA fragment. The DraI site is converted to an EcoRI site through the use of a synthetic DNA linker. The DraI site sits 45 base pairs upstream of the natural gpE start codon and extends the open reading frame at the amino terminus of the protein for 15 amino acids. The synthetic pox promoter/EcoRI DNA linker contributes another 4 amino acids. Therefore, the engineered gpE gene contains 19 additional amino acids fused to the amino terminus of gpE. The nineteen amino acids are Met-Asn-Ser-Gly-Asn-Leu-Gly-Thr-Pro-Ala-Ser-Leu-Ala-His-Thr-Gly-Val-Glu-Thr. Fragment 4 is an approximately 2156 base pair AccI to HindIII subfragment of the SPV HindIII fragment M (23). The AccI sites of fragments 1 and 4 are converted to PstI sites using synthetic DNA linkers.
HOMOLOGY VECTOR 520-90.15. The plasmid 520-90.15 was constructed for the purpose of inserting foreign DNA into SPV. It contains a unique NdeI restriction enzyme site into which foreign DNA may be inserted. When a plasmid, containing a foreign DNA insert at the NdeI site, is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing the foreign DNA will result. Plasmid 520-90.15 was constructed utilizing standard recombinant DNA techniques (22 and 30), by joining two restriction fragments from the following sources. The first fragment is an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). The second fragment is an approximately 1700 base pair HindIII to BamHI restriction subfragment of the SPV HindIII restriction fragment G (23).
EXAMPLES
Example 1
Homology Vector 515-85.1. The homology vector 515-85.1 is a plasmid useful for the insertion of foreign DNA into SPV. Plasmid 515-85.1 contains a unique AccI restriction site into which foreign DNA may be cloned. A plasmid containing such a foreign DNA insert may be used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV to generate a SPV containing the foreign DNA. For this procedure to be successful it is important that the insertion site (AccI) be in a region non-essential to the replication of the SPV and that the site be flanked with swinepox virus DNA appropriate for mediating homologous recombination between virus and plasmid DNAs. We have demonstrated that the AccI site in homology vector 515-85.1 may be used to insert foreign DNA into at least three recombinant SPV (see examples 2-4).
In order to define an appropriate insertion site, a library of SPV HindIII restriction fragments was generated. Several of these restriction fragments (HindIII fragments G, J, and M see FIG. 1) were subjected to restriction mapping analysis. Two restriction sites were identified in each fragment as potential insertion sites. These sites included HpaI and NruI in fragment G, BalI and XbaI in fragment J, and AccI and PstI in fragment M. A .beta.-galactosidase (lacZ) marker gene was inserted in each of the potential sites. The resulting plasmids were utilized in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The generation of recombinant virus was determined by the SCREEN FOR RECOMBINANT SPV EXPRESSING .beta.-GALACTOSIDASE ASSAYS. Four of the six sites were found to generate recombinant virus, however the ability of each of these viruses to be purified away from the parental SPV varied greatly. In one case virus could not be purified above the level of 1%, in another case virus could not be purified above the level of 50%, and in a third case virus could not be purified above the level of 90%. The inability to purify these viruses indicates instability at the insertion site. This makes the corresponding sites inappropriate for insertion of foreign DNA. However the insertion at one site, the AccI site of Homology vector 515-85.1, resulted in a virus which was easily purified to 100% (see example 2), clearly defining an appropriate site for the insertion of foreign DNA.
The homology vector 515-85.1 was further characterized by DNA sequence analysis. Two regions of the homology vector were sequenced. The first region covers a 599 base pair sequence which flanks the unique AccI site (see FIGS. 2A-2B). The second region covers the 899 base pairs upstream of the unique HindIII site (see FIGS. 2A-2B). The sequence of the first region codes for an open reading frame (ORF) which shows homology to amino acids 1 to 115 of the vaccinia virus (VV) 01L open reading frame identified by Goebel et al, 1990 (see FIGS. 3A-3B). The sequence of the second region codes for an open reading frame which shows homology to amino acids 568 to 666 of the same vaccinia virus 01L open reading frame (see FIGS. 3A-3B). These data suggest that the AccI site interrupts the presumptive VV 01L-like ORF at approximately amino acid 41, suggesting that this ORF codes for a gene non-essential for SPV replication. Goebel et al. suggest that the VV 01L ORF contains a leucine zipper motif characteristic of certain eukaryotic transcriptional regulatory proteins, however they indicate that it is not known whether this gene is essential for virus replication.
The DNA sequence located upstream of the VV 01L-like ORF (see FIG. 2A) would be expected to contain a swinepox viral promoter. This swinepox viral promoter will be useful as the control element of foreign DNA introduced into the swinepox genome.
Example 2
S-SPV-003
S-SPV-003 is a swinepox virus that expresses a foreign gene. The gene for E.coli .beta.-galactosidase (lacZ gene) was inserted into the SPV 515-85.1 ORF. The foreign gene (lacZ) is under the control of a synthetic early/late promoter (EP1LP2).
S-SPV-003 was derived from S-SPV-001 (Kasza strain). This was accomplished utilizing the homology vector 520-17.5 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING .beta.-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-003. This virus was assayed for .beta.-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable and expressing the foreign gene. The assays described here were carried out in VERO cells as well as EMSK cells, indicating that VERO cells would be a suitable substrate for the production of SPV recombinant vaccines. S-SPV-003 has been deposited with the ATCC under Accession No. VR 2335.
Example 3
S-SPV-008
S-SPV-008 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli .beta.-galactosidase (lacZ gene) and the gene for pseudorabies virus (PRV) g50 (gpD) (26) were inserted into the SPV 515-85.1 ORF. The lacZ gene is under the control of a synthetic late promoter (LP1) and the g50 (gp)D gene is under the control of a synthetic early/late promoter (EP1LP2).
S-SPV-008 was derived from S-SPV-001 (Kasza strain). This was accomplished utilizing the homology vector 538-46.16 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING .beta.-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-008. This virus was assayed for .beta.-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-SPV-008 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Swine anti-PRV serum was shown to react specifically with S-SPV-008 plaques and not with S-SPV-009 negative control plaques. All S-SPV-008 observed plaques reacted with the swine antiserum indicating that the virus was stably expressing the PRV foreign gene. The black plaque assay was also performed on unfixed monolayers. The SPV plaques on the unfixed monolayers also exhibited specific reactivity with swine anti-PRV serum indicating that the PRV antigen is expressed on the infected cell surface.
To confirm the expression of the PRV g50 (gpD) gene product, cells were infected with SPV and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. The swine anti-PRV serum was used to detect expression of PRV specific proteins. As shown in FIG. 6, the lysate from S-SPV-008 infected cells exhibits a specific band of approximately 48 kd, the reported size of PRV g50 (gpD) (35).
PRV g50 (gpD) is the g50 (gpD) homologue of HSV-1 (26). Several investigators have shown that VV expressing HSV-1 g50 (gpD) will protect mice against challenge with HSV-1 (6 and 34). Therefore the S-SPV-008 should be valuable as a vaccine to protect swine against PRV disease.
It is anticipated that several other PRV glycoproteins will be useful in the creation of recombinant swinepox vaccines to protect against PRV disease. These PRV glycoproteins include gpII (28), gpIII (27), and gpH (19). The PRV gpIII coding region has been engineered behind several synthetic pox promoters. The techniques utilized for the creation of S-SPV-008 will be used to create recombinant swinepox viruses expressing all four of these PRV glycoprotein genes. Such recombinant swinepox viruses will be useful as vaccines against PRV disease. Since the PRV vaccines described here do not express PRV gpX or gpI, they would be compatible with current PRV diagnostic tests (gX HerdChek.RTM., gI HerdChek.RTM. and ClinEase.RTM.) which are utilized to distinguish vaccinated animals from infected animals. S-SPV-008 has been deposited with the ATCC under Accession No. VR 2339.
Example 4
S-SPV-011
S-SPV-011 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ) and the gene for pseudorabies virus gIII (gpC) were inserted into the unique PstI restriction site (PstI linkers inserted into a unique AccI site) of the homology vector 570-33.32. The lac Z gene is under the control of the synthetic late promoter (LP1) and the PRV gIII (gpC) gene is under the control of the synthetic early promoter (EP2).
S-SPV-011 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 570-91.21 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-011. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.
S-SPV-011 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal goat anti-PRV gIII (gpC) antibody was shown to react specifically with S-SPV-011 plaques and not with S-SPV-001 negative control plaques. All S-SPV-011 observed plaques reacted with the swine anti-PRV serum indicating that the virus was stably expressing the PRV foreign gene. The assays described here were carried out in EMSK cells, indicating that EMSK cells would be a suitable substrate for the production of SPV recombinant vaccines.
To confirm the expression of the PRV gIII (gpC) gene product, cells were infected with SPV and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal goat anti-PRV gIII (gpC) antibody was used to detect expression of PRV specific proteins. As shown in FIG. 16, the lysate from S-SPV-011 infected cells exhibits a specific band of approximately 92 kd, the reported size of PRV gIII (gpC) (37).
Recombinant-expressed PRV gIII (gpC) has been shown to elicit a significant immune response in mice and swine (37, 38). Furthermore, when gIII (gpC) is coexpressed with gII (gpB) or g50 (gpD), significant protection from challenge with virulent PRV is obtained (39). Therefore S-SPV011 should be valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gpX or gpI, they would be compatible with current PRV diagnostic tests (gX HerdChek.RTM., gI HerdChek.RTM. and ClinEase.RTM.) which are utilized to distinguish vaccinated animals from infected animals.
Example 5
S-SPV-012
S-SPV-012 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ) and the gene for pseudorabies virus gIII (gpC) were inserted into the unique PstI restriction site (PstI linkers inserted into a unique AccI site) of the homology vector 570-33.32. The lacZ gene is under the control of the synthetic late promoter (LP1) and the PRV gIII (gpC) gene is under the control of the synthetic early late promoter (EP1LP2).
S-SPV-012 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 570-91.41 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-012. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.
S-SPV-012 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal goat anti-PRV gIII (gpC) antibody was shown to react specifically with S-SPV-012 plaques and not with S-SPV-001 negative control plaques. All S-SPV-012 observed plaques reacted with the swine anti-PRV serum, indicating that the virus was stably expressing the PRV foreign gene. The assays described here were carried out in EMSK and VERO cells, indicating that EMSK cells would be a suitable substrate for the production of SPV recombinant vaccines.
To confirm the expression of the PRV gIII (gpC) gene product, cells were infected with S-SPV-012 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal goat anti-PRV gIII (gpC) antibody was used to detect expression of PRV specific proteins. As shown in FIG. 16, the lysate from S-SPV-012 infected cells exhibits two specific bands which are the reported size of PRV gIII (gpC) (37)--a 92 kd mature form and a 74 kd pre-golgi form.
Recombinant-expressed PRV gIII (gpC) has been shown to elicit a significant immune response in mice and swine (37, 38). Furthermore, when gIII (gpC) is coexpressed with gII (gpB) or g50 (gpD), significant protection from challenge with virulent PRV is obtained (39). Therefore S-SPV-012 should be valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gpX or gpI, they would be compatible with current PRV diagnostic tests (gX HerdChek.RTM., gI HerdChek.RTM. and ClinEase.RTM.) which are utilized to distinguish vaccinated animals from infected animals.
Example 6
S-SPV-013
S-SPV-013 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ) and the gene for pseudorabies virus gIII (gpC) were inserted into the unique PstI restriction site (PstI linkers inserted into a unique AccI site) of the homology vector 570-33.32. The lacZ gene is under the control of the synthetic late promoter (LP1) and the PRV gIII (gpC) gene is under the control of the synthetic late early promoter (LP2EP2).
S-SPV-013 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 570-91.64 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-013. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.
S-SPV-013 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal goat anti-PRV gIII (gpC) antibody was shown to react specifically with S-SPV-013 plaques and not with S-SPV-001 negative control plaques. All S-SPV-013 observed plaques reacted with the swine anti-PRV serum indicating that the virus was stably expressing the PRV foreign gene. The assays described here were carried out in EMSK and VERO cells, indicating that EMSK cells would be a suitable substrate for the production of SPV recombinant vaccines.
To confirm the expression of the PRV gIII (gpC) gene product, cells were infected with SPV and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal goat anti-PRV gIII (gpC) antibody was used to detect expression of PRV specific proteins. As shown in FIG. 16, the lysate from S-SPV-013 infected cells exhibits two specific bands which are the reported size of PRV gIII (gpC) (37)--a 92 kd mature form and a 74 kd pre-Golgi form.
Recombinant-expressed PRV gIII (gpC) has been shown to elicit a significant immune response in mice and swine (39, 38). Furthermore, when gIII (gpC) is coexpressed with gII (gpB) or g50 (gpD), significant protection from challenge with virulent PRV is obtained. (39) Therefore S-SPV-013 should be valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gpX or gpI, they would be compatible with current PRV diagnostic tests (gX HerdChek.RTM., gI HerdChek.RTM. and ClinEase.RTM.) which are utilized to distinguish vaccinated animals from infected animals. S-SPV-013 has been deposited with the ATCC under Accession No. VR 2418.
Example 7
S-SPV-015
S-SPV-015 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ gene) and the gene for pseudorabies virus (PRV) gII (gpB) are inserted into the SPV 515-85.1 ORF. The lacZ gene is under the control of a synthetic late promoter (LP1) and the gII (gpB) gene is under the control of a synthetic late/early promoter (LP2EP2).
S-SPV-015 can be derived from S-SPV-001 (Kasza strain). This is accomplished utilizing the lacZ/PRV gII (gpB) homology vector (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock is screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS).
Example 8
Recombinant swinepox virus expressing more than one pseudorabies virus (PRV) glycoproteins, which can elicit production of neutralizing antibodies against pseudorabies virus, is constructed in order to obtain a recombinant swinepox virus with enhanced ability to protect against PRV infection than that which can be obtained by using a recombinant swinepox virus expressing only one of those PRV glycoproteins.
There are several examples of such recombinant swinepox virus expressing more than one PRV glycoproteins: a recombinant swinepox virus expressing PRV g50 (gpD) and gIII (gpC), a recombinant swinepox virus expressing PRV g50 (gpD) and gII (gpB); a recombinant swinepox virus expressing PRV gII (gpB) and gIII (gpC); and a recombinant swinepox virus expressing PRV g50 (gpD), gIII (gpC) and gII (gpB). Each of the viruses cited above is also engineered to contain and express E. coli B-galactosidase (lac Z) gene, which will facilitate the cloning of the recombinant swinepox virus.
Listed below are three examples of a recombinant swinepox virus expressing PRV g50 (gpD), PRV gIII (gpC), PRV gII (gpB) and E. coli B-galactosidase (lacZ):
a) Recombinant swinepox virus containing and expressing PRV g50 (gpD) gene, PRV gIII (gpC) gene, PRV gII (gpB) gene and lacZ gene. All four genes are inserted into the unique AccI restriction endonuclease site within the HindIII M fragment of the swinepox virus genome. PRV g50 (gpD) gene is under the control of a synthetic early/late promoter (EP1LP2), PRV gIII (gpC) gene is under the control of a synthetic early promoter (EP2), PRV gII (gpB) gene is under the control of a synthetic late/early promoter (LP2EP2) and lacZ gene is under the control of a synthetic late promoter (LP1).
b) Recombinant swinepox virus containing and expressing PRV g50 (gpD) gene, PRV gIII (gpC) gene, PRV gII (gpB) gene and lacZ gene. All four genes are inserted into the unique AccI restriction endonuclease site within the HindIII M fragment of the swinepox virus genome. PRV g50 (gpD) gene is under the control of a synthetic early/late promoter (EP1LP2), PRV gIII (gpC) gene is under the control of a synthetic early/late promoter (EP1LP2), PRV gII (gpB) gene is under the control of a synthetic late/early promoter (LP2EP2) and lacZ gene is under the control of a synthetic late promoter (LP1).
c) Recombinant swinepox virus containing and expressing PRV g50 (gpD) gene, PRV gIII (gpC) gene, PRV gII (gpB) gene and lacZ gene. All four genes are inserted into the unique AccI restriction endonuclease site within the HindIII M fragment of the swinepox virus genome. PRV g50 (gpD) gene is under the control of a synthetic early/late promoter (EP1LP2), PRV gIII (gpC) gene is under the control of a synthetic late/early promoter (LP2EP2), PRV gII (gpB) gene is under the control of a synthetic late/early promoter (LP2EP2) and lacZ gene is under the control of a synthetic late promoter (LP1).
Example 9
S-SPV-009
S-SPV-009 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli .beta.-galactosidase (lacZ gene) and the gene for Newcastle's Disease virus hemagglutinin (HN) gene were inserted into the SPV 515-85.1 ORF. The lacZ gene is under the control of a synthetic late promoter (LP1) and the HN gene is under the control of an synthetic early/late promoter (EP1LP2).
S-SPV-009 was derived from S-SPV-001 (Kasza strain). This was accomplished utilizing the homology vector 538-46.26 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING .beta.-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-009. This virus was assayed for .beta.-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-SPV-009 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Rabbit anti-NDV HN serum was shown to react specifically with S-SPV-009 plaques and not with S-SPV-008 negative control plaques. All S-SPV-009 observed plaques reacted with the swine antiserum indicating that the virus was stably expressing the NDV foreign gene. S-SPV-009 has been deposited with the ATCC under Acession No. VR 2344).
To confirm the expression of the NDV HN gene product, cells were infected with SPV and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. The rabbit anti-NDV HN serum was used to detect expression of the HN protein. The lysate from S-SPV-009 infected cells exhibited a specific band of approximately 74 kd, the reported size of NDV HN (29).
Example 10
S-SPV-014
S-SPV-014 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ) and the gene for infectious laryngotracheitis virus glycoprotein G (ILT gpG) were inserted into the SPV 570-33.32 ORF (a unique PstI site has replaced the unique AccI site). The lacZ gene is under the control of the synthetic late promoter (LP1), and the ILT gpG gene is under the control of the synthetic early/late promoter (EP1LP2).
S-SPV-014 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 599-65.25 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-014. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene. The assays described here were carried out in ESK-4 cells, indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant vaccines.
To confirm the expression of the ILT gpG gene product, cells were infected with SPV-014 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Peptide antisera to ILT gG was used to detect expression of ILT specific proteins. The lysate from S-SPV-014 infected cells exhibited a band at 43 kd which is the expected size of the ILT gpG protein and additional bands of higher molecular weight which represent glycosylated forms of the protein which are absent in deletion mutants for ILT gpG.
This virus is used as an expression vector for expressing ILT glycoprotein G (gpG). Such ILT gpG is used as an antigen to identify antibodies directed against the wild-type ILT virus as opposed to antibodies directed against gpG deleted ILT viruses. This virus is also used as an antigen for the production of ILT gpG specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the ILT gpG protein. Monoclonal antibodies are generated in mice utilizing this virus according to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials & Methods).
Example 11
S-SPV-016
S-SPV-016 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ) and the gene for infectious laryngotracheitis virus glycoproteini (ILT gpI) were inserted into the SPV 617-48.1 ORF (a unique NotI restriction site has replaced a unique AccI restriction site). The lacZ gene is under the control of the synthetic late promoter (LP1), and the ILT gpI gene is under the control of the synthetic late/early promoter (LP2EP2).
S-SPV-016 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 624-20.1C (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-016. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.
S-SPV-016 was assayed for expression of ILT gpI- and B-galactosidase-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal chicken anti-ILT antibody was shown to react specifically with S-SPV-016 plaques and not with S-SPV-017 negative control plaques. All S-SPV-016 observed plaques reacted with the chicken antiserum indicating that the virus was stably expressing the ILT foreign gene. The assays described here were carried out in ESK-4 cells, indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant vaccines.
To confirm the expression of the ILT gpI gene product, cells were infected with SPV-016 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal chicken anti-ILT antibody was used to detect expression of ILT specific proteins. The lysate from S-SPV-016 infected cells exhibits a range of bands reactive to the anti-ILT antibody from 40 to 200 kd indicating that the ILT gpI may be heavily modified.
This virus is used as an expression vector for expressing ILT glycoprotein I (gpI). Such ILT gpI is used as an antigen to identify antibodies directed against the wild-type ILT virus as opposed to antibodies directed against gpI deleted ILT viruses. This virus is also used as an antigen for the production of ILT gpI specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the ILT gpI protein. Monoclonal antibodies are generated in mice utilizing this virus according to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials & Methods).
Example 12
S-SPV-017
S-SPV-017 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ) and the gene for infectious bovine rhinotracheitis virus glycoprotein G (IBR gpG) were inserted into the SPV 617-48.1 ORF (a unique NotI restriction site has replaced a unique AccI restriction site). The lacZ gene is under the control of the synthetic late promoter (LP1), and the IBR gpG gene is under the control of the synthetic late/early promoter (LP2EP2).
S-SPV-017 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 614-83.18 (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-017.
This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.
S-SPV-017 was assayed for expression of IBR-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Monoclonal antibodies and peptide antisera to IBR gpG were shown to react specifically with S-SPV-017 plaques and not with S-SPV-016 negative control plaques. All S-SPV-017 observed plaques reacted with the antiserum indicating that the virus was stably expressing the IBR foreign gene. The assays described here were carried out in ESK-4 cells, indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant vaccines.
To confirm the expression of the IBR gpG gene product, cells were infected with SPV-017 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Antisera to IBR gpG was used to detect expression of IBR specific proteins. The lysate from S-SPV-017 infected cells exhibited a band at 43 kd which is the expected size of the IBR gpG protein and additional bands of higher molecular weight which represent glycosylated forms of the protein which are absent in deletion mutants for IBR gpG.
This virus is used as an expression vector for expressing IBR glycoprotein G (gpG). Such IBR gpG is used as an antigen to identify antibodies directed against the wild-type IBR virus as opposed to antibodies directed against gpG deleted IBR viruses. This virus is also used as an antigen for the production of IBR gpG specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the IBR gpG protein. Monoclonal antibodies are generated in mice utilizing this virus according to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials & Methods).
Example 13
S-SPV-019
S-SPV-019 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ gene) and the gene for infectious bovine rhinotracheitits virus (IBR) glycoproteinE (gpE) are inserted into the SPV 515-85.1 ORF. The lacZ gene is under the control of a synthetic late promoter (LP1) and the gpE gene is under the control of a synthetic early/late promoter (LP2EP2).
S-SPV-019 is derived from S-SPV-001 (Kasza strain). This is accomplished utilizing the lacZ/IBR gpE homology vector (see Materials and Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock is screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS).
This virus is used as an expression vector for expressing IBR glycoprotein E (gpE). Such IBR gpE is used as an antigen to identify antibodies directed against the wild-type IBR virus as opposed to antibodies directed against gpE deleted IBR viruses. This virus is also used as an antigen for the production of IBR gpE specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the IBR gpE protein. Monoclonal antibodies are generated in mice utilizing this virus according to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials & Methods).
Example 14
S-SPV-018
S-SPV-018 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli B-galactosidase (lacZ) and the gene for pseudorabies virus glycoprotein E (PRV gpE) are inserted into the SPV 570-33.32 ORF (a unique PstI site has replaced the unique AccI site). The lacZ gene is under the control of the synthetic late promoter (LP1), and the PRV gpE gene is under the control of the synthetic early/late promoter (EP1LP2).
S-SPV-018 is derived from the S-SPV-001 (Kasza Strain). This is accomplished utilizing the final homology vector and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock is screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAYS). Red plaque purification of the recombinant virus is designated S-SPV-018. This virus is assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay described in Materials and Methods. After the initial three rounds of purification, all plaques observed are blue indicating that the virus is pure, stable, and expressing the foreign gene.
This virus is used as an expression vector for expressing PRV glycoprotein E (gpE). Such PRV gpE is used as an antigen to identify antibodies directed against the wild-type PRV virus as opposed to antibodies directed against gpE deleted PRV viruses. This virus is also used as an antigen for the production of PRV gpE specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the PRV gpE protein. Monoclonal antibodies are generated in mice utilizing this virus according to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials & Methods).
Example 15
Homology Vector 520-90.15
The homology vector 520-90.15 is a plasmid useful for the insertion of foreign DNA into SPV. Plasmid 520-90.15 contains a unique NdeI restriction site into which foreign DNA may be cloned. A plasmid containing such a foreign DNA insert has been used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV to generate a SPV 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 SPV and that the site be flanked with swinepox virus DNA appropriate for mediating homologous recombination between virus and plasmid DNAs. The unique NdeI restriction site in plasmid 520-90.15 is located within the coding region of the SPV thymidine kinase gene (32). Therefore, we have shown that the thymidine kinase gene of swinepox virus is non-essential for DNA replication and is an appropriate insertion site.
Example 16
S-PRV-010
S-SPV-010 is a swinepox virus that expresses a foreign gene. The E. coli B-galactosidase (lacZ) gene is inserted into a unique NdeI restriction site within the thymidine kinase gene. The foreign gene (lacZ) is under the control of the synthetic late promoter, LP1. We have shown that the swinepox virus thymidine kinase gene is non-essential for replication of the virus and is an appropriate insertion site.
A 1739 base pair HindIII-BamHI fragment subcloned from the HindIII G fragment contains the swinepox virus thymidine kinase gene and is designated homology vector 520-90.15. The homology vector 520-90.15 was digested with Nde I, and AscI linkers were inserted at this unique site within the thymidine kinase gene. The LP1 promoter-lac Z cassette with AscI linkers was ligated into the Asc I site within the thymidine kinase gene. The recombinant homology vector 561-36.26 was cotransfected with virus S-SPV-001 by the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV and virus plaques expressing B-galactosidase were selected by SCREEN FOR RECOMBINANT SPV EXPRESSING B-GALACTOSIDASE (BLUOGAL AND CPRG ASSAY). The final result of blue and red plaque purification was the recombinant virus designated S-SPV-010. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable and expressing the foreign gene. The assays described here were carried out in ESK-4 cells, indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant vaccines.
Example 17
The development of vaccines utilizing the swinepox virus to express antigens from various disease causing microorganisms can be engineered.
TRANSMISSIBLE GASTROENTERITIS VIRUS
The major neutralizing antigen of the transmissible gastroenteritis virus (TGE), glycoprotein 195, for use in the swinepox virus vector has been cloned. The clone of the neutralizing antigen is disclosed in U.S. Ser. No. 078,519, filed Jul. 27, 1987. It is contemplated that the procedures that have been used to express PRV g50 (gpD) in SPV and are disclosed herein are applicable to TGE.
PORCINE PARVOVIRUS
We have cloned the major capsid protein of the porcine (swine) parvovirus (PPV) for use in the swinepox virus vector. The clone of the capsid protein is disclosed in U.S. Pat. No. 5,068,192 issued Nov. 26, 1991. It is contemplated that the procedures that have been used to express PRV g50 (gpD) in SPV and are disclosed herein are applicable to PPV.
SWINE ROTAVIRUS
We have cloned the major neutralizing antigen of the swine rotavirus, glycoprotein 38, for use in the swinepox virus vector. The clone of glycoprotein 38 is disclosed in U.S. Pat. No. 5,068,192 issued Nov. 26, 1991. It is contemplated that the procedures that have been used to express PRV g50 (gpD) in SPV and are disclosed herein are applicable to SRV.
HOG CHOLERA VIRUS
The major neutralizing antigen of the bovine viral diarrhea (BVD) virus was cloned as disclosed in U.S. Ser. No. 225,032, filed Jul. 27, 1988. Since the BVD and hog cholera viruses are cross protective (31), the BVD virus antigen has been targeted for use in the swinepox virus vector. It is contemplated that the procedures that have been used to express PRV g50 (gpD) in SPV and are disclosed herein are applicable to BVD virus.
SERPULINA HYODYSENTERIAE
A protective antigen of Serpulina hyodysenteriae (3), for use in the swinepox virus vectorhas been cloned. It is contemplated that the procedures that have been used to express PRV gp50 in SPV and are disclosed herein are also applicable to Serpulina hyodysenteriae.
Antigens from the following microorganisms may also be utilized to develop animal vaccines: Swine influenza virus, foot and mouth disease virus, African swine fever virus, hog cholera virus and Mycoplasma hyodysenteriae.
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__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 112(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 599 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 202..597(D) OTHER INFORMATION: /partial/codon.sub.-- start= 202/function= "Potential eukaryotic transcriptionalregulatory protein"/standard.sub.-- name= "515-85.1 ORF"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AATGTATCCAGAGTTGTTGAATGCCTTATCGTACCTAATATTAATATAGAGTTATTAACT60GAATAAGTATATATAAATGATTGTTTTTATAATGTTTGTTATCGCATTTAGTTTTGCTGT120ATGGTTATCATATACATTTTTAAGGCCGTATATGATAAATGAAAATATATAAGCACTTAT180TTTTGTTAGTATAATAACACAATGCCGTCGTATATGTATCCGAAGAACGCA231MetProSerTyrMetTyrProLysAsnAla1510AGAAAAGTAATTTCAAAGATTATATCATTACAACTTGATATTAAAAAA279ArgLysValIleSerLysIleIleSerLeuGlnLeuAspIleLysLys152025CTTCCTAAAAAATATATAAATACCATGTTAGAATTTGGTCTACATGGA327LeuProLysLysTyrIleAsnThrMetLeuGluPheGlyLeuHisGly303540AATCTACCAGCTTGTATGTATAAAGATGCCGTATCATATGATATAAAT375AsnLeuProAlaCysMetTyrLysAspAlaValSerTyrAspIleAsn455055AATATAAGATTTTTACCTTATAATTGTGTTATGGTTAAAGATTTAATA423AsnIleArgPheLeuProTyrAsnCysValMetValLysAspLeuIle606570AATGTTATAAAATCATCATCTGTAATAGATACTAGATTACATCAATCT471AsnValIleLysSerSerSerValIleAspThrArgLeuHisGlnSer75808590GTATTAAAACATCGTAGAGCGTTAATAGATTACGGCGATCAAGACATT519ValLeuLysHisArgArgAlaLeuIleAspTyrGlyAspGlnAspIle95100105ATCACTTTAATGATCATTAATAAGTTACTATCGATAGATGATATATCC567IleThrLeuMetIleIleAsnLysLeuLeuSerIleAspAspIleSer110115120TATATATTAGATAAAAAAATAATTCATGTAAC599TyrIleLeuAspLysLysIleIleHisVal125130(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 132 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetProSerTyrMetTyrProLysAsnAlaArgLysValIleSerLys151015IleIleSerLeuGlnLeuAspIleLysLysLeuProLysLysTyrIle202530AsnThrMetLeuGluPheGlyLeuHisGlyAsnLeuProAlaCysMet354045TyrLysAspAlaValSerTyrAspIleAsnAsnIleArgPheLeuPro505560TyrAsnCysValMetValLysAspLeuIleAsnValIleLysSerSer65707580SerValIleAspThrArgLeuHisGlnSerValLeuLysHisArgArg859095AlaLeuIleAspTyrGlyAspGlnAspIleIleThrLeuMetIleIle100105110AsnLysLeuLeuSerIleAspAspIleSerTyrIleLeuAspLysLys115120125IleIleHisVal130(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 899 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 3..662(D) OTHER INFORMATION: /partial/codon.sub.-- start= 3/function= "Potential eukaryotic transcriptionalregulatory protein"/standard.sub.-- name= "515-85.1 ORF"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GAGATATTAAATCATGTAAATGCTCGATATGTTCCGACTCTATAACA47AspIleLysSerCysLysCysSerIleCysSerAspSerIleThr151015CATCATATATATGAAACAACATCATGTATAAATTATAAATCTACCGAT95HisHisIleTyrGluThrThrSerCysIleAsnTyrLysSerThrAsp202530AATGATCTTATGATAGTATTGTTCAATCTAACTAGATATTTAATGCAT143AsnAspLeuMetIleValLeuPheAsnLeuThrArgTyrLeuMetHis354045GGGATGATACATCCTAATCTTATAAGCGTAAAAGGATGGGGTCCCCTT191GlyMetIleHisProAsnLeuIleSerValLysGlyTrpGlyProLeu505560ATTGGATTATTAACGGGTGATATAGGTATTAATTTAAAACTATATTCC239IleGlyLeuLeuThrGlyAspIleGlyIleAsnLeuLysLeuTyrSer657075ACCATGAATATAAATGGGCTACGGTATGGAGATATTACGTTATCTTCA287ThrMetAsnIleAsnGlyLeuArgTyrGlyAspIleThrLeuSerSer80859095TACGATATGAGTAATAAATTAGTCTCTATTATTAATACACCCATATAT335TyrAspMetSerAsnLysLeuValSerIleIleAsnThrProIleTyr100105110GAGTTAATACCGTTTACTACATGTTGTTCACTCAATGAATATTATTCA383GluLeuIleProPheThrThrCysCysSerLeuAsnGluTyrTyrSer115120125AAAATTGTGATTTTAATAAATGTTATTTTAGAATATATGATATCTATT431LysIleValIleLeuIleAsnValIleLeuGluTyrMetIleSerIle130135140ATATTATATAGAATATTGATCGTAAAAAGATTTAATAACATTAAAGAA479IleLeuTyrArgIleLeuIleValLysArgPheAsnAsnIleLysGlu145150155TTTATTTCAAAAGTCGTAAATACTGTACTAGAATCATCAGGCATATAT527PheIleSerLysValValAsnThrValLeuGluSerSerGlyIleTyr160165170175TTTTGTCAGATGCGTGTACATGAACAAATTGAATTGGAAATAGATGAG575PheCysGlnMetArgValHisGluGlnIleGluLeuGluIleAspGlu180185190CTCATTATTAATGGATCTATGCCTGTACAGCTTATGCATTTACTTCTA623LeuIleIleAsnGlySerMetProValGlnLeuMetHisLeuLeuLeu195200205AAGGTAGCTACCATAATATTAGAGGAAATCAAAGAAATATAACGTATTT672LysValAlaThrIleIleLeuGluGluIleLysGluIle210215220TTTCTTTTAAATAAATAAAAATACTTTTTTTTTTAAACAAGGGGTGCTACCTTGTCTAAT732TGTATCTTGTATTTTGGATCTGATGCAAGATTATTAAATAATCGTATGAAAAAGTAGTAG792ATATAGTTTATATCGTTACTGGACATGATATTATGTTTAGTTAATTCTTCTTTGGCATGA852ATTCTACACGTCGGANAAGGTAATGTATCTATAATGGTATAAAGCTT899(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 220 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AspIleLysSerCysLysCysSerIleCysSerAspSerIleThrHis151015HisIleTyrGluThrThrSerCysIleAsnTyrLysSerThrAspAsn202530AspLeuMetIleValLeuPheAsnLeuThrArgTyrLeuMetHisGly354045MetIleHisProAsnLeuIleSerValLysGlyTrpGlyProLeuIle505560GlyLeuLeuThrGlyAspIleGlyIleAsnLeuLysLeuTyrSerThr65707580MetAsnIleAsnGlyLeuArgTyrGlyAspIleThrLeuSerSerTyr859095AspMetSerAsnLysLeuValSerIleIleAsnThrProIleTyrGlu100105110LeuIleProPheThrThrCysCysSerLeuAsnGluTyrTyrSerLys115120125IleValIleLeuIleAsnValIleLeuGluTyrMetIleSerIleIle130135140LeuTyrArgIleLeuIleValLysArgPheAsnAsnIleLysGluPhe145150155160IleSerLysValValAsnThrValLeuGluSerSerGlyIleTyrPhe165170175CysGlnMetArgValHisGluGlnIleGluLeuGluIleAspGluLeu180185190IleIleAsnGlySerMetProValGlnLeuMetHisLeuLeuLeuLys195200205ValAlaThrIleIleLeuGluGluIleLysGluIle210215220(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 129 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: YES(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Vaccinia virus(B) STRAIN: Copenhagen(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:MetPheMetTyrProGluPheAlaArgLysAlaLeuSerLysLeuIle151015SerLysLysLeuAsnIleGluLysValSerSerLysHisGlnLeuVal202530LeuLeuAspTyrGlyLeuHisGlyLeuLeuProLysSerLeuTyrLeu354045GluAlaIleAsnSerAspIleLeuAsnValArgPhePheProProGlu505560IleIleAsnValThrAspIleValLysAlaLeuGlnAsnSerCysArg65707580ValAspGluTyrLeuLysAlaValSerLeuTyrHisLysAsnSerLeu859095MetValSerGlyProAsnValValLysLeuMetIleGluTyrAsnLeu100105110LeuThrHisSerAspLeuGluTrpLeuIleAsnGluAsnValValLys115120125Ala(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 132 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: YES(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetProSerTyrMetTyrProLysAsnAlaArgLysValIleSerLys151015IleIleSerLeuGlnLeuAspIleLysLysLeuProLysLysTyrIle202530AsnThrMetLeuGluPheGlyLeuHisGlyAsnLeuProAlaCysMet354045TyrLysAspAlaValSerTyrAspIleAsnAsnIleArgPheLeuPro505560TyrAsnCysValMetValLysAspLeuIleAsnValIleLysSerSer65707580SerValIleAspThrArgLeuHisGlnSerValLeuLysHisArgArg859095AlaLeuIleAspTyrGlyAspGlnAspIleIleThrLeuMetIleIle100105110AsnLysLeuLeuSerIleAspAspIleSerTyrIleLeuAspLysLys115120125IleIleHisVal130(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 101 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: YES(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: C-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Vaccinia virus(B) STRAIN: Copenhagen(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:ValLeuAsnAspGlnTyrAlaLysIleValIlePhePheAsnThrIle151015IleGluTyrIleIleAlaThrIleTyrTyrArgLeuThrValLeuAsn202530AsnTyrThrAsnValLysHisPheValSerLysValLeuHisThrVal354045MetGluAlaCysGlyValLeuPheSerTyrIleLysValAsnAspLys505560IleGluHisGluLeuGluGluMetValAspLysGlyThrValProSer65707580TyrLeuTyrHisLeuSerIleAsnValIleSerIleIleLeuAspAsp859095IleAsnGlyThrArg100(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 100 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: YES(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: C-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:SerLeuAsnGluTyrTyrSerLysIleValIleLeuIleAsnValIle151015LeuGluTyrMetIleSerIleIleLeuTyrArgIleLeuIleValLys202530ArgPheAsnAsnIleLysGluPheIleSerLysValValAsnThrVal354045LeuGluSerSerGlyIleTyrPheCysGlnMetArgValHisGluGln505560IleGluLeuGluIleAspGluLeuIleIleAsnGlySerMetProVal65707580GlnLeuMetHisLeuLeuLeuLysValAlaThrIleIleLeuGluGlu859095IleLysGluIle100(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 102 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 520-17.5 (Junction A)(x) PUBLICATION INFORMATION:(A) AUTHORS: Ferrari, Franco ATrach, KathleenHoch, James A(B) TITLE: Sequence Analysis of the spo0B Locus Revels aPolycistronic Transcription Unit(C) JOURNAL: J. Bacteriol.(D) VOLUME: 161(E) ISSUE: 2(F) PAGES: 556-562(G) DATE: Feb.-1985(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:CACATACGATTTAGGTGACACTATAGAATACAAGCTTTATACCATTATAGATACATTACC60TTGTCCGACGTGTAGAATTCATGCCAAAGAAGAATTAACTAA102(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 102 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 520-17.5 (Junction B)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 85..99(D) OTHER INFORMATION: /codon.sub.-- start= 85/function= "Translational start of hybrid protein"/product= "N-terminal peptide"/number= 1/standard.sub.-- name= "Translation of synthetic DNAsequence"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 100..102(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 100/function= "marker enzyme"/product= "Beta-Galactosidase"/evidence= EXPERIMENTAL/gene= "lacZ"/number= 2/citation= (�1!)(x) PUBLICATION INFORMATION:(A) AUTHORS: Ferrari, Franco ATrach, KathleenHoch, James A(B) TITLE: Seqquence Analysis of the spo0B Locus Revealsa Polycistronic Transcription Unit(C) JOURNAL: J. Bacteriol.(D) VOLUME: 161(E) ISSUE: 2(F) PAGES: 556-562(G) DATE: Feb.-1985(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:GTAGTCGACTCTAGAAAAAATTGAAAAACTATTCTAATTTATTGCACGGAGATCTTTTTT60TTTTTTTTTTTTTTTGGCATATAAATGAATTCGGATCCCGTC102MetAsnSerAspProVal151(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:MetAsnSerAspPro15(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:Val(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 103 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 520-17.5 (Junction C)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..72(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 1/function= "marker enzyme"/product= "Beta-galactosidase"/evidence= EXPERIMENTAL/gene= "lacZ"/number= 1/citation= (�1!)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 73..78(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /codon.sub.-- start= 73/function= "Translational finish of hybridprotein"/product= "C-terminal peptide"/evidence= EXPERIMENTAL/number= 2/standard.sub.-- name= "Translation of synthetic DNAsequence"(x) PUBLICATION INFORMATION:(A) AUTHORS: Ferrari, Franco ATrach, KathleenHoch, James A(B) TITLE: Seqquence Analysis of the spo0B Locus Revealsa Polycistronic Transcription Unit(C) JOURNAL: J. Bacteriol.(D) VOLUME: 161(E) ISSUE: 2(F) PAGES: 556-562(G) DATE: Feb.-1985(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:AGCCCGTCAGTATCGGCGGAAATCCAGCTGAGCGCCGGTCGCTACCAT48SerProSerValSerAlaGluIleGlnLeuSerAlaGlyArgTyrHis151015TACCAGTTGGTCTGGTGTCAAAAAGATCCATAATTAATTAACCCGGGTCG98TyrGlnLeuValTrpCysGlnLysAspPro201AAGAC103(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:SerProSerValSerAlaGluIleGlnLeuSerAlaGlyArgTyrHis151015TyrGlnLeuValTrpCysGlnLys20(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:AspPro1(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 48 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 520-17.5 (Junction D)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:AGATCCCCGGGCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTCC48(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.26 (Junction A)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:CACATACGATTTAGGTGACACTATAGAATACAAGCTTTATACCATTATAGATACATT57(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 102 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.16 (Junction B)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 91..102(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 91/function= "marker enzyme"/product= "Beta-Galactosidase"/evidence= EXPERIMENTAL/gene= "lacZ"/number= 2/citation= (�1!)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 76..90(D) OTHER INFORMATION: /partial/codon.sub.-- start= 76/function= "Translational start of hybrid protein"/product= "N-terminal peptide"/number= 1/standard.sub.-- name= "Translation of synthetic DNAsequence"(x) PUBLICATION INFORMATION:(A) AUTHORS: Ferrari, Franco ATrach, KathleenHoch, James A(B) TITLE: Seqquence Analysis of the spo0B Locus Revealsa Polycistronic Transcription Unit(C) JOURNAL: J. Bacteriol.(D) VOLUME: 161(E) ISSUE: 2(F) PAGES: 556-562(G) DATE: Feb.-1985(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:AAGCTGGTAGATTTCCATGTAGGGCCGCCTGCAGGTCGACTCTAGAATTTCATTTTGTTT60TTTTCTATGCTATAAATGAATTCGGATCCCGTCGTTTTACAA102MetAsnSerAspProValValLeuGln151(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:MetAsnSerAspPro15(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:ValValLeuGln1(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 206 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.16 (Junction C)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 1/function= "marker enzyme"/product= "Beta-galactosidase"/evidence= EXPERIMENTAL/number= 1/citation= (�1!)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 64..69(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /codon.sub.-- start= 64/function= "Translational finish of hybridprotein"/product= "C-terminal peptide"/evidence= EXPERIMENTAL/standard.sub.-- name= "Translation of synthetic DNAsequence"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 177..185(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /codon.sub.-- start= 177/function= "Translational start of hybrid protein"/product= "N-terminal peptide"/evidence= EXPERIMENTAL/standard.sub.-- name= "Translation of synthetic DNAsequence"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 186..206(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 186/function= "glycoprotein"/product= "PRV gp50"/evidence= EXPERIMENTAL/gene= "gp50"/number= 3/citation= (�2!)(x) PUBLICATION INFORMATION:(A) AUTHORS: Ferrari, Franco ATrach, KathleenHoch, James A(B) TITLE: Seqquence Analysis of the spo0B Locus Revealsa Polycistronic Transcription Unit(C) JOURNAL: J. Bacteriol.(D) VOLUME: 161(E) ISSUE: 2(F) PAGES: 556-562(G) DATE: Feb.-1985(x) PUBLICATION INFORMATION:(A) AUTHORS: Petrovskis, Erik ATimmins, James GArmentrout, Marty AMarchioli, Carmine CJr. Yancy, Robert JPost, Leonard E(B) TITLE: DNA Sequence of the Gene for PseudorabiesVirus gp50, a Glycoprotein without N-LinkedGlycosylation(C) JOURNAL: J. Virol.(D) VOLUME: 59(E) ISSUE: 2(F) PAGES: 216-223(G) DATE: Aug.-1986(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:GTATCGGCGGAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTG48ValSerAlaGluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeu151015GTCTGGTGTCAAAAAGATCCATAATTAATTAACCCGGCCGCCTGCAGGTCG99ValTrpCysGlnLysAspPro201ACTCTAGAAAAAATTGAAAAACTATTCTAATTTATTGCACGGAGATCTTTTTTTTTTTTT159TTTTTTTTGGCATATAAATGAATTCGCTCGCAGCGCTATTGGCGGCG206MetAsnSerLeuAlaAlaLeuLeuAlaAla115(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:ValSerAlaGluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeu151015ValTrpCysGlnLys20(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:AspPro1(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:MetAsnSer1(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:LeuAlaAlaLeuLeuAlaAla15(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 101 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.16 (Junction D)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..15(D) OTHER INFORMATION: /partial/codon.sub.-- start= 1/function= "glycoprotein"/product= "PRV gp63"/gene= "gp63"/number= 1/citation= (�1!)(x) PUBLICATION INFORMATION:(A) AUTHORS: Petrovskis, Erik ATimmins, James GPost, Lenoard E(B) TITLE: Use of Lambda-gt11 To Isolate Genes for twoPseudorabies Virus Glycoproteins with homology toHerpes Simplex Virus and Varicella-Zoster VirusGlycoproteins(C) JOURNAL: J. Virol.(D) VOLUME: 60(E) ISSUE: 1(F) PAGES: 185-193(G) DATE: Oct.-1986(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:CGCGTGCACCACGAGGGACTCTAGAGGATCCATAATTAATTAATTAATTTTTATC55ArgValHisHisGlu15CCGGGTCGACCTGCAGGCGGCCGGGTCGACCTGCAGGCGGCCAGAC101(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:ArgValHisHisGlu15(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.16 (Junction E)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:AGATCCCCGGGCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA57(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1907 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA to mRNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Newcastle disease virus(B) STRAIN: B1(vii) IMMEDIATE SOURCE:(B) CLONE: 137-23.803 (PSY1142)(viii) POSITION IN GENOME:(B) MAP POSITION: 50%(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 92..1822(D) OTHER INFORMATION: /codon.sub.-- start= 92/product= "NDV heamagglutinin-Neuraminidase"/gene= "HN"/number= 1(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:ACGGGTAGAACGGTAAGAGAGGCCGCCCCTCAATTGCGAGCCAGACTTCACAACCTCCGT60TCTACCGCTTCACCGACAACAGTCCTCAATCATGGACCGCGCCGTTAGCCAA112MetAspArgAlaValSerGln15GTTGCGTTAGAGAATGATGAAAGAGAGGCAAAAAATACATGGCGCTTG160ValAlaLeuGluAsnAspGluArgGluAlaLysAsnThrTrpArgLeu101520ATATTCCGGATTGCAATCTTATTCTTAACAGTAGTGACCTTGGCTATA208IlePheArgIleAlaIleLeuPheLeuThrValValThrLeuAlaIle253035TCTGTAGCCTCCCTTTTATATAGCATGGGGGCTAGCACACCTAGCGAT256SerValAlaSerLeuLeuTyrSerMetGlyAlaSerThrProSerAsp40455055CTTGTAGGCATACCGACTAGGATTTCCAGGGCAGAAGAAAAGATTACA304LeuValGlyIleProThrArgIleSerArgAlaGluGluLysIleThr606570TCTACACTTGGTTCCAATCAAGATGTAGTAGATAGGATATATAAGCAA352SerThrLeuGlySerAsnGlnAspValValAspArgIleTyrLysGln758085GTGGCCCTTGAGTCTCCATTGGCATTGTTAAATACTGAGACCACAATT400ValAlaLeuGluSerProLeuAlaLeuLeuAsnThrGluThrThrIle9095100ATGAACGCAATAACATCTCTCTCTTATCAGATTAATGGAGCTGCAAAC448MetAsnAlaIleThrSerLeuSerTyrGlnIleAsnGlyAlaAlaAsn105110115AACAGCGGGTGGGGGGCACCTATTCATGACCCAGATTATATAGGGGGG496AsnSerGlyTrpGlyAlaProIleHisAspProAspTyrIleGlyGly120125130135ATAGGCAAAGAACTCATTGTAGATGATGCTAGTGATGTCACATCATTC544IleGlyLysGluLeuIleValAspAspAlaSerAspValThrSerPhe140145150TATCCCTCTGCATTTCAAGAACATCTGAATTTTATCCCGGCGCCTACT592TyrProSerAlaPheGlnGluHisLeuAsnPheIleProAlaProThr155160165ACAGGATCAGGTTGCACTCGAATACCCTCATTTGACATGAGTGCTACC640ThrGlySerGlyCysThrArgIleProSerPheAspMetSerAlaThr170175180CATTACTGCTACACCCATAATGTAATATTGTCTGGATGCAGAGATCAC688HisTyrCysTyrThrHisAsnValIleLeuSerGlyCysArgAspHis185190195TCACACTCACATCAGTATTTAGCACTTGGTGTGCTCCGGACATCTGCA736SerHisSerHisGlnTyrLeuAlaLeuGlyValLeuArgThrSerAla200205210215ACAGGGAGGGTATTCTTTTCTACTCTGCGTTCCATCAACCTGGACGAC784ThrGlyArgValPhePheSerThrLeuArgSerIleAsnLeuAspAsp220225230ACCCAAAATCGGAAGTCTTGCAGTGTGAGTGCAACTCCCCTGGGTTGT832ThrGlnAsnArgLysSerCysSerValSerAlaThrProLeuGlyCys235240245GATATGCTGTGCTCGAAAGCCACGGAGACAGAGGAAGAAGATTATAAC880AspMetLeuCysSerLysAlaThrGluThrGluGluGluAspTyrAsn250255260TCAGCTGTCCCTACGCGGATGGTACATGGGAGGTTAGGGTTCGACGGC928SerAlaValProThrArgMetValHisGlyArgLeuGlyPheAspGly265270275CAATATCACGAAAAGGACCTAGATGTCACAACATTATTCGGGGACTGG976GlnTyrHisGluLysAspLeuAspValThrThrLeuPheGlyAspTrp280285290295GTGGCCAACTACCCAGGAGTAGGGGGTGGATCTTTTATTGACAGCCGC1024ValAlaAsnTyrProGlyValGlyGlyGlySerPheIleAspSerArg300305310GTGTGGTTCTCAGTCTACGGAGGGTTAAAACCCAATACACCCAGTGAC1072ValTrpPheSerValTyrGlyGlyLeuLysProAsnThrProSerAsp315320325ACTGTACAGGAAGGGAAATATGTGATATACAAGCGATACAATGACACA1120ThrValGlnGluGlyLysTyrValIleTyrLysArgTyrAsnAspThr330335340TGCCCAGATGAGCAAGACTACCAGATTCGAATGGCCAAGTCTTCGTAT1168CysProAspGluGlnAspTyrGlnIleArgMetAlaLysSerSerTyr345350355AAGCCTGGACGGTTTGGTGGGAAACGCATACAGCAGGCTATCTTATCT1216LysProGlyArgPheGlyGlyLysArgIleGlnGlnAlaIleLeuSer360365370375ATCAAAGTGTCAACATCCTTAGGCGAAGACCCGGTACTGACTGTACCG1264IleLysValSerThrSerLeuGlyGluAspProValLeuThrValPro380385390CCCAACACAGTCACACTCATGGGGGCCGAAGGCAGAATTCTCACAGTA1312ProAsnThrValThrLeuMetGlyAlaGluGlyArgIleLeuThrVal395400405GGGACATCCCATTTCTTGTATCAGCGAGGGTCATCATACTTCTCTCCC1360GlyThrSerHisPheLeuTyrGlnArgGlySerSerTyrPheSerPro410415420GCGTTATTATATCCTATGACAGTCAGCAACAAAACAGCCACTCTTCAT1408AlaLeuLeuTyrProMetThrValSerAsnLysThrAlaThrLeuHis425430435AGTCCTTATACATTCAATGCCTTCACTCGGCCAGGTAGTATCCCTTGC1456SerProTyrThrPheAsnAlaPheThrArgProGlySerIleProCys440445450455CAGGCTTCAGCAAGATGCCCCAACTCATGTGTTACTGGAGTCTATACA1504GlnAlaSerAlaArgCysProAsnSerCysValThrGlyValTyrThr460465470GATCCATATCCCCTAATCTTCTATAGAAACCACACCTTGCGAGGGGTA1552AspProTyrProLeuIlePheTyrArgAsnHisThrLeuArgGlyVal475480485TTCGGGACAATGCTTGATGGTGAACAAGCAAGACTTAACCCTGCGTCT1600PheGlyThrMetLeuAspGlyGluGlnAlaArgLeuAsnProAlaSer490495500GCAGTATTCGATAGCACATCCCGCAGTCGCATAACTCGAGTGAGTTCA1648AlaValPheAspSerThrSerArgSerArgIleThrArgValSerSer505510515AGCAGCATCAAAGCAGCATACACAACATCAACTTGTTTTAAAGTGGTC1696SerSerIleLysAlaAlaTyrThrThrSerThrCysPheLysValVal520525530535AAGACCAATAAGACCTATTGTCTCAGCATTGCTGAAATATCTAATACT1744LysThrAsnLysThrTyrCysLeuSerIleAlaGluIleSerAsnThr540545550CTCTTCGGAGAATTCAGAATCGTCCCGTTACTAGTTGAGATCCTCAAA1792LeuPheGlyGluPheArgIleValProLeuLeuValGluIleLeuLys555560565GATGACGGGGTTAGAGAAGCCAGGTCTGGCTAGTTGAGTCAACTATGAAA1842AspAspGlyValArgGluAlaArgSerGly570575GAGTTGGAAAGATGGCATTGTATCACCTATCTTCTGCGACATCAAGAATCAAACCGAATG1902CCGGC1907(2) INFORMATION FOR SEQ ID NO:30:(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:30:MetAspArgAlaValSerGlnValAlaLeuGluAsnAspGluArgGlu151015AlaLysAsnThrTrpArgLeuIlePheArgIleAlaIleLeuPheLeu202530ThrValValThrLeuAlaIleSerValAlaSerLeuLeuTyrSerMet354045GlyAlaSerThrProSerAspLeuValGlyIleProThrArgIleSer505560ArgAlaGluGluLysIleThrSerThrLeuGlySerAsnGlnAspVal65707580ValAspArgIleTyrLysGlnValAlaLeuGluSerProLeuAlaLeu859095LeuAsnThrGluThrThrIleMetAsnAlaIleThrSerLeuSerTyr100105110GlnIleAsnGlyAlaAlaAsnAsnSerGlyTrpGlyAlaProIleHis115120125AspProAspTyrIleGlyGlyIleGlyLysGluLeuIleValAspAsp130135140AlaSerAspValThrSerPheTyrProSerAlaPheGlnGluHisLeu145150155160AsnPheIleProAlaProThrThrGlySerGlyCysThrArgIlePro165170175SerPheAspMetSerAlaThrHisTyrCysTyrThrHisAsnValIle180185190LeuSerGlyCysArgAspHisSerHisSerHisGlnTyrLeuAlaLeu195200205GlyValLeuArgThrSerAlaThrGlyArgValPhePheSerThrLeu210215220ArgSerIleAsnLeuAspAspThrGlnAsnArgLysSerCysSerVal225230235240SerAlaThrProLeuGlyCysAspMetLeuCysSerLysAlaThrGlu245250255ThrGluGluGluAspTyrAsnSerAlaValProThrArgMetValHis260265270GlyArgLeuGlyPheAspGlyGlnTyrHisGluLysAspLeuAspVal275280285ThrThrLeuPheGlyAspTrpValAlaAsnTyrProGlyValGlyGly290295300GlySerPheIleAspSerArgValTrpPheSerValTyrGlyGlyLeu305310315320LysProAsnThrProSerAspThrValGlnGluGlyLysTyrValIle325330335TyrLysArgTyrAsnAspThrCysProAspGluGlnAspTyrGlnIle340345350ArgMetAlaLysSerSerTyrLysProGlyArgPheGlyGlyLysArg355360365IleGlnGlnAlaIleLeuSerIleLysValSerThrSerLeuGlyGlu370375380AspProValLeuThrValProProAsnThrValThrLeuMetGlyAla385390395400GluGlyArgIleLeuThrValGlyThrSerHisPheLeuTyrGlnArg405410415GlySerSerTyrPheSerProAlaLeuLeuTyrProMetThrValSer420425430AsnLysThrAlaThrLeuHisSerProTyrThrPheAsnAlaPheThr435440445ArgProGlySerIleProCysGlnAlaSerAlaArgCysProAsnSer450455460CysValThrGlyValTyrThrAspProTyrProLeuIlePheTyrArg465470475480AsnHisThrLeuArgGlyValPheGlyThrMetLeuAspGlyGluGln485490495AlaArgLeuAsnProAlaSerAlaValPheAspSerThrSerArgSer500505510ArgIleThrArgValSerSerSerSerIleLysAlaAlaTyrThrThr515520525SerThrCysPheLysValValLysThrAsnLysThrTyrCysLeuSer530535540IleAlaGluIleSerAsnThrLeuPheGlyGluPheArgIleValPro545550555560LeuLeuValGluIleLeuLysAspAspGlyValArgGluAlaArgSer565570575Gly(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.26 (Junction A)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:CACATACGATTTAGGTGACACTATAGAATACAAGCTTTATACCATTATAGATACATT57(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 108 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.26 (Junction B)(ix) FEATURE:(A) NAME/KEY: exon(B) LOCATION: 88..102(D) OTHER INFORMATION: /codon.sub.-- start= 88/function= "Translational start of hybrid protein"/product= "N-terminal peptide"/number= 1/standard.sub.-- name= "Translation of synthetic DNAsequence"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 103..108(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 103/product= "NDV Heamagglutinin-Neuraminidase"/evidence= EXPERIMENTAL/gene= "HN"/number= 2(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:CATGTAGTCGACTCTAGAAAAAATTGAAAAACTATTCTAATTTATTGCACGGAGATCTTT60TTTTTTTTTTTTTTTTTTGGCATATAAATGAATTCGGATCCGGACCGC108AspArg1(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:AspArg1(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 108 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.26 (Junction C)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 70..84(D) OTHER INFORMATION: /codon.sub.-- start= 70/function= "Translational start of hybrid protein"/product= "N-terminal peptide"/number= 1/standard.sub.-- name= "Translation of synthetic DNAsequence"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 85..108(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 85/function= "marker enzyme"/product= "Beta-galactosidase"/evidence= EXPERIMENTAL/gene= "lacZ"/number= 2/citation= (�1!)(x) PUBLICATION INFORMATION:(A) AUTHORS: Ferrari, Franco ATrach, KathleenHoch, James A(B) TITLE: Sequence Analysis of the spo0B Locus Revealsa Polycistronic Transcription Unit(C) JOURNAL: J. Bacteriol.(D) VOLUME: 161(E) ISSUE: 2(F) PAGES: 556-562(G) DATE: Feb.-1985(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:TGCGACATCAAGAATCAAACCGAATGCCCTCGACTCTAGAATTTCATTTTGTTTTTTTCT60ATGCTATAAATGAATTCGGATCCCGTCGTTTTACAACGTCGTGACTGG108MetAsnSerAspProValValLeuGlnArgArgAspTrp1515(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:MetAsnSerAspPro15(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:ValValLeuGlnArgArgAspTrp15(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 108 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.26(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..54(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /partial/codon.sub.-- start= 1/function= "marker enzyme"/product= "Beta-galactosidase"/evidence= EXPERIMENTAL/gene= "lacZ"/number= 1/citation= (�1!)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 55..63(C) IDENTIFICATION METHOD: experimental(D) OTHER INFORMATION: /codon.sub.-- start= 55/function= "Translational finish of hybridprotein"/product= "C-terminal peptide"/evidence= EXPERIMENTAL/number= 2/standard.sub.-- name= "Translation of synthetic DNAsequence"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGGTCGAGGGTCGAAGACCAAATTCT100GlnLysAspPro1AACATGGT108(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLys(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:AspPro1(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Plasmid(vii) IMMEDIATE SOURCE:(B) CLONE: 538-46.26 (Junction E)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:AGATCCCCGGGCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA57(2) INFORMATION FOR SEQ ID NO:41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: N(iv) ANTI-SENSE: N(vi) ORIGINAL SOURCE:(A) ORGANISM: Pseudorabies virus \ Synthetic oligonucleotideprimer(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:CGCGAATTCGCTCGCAGCGCTATTGGC27(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: N(iv) ANTI-SENSE: N(vi) ORIGINAL SOURCE:(A) ORGANISM: Pseudorabies virus \ Synthetic oligonucleotideprimer(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:GTAGGAGTGGCTGCTGAAG19(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 70 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:AAAAATTGAAAAACTATTCTAATTTATTGCACGGAGATCTTTTTTTTTTTTTTTTTTTTG60GCATATAAAT70(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 74 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:TTTTTTTTTTTTTTTTTTTTGGCATATAAATAGATCTGTATCCTAAAATTGAATTGTAAT60TATCGATAATAAAT74(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 37 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:GTATCCTAAAATTGAATTGTAATTATCGATAATAAAT37(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 41 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:CGACTCTAGAATTTCATTTTGTTTTTTTCTATGCTATAAAT41(2) INFORMATION 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(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:CACATACGATTTAGGTGACACTATAGAATACAAGCTTTGAGTCTATTGGTTATTTATACG60(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 123 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 100..123(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:TGAATATATAGCAAATAAAGGAAAAATTGTTATCGTTGCTGCATTAGATGGAACATAGGT60CGACTCTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATGAATTCGGATCCC114MetAsnSerAspPro15GTCGTTTTA123ValValLeu(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:50:(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(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGGTCGACCTATGAACGTAAACCATT100GlnLysAspPro20TGGTAATATTCTTAATCTTATACCATTATCGG132(2) INFORMATION FOR SEQ ID NO:51:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:52:(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(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:TCTACTATTGTATATATAGGATCCCCGGGCGAGCTCGAATTCGTAATCATGGTCATAGCT60GTTTCC66(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:ACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGCCCGGGGATCT51(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 104 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 81..104(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:AAATATATAAATACCATGTTAGAATTTGGTCTGCTGCAGGTCGACTCTAGAATTTCATTT60TGTTTTTTTCTATGCTATAAATGAATTCGGATCCCGTCGTTTTA104MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:55:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 150 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 130..150(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGTCGACTCTAGAAAGATCTGTATCC100GlnLysAspPro20TAAAATTGAATTGTAATTATCGATAATAAATGAATTCCGGCATGGCCTCG150MetAsnSerGlyMetAlaSer15(2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:58:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:MetAsnSerGlyMetAlaSer15(2) INFORMATION FOR SEQ ID NO:59:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 109 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:CCATGCTCTAGAGGATCCCCGGGCGAGCTCGAATTCGGATCCATAATTAATTAATTAATT60TTTATCCCGGGTCGACCGGGTCGACCTGCAGCCTACATGGAAATCTACC109(2) INFORMATION FOR SEQ ID NO:60:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:TAATGTATCTATAATGGTATAAAGCTTGTATTCTATAGTGTCACCTAAATC51(2) INFORMATION FOR SEQ ID NO:61:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:ACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGCCCGGGGATCT51(2) INFORMATION FOR SEQ ID NO:62:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 104 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 81..104(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:AAATATATAAATACCATGTTAGAATTTGGTCTGCTGCAGGTCGACTCTAGAATTTCATTT60TGTTTTTTTCTATGCTATAAATGAATTCGGATCCCGTCGTTTTA104MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:63:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:64:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 182 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 156..182(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGTCGACTCTAGAAAAAATTGAAAAA100GlnLysAspPro20CTATTCTAATTTATTGCACGGAGATCTTTTTTTTTTTTTTTTTTTTGGCATATAAATG158Met1AATTCCGGCATGGCCTCGCTCGCG182AsnSerGlyMetAlaSerLeuAla5(2) INFORMATION FOR SEQ ID NO:65:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:66:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:MetAsnSerGlyMetAlaSerLeuAla15(2) INFORMATION FOR SEQ ID NO:67:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 109 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:CCATGCTCTAGAGGATCCCCGGGCGAGCTCGAATTCGGATCCATAATTAATTAATTAATT60TTTATCCCGGGTCGACCGGGTCGACCTGCAGCCTACATGGAAATCTACC109(2) INFORMATION FOR SEQ ID NO:68:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:TAATGTATCTATAATGGTATAAAGCTTGTATTCTATAGTGTCACCTAAATC51(2) INFORMATION FOR SEQ ID NO:69:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:ACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGCCCGGGGATCT51(2) INFORMATION FOR SEQ ID NO:70:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 104 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 81..104(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:AAATATATAAATACCATGTTAGAATTTGGTCTGCTGCAGGTCGACTCTAGAATTTCATTT60TGTTTTTTTCTATGCTATAAATGAATTCGGATCCCGTCGTTTTA104MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:71:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:72:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 180 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 160..180(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGTCGACTCTAGATTTTTTTTTTTTT100GlnLysAspPro20TTTTTTTGGCATATAAATAGATCTGTATCCTAAAATTGAATTGTAATTATCGATAATAA159ATGAATTCCGGCATGGCCTCG180MetAsnSerGlyMetAlaSer15(2) INFORMATION FOR SEQ ID NO:73:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:74:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:MetAsnSerGlyMetAlaSer15(2) INFORMATION FOR SEQ ID NO:75:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 109 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:CCATGCTCTAGAGGATCCCCGGGCGAGCTCGAATTCGGATCCATAATTAATTAATTAATT60TTTATCCCGGGTCGACCGGGTCGACCTGCAGCCTACATGGAAATCTACC109(2) INFORMATION FOR SEQ ID NO:76:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:TAATGTATCTATAATGGTATAAAGCTTGTATTCTATAGTGTCACCTAAATC51(2) INFORMATION FOR SEQ ID NO:77:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:ACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGCCCGGGGATCT51(2) INFORMATION FOR SEQ ID NO:78:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 111 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 88..111(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:CTGCAGGTCGACTCTAGAAAAAATTGAAAAACTATTCTAATTTATTGCACGGAGATCTTT60TTTTTTTTTTTTTTTTTTGGCATATAAATGAATTCCGGCTTCAGTAACATA111MetAsnSerGlyPheSerAsnIle15(2) INFORMATION FOR SEQ ID NO:79:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:MetAsnSerGlyPheSerAsnIle15(2) INFORMATION FOR SEQ ID NO:80:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 126 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 103..126(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:CGCAACATACCTAACTGCTTCATTTCTGATCCATAATTAATTAATTTTTATCCCGGCGCG60CCTCGACTCTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATGAATTCGGAT114MetAsnSerAsp1CCCGTCGTTTTA126ProValValLeu5(2) INFORMATION FOR SEQ ID NO:81:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:82:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 88 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGGTCGACCTGCAG88GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:83:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:84:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:TAATGTATCTATAATGGTATAAAGCTTGTATTCTATAGTGTCACCTAAATC51(2) INFORMATION FOR SEQ ID NO:85:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:ACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGCCCGGGGATCT51(2) INFORMATION FOR SEQ ID NO:86:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 119 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 99..119(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:GCGGCCGCCTGCAGGTCGACTCTAGATTTTTTTTTTTTTTTTTTTTGGCATATAAATAGA60TCTGTATCCTAAAATTGAATTGTAATTATCGATAATAAATGAATTCGCTACTT113MetAsnSerLeuLeu15GGAACT119GlyThr(2) INFORMATION FOR SEQ ID NO:87:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:MetAsnSerLeuLeuGlyThr15(2) INFORMATION FOR SEQ ID NO:88:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 126 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..12(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 103..126(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:ATAAAAATGTGATTAAGTCTGAATGTGGATCCATAATTAATTAATTTTT49IleLysMet1ATCCCGGCGCGCCTCGACTCTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATG105Met1AATTCGGATCCCGTCGTTTTA126AsnSerAspProValValLeu5(2) INFORMATION FOR SEQ ID NO:89:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:IleLysMet1(2) INFORMATION FOR SEQ ID NO:90:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:91:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 111 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGGTCGAGGCGCGCCGGGTCGACCTG100GlnLysAspPro20CAGGCGGCCGC111(2) INFORMATION FOR SEQ ID NO:92:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:93:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:TAATGTATCTATAATGGTATAAAGCTTGTATTCTATAGTGTCACCTAAATC51(2) INFORMATION FOR SEQ ID NO:94:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:ACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGCCCGGGGATCT51(2) INFORMATION FOR SEQ ID NO:95:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 119 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 99..119(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:GCGGCCGCCTGCAGGTCGACTCTAGATTTTTTTTTTTTTTTTTTTTGGCATATAAATAGA60TCTGTATCCTAAAATTGAATTGTAATTATCGATAATAAATGAATTCCCCTGCC113MetAsnSerProAla15GCCCGG119AlaArg(2) INFORMATION FOR SEQ ID NO:96:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:MetAsnSerProAlaAlaArg15(2) INFORMATION FOR SEQ ID NO:97:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 126 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..36(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 103..126(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:CTCCAGGAGCCCGCTCGCCTCGAGCGGGATCCATAATTAATTAATTTTTATCC53LeuGlnGluProAlaArgLeuGluArgAspPro1510CGGCGCGCCTCGACTCTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATGAAT108MetAsn1TCGGATCCCGTCGTTTTA126SerAspProValValLeu5(2) INFORMATION FOR SEQ ID NO:98:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:LeuGlnGluProAlaArgLeuGluArgAspPro1510(2) INFORMATION FOR SEQ ID NO:99:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:MetAsnSerAspProValValLeu15(2) INFORMATION FOR SEQ ID NO:100:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 111 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..63(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:GAAATCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGT48GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015CAAAAAGATCCATAATTAATTAACCCGGGTCGAGGCGCGCCGGGTCGACCTG100GlnLysAspPro20CAGGCGGCCGC111(2) INFORMATION FOR SEQ ID NO:101:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:GluIleGlnLeuSerAlaGlyArgTyrHisTyrGlnLeuValTrpCys151015GlnLysAspPro20(2) INFORMATION FOR SEQ ID NO:102:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:TAATGTATCTATAATGGTATAAAGCTTGTATTCTATAGTGTCACCTAAATC51(2) INFORMATION FOR SEQ ID NO:103:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:CCGAATTCCGGCTTCAGTAACATAGGATCG30(2) INFORMATION FOR SEQ ID NO:104:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:GTACCCATACTGGTCGTGGC20(2) INFORMATION FOR SEQ ID NO:105:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:CCGGAATTCGCTACTTGGAACTCTGG26(2) INFORMATION FOR SEQ ID NO:106:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:CATTGTCCCGAGACGGACAG20(2) INFORMATION FOR SEQ ID NO:107:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:CGCGATCCAACTATCGGTG19(2) INFORMATION FOR SEQ ID NO:108:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:GCGGATCCACATTCAGACTTAATCAC26(2) INFORMATION FOR SEQ ID NO:109:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:ATGAATTCCCCTGCCGCCCGGACCGGCACC30(2) INFORMATION FOR SEQ ID NO:110:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:CATGGATCCCGCTCGAGGCGAGCGGGCTCC30(2) INFORMATION FOR SEQ ID NO:111:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 42 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:CTGGTTCGGCCCAGAATTCTATGGGTCTCGCGCGGCTCGTGG42(2) INFORMATION FOR SEQ ID NO:112:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 42 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Swinepox virus(B) STRAIN: Kasza(C) INDIVIDUAL ISOLATE: S-SPV- 001(vii) IMMEDIATE SOURCE:(B) CLONE: 515-85.1(viii) POSITION IN GENOME:(B) MAP POSITION: 23.2(C) UNITS: %G(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:CTCGCTCGCCCAGGATCCCTAGCGGAGGATGGACTTGAGTCG42__________________________________________________________________________

Number	Name	Date	Kind
5338683	Paoletti	Aug 1994
5382425	Cochran et al.	Jan 1995

Recombinant swinepox virus

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